Building a Comprehensive Mill-Level Database for the Industrial Sectors Integrated Solutions (ISIS) Model of the U.S. Pulp and Paper Sector

Air emissions from the U.S. pulp and paper sector have been federally regulated since 1978; however, regulations are periodically reviewed and revised to improve efficiency and effectiveness of existing emission standards. The Industrial Sectors Integrated Solutions (ISIS) model for the pulp and paper sector is currently under development at the U.S. Environmental Protection Agency (EPA), and can be utilized to facilitate multi-pollutant, sector-based analyses that are performed in conjunction with regulatory development. The model utilizes a multi-sector, multi-product dynamic linear modeling framework that evaluates the economic impact of emission reduction strategies for multiple air pollutants. The ISIS model considers facility-level economic, environmental, and technical parameters, as well as sector-level market data, to estimate the impacts of environmental regulations on the pulp and paper industry. Specifically, the model can be used to estimate U.S. and global market impacts of new or more stringent air regulations, such as impacts on product price, exports and imports, market demands, capital investment, and mill closures. One major challenge to developing a representative model is the need for an extensive amount of data. This article discusses the collection and processing of data for use in the model, as well as the methods used for building the ISIS pulp and paper database that facilitates the required analyses to support the air quality management of the pulp and paper sector.     

Mechanism of formation of amyloid protofibrils of barstar from soluble oligomers: evidence for multiple steps and lateral association coupled to conformational conversion

Understanding the heterogeneity of the soluble oligomers and protofibrillar structures that form initially during the process of amyloid fibril formation is a critical aspect of elucidating the mechanism of amyloid fibril formation by proteins. The small protein barstar offers itself as a good model protein for understanding this aspect of amyloid fibril formation, because it forms a stable soluble oligomer, the A form, at low pH, which can transform into protofibrils. The mechanism of formation of protofibrils from soluble oligomer has been studied by multiple structural probes, including binding to the fluorescent dye thioflavin T, circular dichroism and dynamic light scattering, and at different temperatures and different protein concentrations. The kinetics of the increase in any probe signal are single exponential, and the rate measured depends on the structural probe used to monitor the reaction. Fastest is the rate of increase in the mean hydrodynamic radius, which grows from a value of 6 nm for the A form to 20 nm for the protofibril. Slower is the rate of increase in thioflavin T binding capacity, and slowest is the rate of increase in circular dichroism at 216 nm, which occurs at about the same rate as that of the increase in light scattering intensity. The dynamic light scattering measurements suggest that the A form transforms completely into larger size aggregates at an early stage during the aggregation process. It appears that structural changes within the aggregates occur at the late stages of assembly into protofibrils. For all probes, and at all temperatures, no initial lag phase in protofibril growth is observed for protein concentrations in the range of 1 microM to 50 microM. The absence of a lag phase in the increase of any probe signal suggests that aggregation of the A form to protofibrils is not nucleation dependent. In addition, the absence of a lag phase in the increase of light scattering intensity, which changes the slowest, suggests that protofibril formation occurs through more than one pathway. The rate of aggregation increases with increasing protein concentration, but saturates at high concentrations. An analysis of the dependence of the apparent rates of protofibril formation, determined by the four structural probes, indicates that the slowest step during protofibil formation is lateral association of linear aggregates. Conformational conversion occurs concurrently with lateral association, and does so in two steps leading to the creation of thioflavin T binding sites and then to an increase in beta-sheet structure. Overall, the study indicates that growth during protofibril formation occurs step-wise through progressively larger and larger aggregates, via multiple pathways, and finally through lateral association of critical aggregates.     

