Green Monomer Research Articles

Acoustic Study Based on Sustainable Green Polymer Treated with H<sub>2</sub>O

Green polymer foam was prepared by the reaction of green monomer based on vegetable oil with commercial Polymethane Polyphenyl Isocyanate (Modified Polymeric-MDI) and distilled water (H2O). The morphological study of green polymer foam was examined by Scanning Electron Microscope (SEM) and acoustic property by means of H2O composition ratio equivalent to weight of polyol. It was found that the cell size of green polymer foam has significantly increment as well as H2O loading increased. Increasing of H2O more than 50% equivalent to weight of polyol shows nonuniform pore distribution, large average pore size and smallest number of pore. Furthermore, the cell size of neat green polymer foam gives 400μm up to 1833.3μm with high loading of H2O. In addition, the cell size of green polymer foam influences by the increasing amount of H2O loading and enhanced the sound absorption property at low frequency level.

Enzyme-catalyzed synthesis of unsaturated aliphatic polyesters based on green monomers from renewable resources.

Bio-based commercially available succinate, itaconate and 1,4-butanediol are enzymatically co-polymerized in solution via a two-stage method, using Candida antarctica Lipase B (CALB, in immobilized form as Novozyme® 435) as the biocatalyst. The chemical structures of the obtained products, poly(butylene succinate) (PBS) and poly(butylene succinate-co-itaconate) (PBSI), are confirmed by 1H- and 13C-NMR. The effects of the reaction conditions on the CALB-catalyzed synthesis of PBSI are fully investigated, and the optimal polymerization conditions are obtained. With the established method, PBSI with tunable compositions and satisfying reaction yields is produced. The 1H-NMR results confirm that carbon-carbon double bonds are well preserved in PBSI. The differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA) results indicate that the amount of itaconate in the co-polyesters has no obvious effects on the glass-transition temperature and the thermal stability of PBS and PBSI, but has significant effects on the melting temperature.

Poly(brilliant green)/carbon nanotube-modified carbon film electrodes and application as sensors

Poly(brilliant green) (PBG) films were formed on carbon film electrodes (CFE) by electropolymerisation of brilliant green monomer using potential cycling or at fixed potential from different pH solutions. The modified electrodes, PBG/CFE, were characterised by cyclic voltammetry (CV) in electrolytes of different pH by electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). In order to increase the stability of the polymer film and enhance the response, multi-walled carbon nanotubes (MWCNTs) were first deposited on CFE and then PBG was formed on top, PBG/CNT/CFE. The modified electrodes were applied to the amperometric determination of ascorbic acid (AA) in phosphate buffer pH 7.0 at 0.0 V vs. saturated calomel electrode (SCE) and the results were compared, the presence of CNT leading to a significant increase in sensitivity. An interference study was carried out and good separation between AA and dopamine (DA) peaks was achieved that led to the successful determination of DA without interferences. Other interferents: aspirin, acetaminophen, salicylic acid and uric acid exhibited no response on the PBG/CNT/CFE. Determination of AA in pharmaceutical samples was successfully performed.

Revealing the Excited-State Dynamics of the Fluorescent Protein Dendra2

Green-to-red photoconversion is a reaction that occurs in a limited number of fluorescent proteins and that is currently mechanistically debated. In this contribution, we report on our investigation of the photoconvertible fluorescent protein Dendra2 by employing a combination of pump-probe, up-conversion and single photon timing spectroscopic techniques. Our findings indicate that upon excitation of the neutral green state an excited state proton transfer proceeds with a time constant of 3.4 ps between the neutral green and the anionic green states. In concentrated solution we detected resonance energy transfer (25 ps time constant) between green and red monomers. The time-resolved emission spectra suggest also the formation of a super-red species, first observed for DsRed (a red fluorescent protein from the corallimorph species Discosoma) and consistent with peculiar structural details present in both proteins.

Improvement of the amperometric response to l-lactate by using a cationic bioinspired thymine polycation in a bioelectrode with immobilized lactate oxidase

We report the electroanalytical properties of an amperometric bioelectrode containing l-lactate oxidase (LOx) immobilized on glassy carbon electrode with a hydrogel film composed of laponite and different amounts of a novel bioinspired polycation obtained by copolymerization of 4-vinylbenzyl thymine (VBT) and 4-vinylbenzyl triethylammonium chloride (VBA) in a molar ratio 1:4, respectively. The electrochemical behavior of the redox couple probe [Fe(CN)6]3−/4− of these VBT–VBA bioelectrodes was compared with that observed for a bioelectrode containing the classical polycation polydiallyldimethylammonium chloride (PDDA). The best response was obtained for a bioelectrode containing a VBT–VBA/laponite mass ratio double than the cationic exchange capacity of the clay, demonstrating that under this condition the polycation induces an optimal microenvironment in the interlamellar space of the clay, both for the position and the functionality of LOx.The VBT–VBA bioelectrode displayed a very high sensitivity (7.2±0.2)×102μAmM−1cm−2, a short time response (<5s), a wide linear response range (e.g. 0.01–1.0mM of l-lactate) and an excellent stability over a storage period of 60 days, when sensing l-lactate. The analytical response of the bioelectrode was tested in real food samples, e.g. milk, white wine, and beer, as well as during milk fermentation at 37°C. No effect of molecular interferences in the food matrices was detected, and the quantification of l-lactate was in complete agreement with standard assays reported values.Current results indicate that polycations containing the multifunctional green monomer VBT have high potential for their use in hydrogel film formation producing more responsive and stable electrochemical biosensors.

Green Polymer Chemistry and Bio‐based Plastics: Dreams and Reality

AbstractDwindling fossil resources, surging energy demand and global warming stimulate growing demand for renewable polymer products with low carbon footprint. Going well beyond the limited scope of natural polymers, biomass conversion in biorefineries and chemical carbon dioxide fixation are teamed up with highly effective tailoring, processing and recycling of polymers. “Green monomers” from biorefineries, and “renewable oil”, gained from plastics' and bio wastes, render synthetic polymers renewable without impairing their property profiles and recycling. In context of biofuel production, limitations of the green economy concepts are clearly visible. Dreams and reality of “green polymers” are highlighted. Regardless of their new greenish touch, highly versatile and cost‐effective polymers play an essential role in sustainable development.

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3. mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function. Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established(1), accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca(2+) homeostasis(2), bioenergetics and redox signaling( 3). mtPTP opening is triggered by matrix Ca(2+) but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids(4). In vitro, mtPTP opening can be achieved by increasing Ca(2+) concentration inside the mitochondrial matrix through exogenous additions of Ca(2+) (calcium retention capacity). When Ca(2+) levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca(2+) release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function. Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca(2+) handling (uptake and release, as measured by Ca(2+) concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca(2+) measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca(2+), and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

Lyophilization and enhanced stability of fluorescent protein nanoparticles

Protein nanoparticles (PNPs) that are nanostructured biomaterials with intrinsic biological function have been widely employed as three-dimensional nanobiomaterials for sensitive bioassays, MRI contrast, semiconductor devices, template for hybrid materials, etc., and stable and long-term maintenance of PNPs seems to be of crucial importance. We evaluated the stability of PNPs and the efficacy of lyophilization for the long-term stability of PNPs, especially using green fluorescent protein nanoparticles (gFPNPs) as a model PNP. Fluorescence intensities and TEM images of gFPNPs were analyzed to monitor their functional and structural stabilities. Unlike the green fluorescent protein monomers (eGFP) that were gradually inactivated in aqueous solution, gFPNP in the same aqueous solution retained the initial fluorescence activity and spherical nanoparticle structure even for 2 weeks at 4 °C. To ensure stable and long-term maintenance of gFPNPs, gFPNPs in aqueous solution were converted to the dried solid forms through lyophilization. It is notable that fluorescence activity and nanoparticle structure of gFPNPs that were lyophilized with both Tween 80 and sucrose were very stably maintained even for 10 weeks at various storage temperatures (−20 °C, 4 °C, 25 °C, and 37 °C). During the period of 10 weeks, the fluorescence of gFPNP was always more than 80% level of initial fluorescence at a wide range of temperature. Although this stability study was focused on gFPNPs, the developed optimal lyophilization conditions for gFPNPs can be applied in general to stable and long-term maintenance of many other PNP-derived biomaterials.

Visualization of Actin Polymerization in Invasive Structures of Macrophages and Carcinoma Cells Using Photoconvertible β-Actin – Dendra2 Fusion Proteins

Actin polymerization controls a range of cellular processes, from intracellular trafficking to cell motility and invasion. Generation and elongation of free barbed ends defines the regions of actively polymerizing actin in cells and, consequently, is of importance in the understanding of the mechanisms through which actin dynamics are regulated. Herein we present a method that does not involve cell permeabilization and provides direct visualization of growing barbed ends using photoswitchable β-actin - Dendra2 constructs expressed in murine macrophage and rat mammary adenocarcinoma cell lines. The method exploits the ability of photoconverted (red) G-actin species to become incorporated into pre-existing (green) actin filaments, visualized in two distinct wavelengths using TIRF microscopy. In growing actin filaments, photoconverted (red) monomers are added to the barbed end while only green monomers are recycled from the pointed end. We demonstrate that incorporation of actin into intact podosomes of macrophages occurs constitutively and is amenable to inhibition by cytochalasin D indicating barbed end incorporation. Additionally, actin polymerization does not occur in quiescent invadopodial precursors of carcinoma cells suggesting that the filaments are capped and following epidermal growth factor stimulation actin incorporation occurs in a single but extended peak. Finally, we show that Dendra2 fused to either the N- or the C-terminus of β-actin profoundly affects its localization and incorporation in distinct F-actin structures in carcinoma cells, thus influencing the ability of monomers to be photoconverted. These data support the use of photoswitchable actin-Dendra2 constructs as powerful tools in the visualization of free barbed ends in living cells.

The Antioxidant Transcription Factor Nrf2 Negatively Regulates Autophagy and Growth Arrest Induced by the Anticancer Redox Agent Mitoquinone

Mitoquinone (MitoQ) is a synthetically modified, redox-active ubiquinone compound that accumulates predominantly in mitochondria. We found that MitoQ is 30-fold more cytotoxic to breast cancer cells than to healthy mammary cells. MitoQ treatment led to irreversible inhibition of clonogenic growth of breast cancer cells through a combination of autophagy and apoptotic cell death mechanisms. Relatively limited cytotoxicity was seen with the parent ubiquinone coenzyme Q(10.) Inhibition of cancer cell growth by MitoQ was associated with G(1)/S cell cycle arrest and phosphorylation of the checkpoint kinases Chk1 and Chk2. The possible role of oxidative stress in MitoQ activity was investigated by measuring the products of hydroethidine oxidation. Increases in ethidium and dihydroethidium levels, markers of one-electron oxidation of hydroethidine, were observed at cytotoxic concentrations of MitoQ. Keap1, an oxidative stress sensor protein that regulates the antioxidant transcription factor Nrf2, underwent oxidation, degradation, and dissociation from Nrf2 in MitoQ-treated cells. Nrf2 protein levels, nuclear localization, and transcriptional activity also increased following MitoQ treatment. Knockdown of Nrf2 caused a 2-fold increase in autophagy and an increase in G(1) cell cycle arrest in response to MitoQ but had no apparent effect on apoptosis. The Nrf2-regulated enzyme NQO1 is partly responsible for controlling the level of autophagy. Keap1 and Nrf2 act as redox sensors for oxidative perturbations that lead to autophagy. MitoQ and similar compounds should be further evaluated for novel anticancer activity.

Oligocarbonate diols from ethylene carbonate—Optimization of the synthesis process

AbstractOligocarbonate diols due to their resistance to oxidation and hydrolysis are particularly valuable components of polyurethanes for biomedical applications. It was shown that for their synthesis “green monomer,” ethylene carbonate can be used in the reaction with 1,6‐hexanediol, instead of usually applied toxic and harmful phosgene. Depending on reaction conditions, besides ester exchange leading to the desired product, competitive etherification is often observed. To optimize the reaction conditions leading to oligocarbonates of high molecular weight without oxyethylene fragments, the method of an experimental design was applied. Such approach enabled the estimation of the influence of reaction temperature, ethylene carbonate to 1,6‐hexanediol molar ratio and catalyst (NaCl) concentration on the molar mass of oligocarbonate diol, content of ether bonds and reaction time. Application of central composite method as an experimental design allowed not only to choose the optimal set of conditions, but also the coefficients of the regression equation were interpreted in a chemical way. Oligocarbonate diols obtained under optimal conditions were used for synthesis poly(urethane‐urea)s which exhibited very good mechanical properties (tensile strength 45–50 MPa and elongation at break up to 500%). © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

Plasma Membrane Ca2+-ATPase Overexpression Depletes Both Mitochondrial and Endoplasmic Reticulum Ca2+ Stores and Triggers Apoptosis in Insulin-secreting BRIN-BD11 Cells

Ca(2+) may trigger apoptosis in β-cells. Hence, the control of intracellular Ca(2+) may represent a potential approach to prevent β-cell apoptosis in diabetes. Our objective was to investigate the effect and mechanism of action of plasma membrane Ca(2+)-ATPase (PMCA) overexpression on Ca(2+)-regulated apoptosis in clonal β-cells. Clonal β-cells (BRIN-BD11) were examined for the effect of PMCA overexpression on cytosolic and mitochondrial [Ca(2+)] using a combination of aequorins with different Ca(2+) affinities and on the ER and mitochondrial pathways of apoptosis. β-cell stimulation generated microdomains of high [Ca(2+)] in the cytosol and subcellular heterogeneities in [Ca(2+)] among mitochondria. Overexpression of PMCA decreased [Ca(2+)] in the cytosol, the ER, and the mitochondria and activated the IRE1α-XBP1s but inhibited the PRKR-like ER kinase-eIF2α and the ATF6-BiP pathways of the ER-unfolded protein response. Increased Bax/Bcl-2 expression ratio was observed in PMCA overexpressing β-cells. This was followed by Bax translocation to the mitochondria with subsequent cytochrome c release, opening of the permeability transition pore, and apoptosis. In conclusion, clonal β-cell stimulation generates microdomains of high [Ca(2+)] in the cytosol and subcellular heterogeneities in [Ca(2+)] among mitochondria. PMCA overexpression depletes intracellular [Ca(2+)] stores and, despite a decrease in mitochondrial [Ca(2+)], induces apoptosis through the mitochondrial pathway. These data open the way to new strategies to control cellular Ca(2+) homeostasis that could decrease β-cell apoptosis in diabetes.

Synthesis of oligocarbonate diols from a "green monomer" – dimethyl carbonate – as soft segments for poly(urethane-urea) elastomers

Summary — Results from the investigation of a two-step synthesis of oligocarbonate diols from a “green monomer” — dimethyl carbonate (DMC) are presented and discussed. In the first step 1,6-hexanediol or 1,10-decanediol was reacted with DMC to obtain bis(methylcarbonate)hexamethylene (1h) or bis(methylcarbonate)decamethylene (1d), respectively which were further reacted in the next step with appropriate diol at a intended molar ratio to obtain the final product. The solvent — 1,4-dioxane — served as a suppressant of the evaporation of both the diol and low molecular weight oligomers, while facilitating the removal of residual amount of methanol and full conversion of methylcarbonate groups. This method allows for the synthesis of oligocarbonate diols without ether linkages containing exclusively terminal hydroxyl groups and of desired molecular weights. It was shown that such oligomerols can be applied for the preparation of poly(urethane-urea) elastomers. The obtained elastomers based on oligocarbonate diols of molecular weights ranging from 1700 to 2700 exhibited very good mechanical properties.

Green Chemistry: Sunlight-Induced Cationic Polymerization of Renewable Epoxy Monomers Under Air

The recently developed silyl radical chemistry is used here for the free-radical-promoted cationic polymerization process (FRPCP) of two epoxy monomers (epoxidized soybean oil and limonene dioxide) that are representative of green monomers. The new developed photoinitiating systems are highly efficient under air upon a solar irradiation in autumn French weather and, for example, lead to 60% conversion after 25 mn and form a completely tack-free and uncolored coating after 1 h. Polymerization profiles obtained upon a visible light irradiation delivered by a xenon lamp or a 405 nm diode laser under air show that 70% conversion can be easily achieved after 400 s of exposure. Excited-state processes are investigated and discussed.

Novel Function of PERK as a Mediator of Force-induced Apoptosis

Induction of apoptosis by tensile forces is an important determinant of connective tissue destruction in osteoarthritis and periodontal diseases. We examined the role of molecular components of the unfolded protein response in force-induced apoptosis. Magnetic fields were used to apply tensile force through integrins to cultured fibroblasts bound with collagen-coated magnetite beads. Tensile force induced caspase 3 cleavage, DNA fragmentation, depolarization of mitochondria, and induction of CHOP10, all indicative of activation of apoptosis. Immunoblotting, immunocytochemistry, and release of Ca(2+) from the endoplasmic reticulum showed evidence for both physical and functional associations between bound beads and the endoplasmic reticulum. Force-induced apoptosis was not detected in PERK null cells, but reconstitution of wild-type PERK in PERK null cells restored the apoptotic response. Force-induced apoptosis did not require PKR, GCN2, eIF2alpha, or CHOP10. Furthermore, force more than 24 h did not activate other initiators of the unfolded protein response including IRE-1 and ATF6. However, force-induced activation of caspase 3 was dependent on caspase 9 but was independent of mitochondria. We conclude that force-induced apoptosis depends on a novel function of PERK that occurs in addition to its canonical role in the unfolded protein response.

Systematic Colocalization Errors between Acridine Orange and EGFP in Astrocyte Vesicular Organelles

Dual-color imaging of acridine orange (AO) and EGFP fused to a vesicular glutamate transporter or the vesicle-associated membrane proteins 2 or 3 has been used to visualize a supposedly well-defined subpopulation of glutamatergic astrocytic secretory vesicles undergoing regulated exocytosis. However, AO metachromasy results in the concomitant emission of green and red fluorescence from AO-stained tissue. Therefore, the question arises whether AO and EGFP fluorescence can be distinguished reliably. We used evanescent-field imaging with spectral fluorescence detection as well as fluorescence lifetime imaging microscopy to demonstrate that green fluorescent AO monomers inevitably coexist with red fluorescing AO dimers, at the level of single astroglial vesicles. The green monomer emission spectrally overlaps with that of EGFP and produces a false apparent colocalization on dual-color images. On fluorophore abundance maps calculated from spectrally resolved and unmixed single-vesicle spectral image stacks, EGFP is obscured by the strong green monomer fluorescence, precluding the detection of EGFP. Hence, extreme caution is required when deriving quantitative colocalization information from images of dim fluorescing EGFP-tagged organelles colabeled with bright and broadly emitting dyes like AO. We finally introduce FM4-64/EGFP dual-color imaging as a remedy for imaging a distinct population of astroglial fusion-competent secretory vesicles.

Synthesis, characterization, electrochemical and solvatochromic investigations of novel monomeric and polymeric vanadyl Schiff-base complexes

This article describes the synthesis and characterization of oxovanadium(IV) complexes containing tetradentate Schiff-base ligands derived from condensation of ethylenediamine, meso-1,2-diphenyl-1,2-ethylenediamine, 1,2-orthophenylenediamine and 1,2-cyclohexanediamine with 5-bromo-3-nitro-2-hydroxybenzaldehyde. The novel VOL1:[VO(5-Br-3-NO2salen)], VOL2:[VO(5-Br-3-NO2saloph)] and VOL3:[VO(5-Br-3-NO2salchxn)] complexes were obtained in orange polymeric form with (V=O) stretching bands at 878, 884 and 884 cm−1, respectively, but the VOL4 complex was obtained as a green monomer with (V=O) stretching band at 978 cm−1. The redox process in acetonitrile was reversible for VOL4 and E° was determined to be approximately 950 mV but for VOL1–3 this process was irreversible or quasi-reversible. The VOL4 complex is considerably soluble in a wide variety of solvents and shows solvatochromic behavior. The 2 B 2 →2 E(I) transition wavenumber shows a linear correlation to the D.N. of the solvent.

Quantitative Analysis of Mitochondrial Membrane Potential Measurements with JC‐1

To guide quantitative investigations into the regulation of mitochondrial metabolism, a computational model that simulates underlying chemical kinetics and thermodynamics of the mitochondrial respiratory chain and oxidative phosphorylation was developed It has been successfully extended to analyze mitochondrial membrane potential measurements with rhodamine 123, one of the most used potential sensitive probes Another dye that is rapidly gaining popularity is JC-1. Its wide application is attributed to its visually appealing dual color detection due to fluorescence emission of both green monomer and red J-aggregates in differential response to membrane potential changes. Although response of the fluorescence signal to membrane potential changes has been calibrated, the lack of consistency, of biophysical understanding of how membrane potential and measured fluorescence are related and how the relationship is impacted by different factors, has limited the quantitative application of the dye. Here we integrate the JC-1 transport, aggregation, quenching and partition into the membrane into the mitochondrial model. The model simulation is consistent with experimental observations. This study provides a useful theoretical framework for experimental design and assessment of both static and transient membrane potential using JC-1. Beard DA. A biophysical model of the mitochondrial respiratory chain and oxidative phosphorylation. PLOS Computational Biology, 2005 Huang M, Camara A, DF Stowe, Qi F, Beard DA. Mitochondrial Inner Membrane Electrophysiology Assessed by Rhodamine-123 Transport and Fluorescence. (in review; available on-line). This work is supported by NIH grant HL072011

Structure of Chlorobium tepidum Sepiapterin Reductase Complex Reveals the Novel Substrate Binding Mode for Stereospecific Production of l-threo-Tetrahydrobiopterin

Sepiapterin reductase (SR) is involved in the last step of tetrahydrobiopterin (BH(4)) biosynthesis by reducing the di-keto group of 6-pyruvoyl tetrahydropterin. Chlorobium tepidum SR (cSR) generates a distinct BH(4) product, L-threo-BH(4) (6R-(1'S,2'S)-5,6,7,8-BH(4)), whereas animal enzymes produce L-erythro-BH(4) (6R-(1'R,2'S)-5,6,7,8-BH(4)) although it has high amino acid sequence similarities to the other animal enzymes. To elucidate the structural basis for the different reaction stereospecificities, we have determined the three-dimensional structures of cSR alone and complexed with NADP and sepiapterin at 2.1 and 1.7 A resolution, respectively. The overall folding of the cSR, the binding site for the cofactor NADP(H), and the positions of active site residues were quite similar to the mouse and the human SR. However, significant differences were found in the substrate binding region of the cSR. In comparison to the mouse SR complex, the sepiapterin in the cSR is rotated about 180 degrees around the active site and bound between two aromatic side chains of Trp-196 and Phe-99 so that its pterin ring is shifted to the opposite side, but its side chain position is not changed. The swiveled sepiapterin binding results in the conversion of the side chain configuration, exposing the opposite face for hydride transfer from NADPH. The different sepiapterin binding mode within the conserved catalytic architecture presents a novel strategy of switching the reaction stereospecificities in the same protein fold.