Effects of PKA phosphorylation of cardiac troponin I and strong crossbridge on conformational transitions of the N-domain of cardiac troponin C in regulated thin filaments

Regulation of cardiac muscle function is initiated by binding of Ca2+ to troponin C (cTnC) which induces a series of structural changes in cTnC and other thin filament proteins. These structural changes are further modulated by crossbridge formation and fine-tuned by phosphorylation of cTnI. The objective of the present study is to use a new F?rster resonance energy transfer-based structural marker to distinguish structural and kinetic effects of Ca2+ binding, crossbridge interaction, and protein kinase A phosphorylation of cTnI on the conformational changes of the cTnC N-domain. The FRET-based structural marker was generated by attaching AEDANS to one cysteine of a double-cysteine mutant cTnC(13C/51C) as a FRET donor and attaching DDPM to the other cysteine as the acceptor. The doubly labeled cTnC mutant was reconstituted into the thin filament by adding cTnI, cTnT, tropomyosin, and actin. Changes in the distance between Cys13 and Cys51 induced by Ca2+ binding/dissociation were determined by FRET-sensed Ca2+ titration and stopped-flow studies, and time-resolved fluorescence measurements. The results showed that the presence of both Ca2+ and strong binding of myosin head to actin was required to achieve a fully open structure of the cTnC N-domain in regulated thin filaments. Equilibrium and stopped-flow studies suggested that strongly bound myosin head significantly increased the Ca2+ sensitivity and changed the kinetics of the structural transition of the cTnC N-domain. PKA phosphorylation of cTnI impacted the Ca2+ sensitivity and kinetics of the structural transition of the cTnC N-domain but showed no global structural effect on cTnC opening. These results provide an insight into the modulation mechanism of strong crossbridge and cTnI phosphorylation in cardiac thin filament activation/relaxation processes.     

Production and partial characterization of two types of phytase from Aspergillus niger NCIM 563 under submerged fermentation conditions

Novel extracellular phytase was produced by Aspergillus niger NCIM 563 under submerged fermentation conditions at 30 °C in medium containing dextrin and glucose as carbon sources along with sodium nitrate as nitrogen source. Maximum phytase activity (41.47 IU/mL at pH 2.5 and 10.71 IU/mL at pH 4.0) was obtained when dextrin was used as carbon source along with glucose and sodium nitrate as nitrogen source. Nearly 13 times increase in phytase activity was observed when phosphate in the form of KH₂PO₄ (0.004 g/100 mL) was added in the fermentation medium. Physic-chemical properties of partially purified enzyme indicate the possibility of two distinct forms of phytases, Phy I and Phy II. Optimum pH and temperature for Phy I was 2.5 and 60 °C while Phy II was 4.0 and 60 °C, respectively. Phy I was stable in the pH range 1.5–3.5 while Phy II was stable in the wider pH range, 2.0–7.0. Molecular weight of Phy I and Phy II on Sephacryl S-200 was approximately 304 kDa and 183 kDa, respectively. Phy I activity was moderately stimulated in the presence of 1 mM Mg²⁺, Mn²⁺, Ca²⁺ and Fe³⁺ ions and inhibited by Zn²⁺ and Cd²⁺ ions while Phy II activity was moderately stimulated by Fe³⁺ ions and was inhibited by Hg²⁺, Mn²⁺ and Zn²⁺ ions at 1 mM concentration in reaction mixture. The Km for Phy I and II was 3.18 and 0.514 mM while Vmax was 331.16 and 59.47 μmols/min/mg protein, respectively.     

Heterologous expression, purification and characterization of heterodimeric monellin

Monellin is an intensely sweet-tasting protein present in the berry of Dioscoreophyllum cumminsii. Naturally occurring monellin (double chain monellin) is a heterodimer of two subunits commonly referred to as chain A and chain B. Monellin is a good model system for structural and dynamic studies of proteins. Single chain monellin has been generated by covalently linking the two subunits of naturally occurring double chain monellin, and has been used extensively for folding and unfolding studies, as well as for protein aggregation studies. There are, however, relatively few reports on such studies with double chain monellin. The primary difficulty associated with studies using double chain monellin appears to be the lack of a standard purification method. Here, a simple method for the purification of double chain monellin is presented. The genes encoding the two chains of monellin were cloned into a modified pETDUET vector under separate T7 promoters. The expression vector containing the genes of the two chains was expressed in E. coli BL21 Star (DE3). The expressed protein was purified using two steps of chromatography, ion exchange chromatography and gel filtration chromatography. This expression system consistently produced 40 mg of pure double chain monellin per litre of E. coli culture, in the correctly folded native state. The purity of the protein was confirmed by mass spectrometry and SDS-PAGE analysis. The purified protein was characterized using different spectroscopic methods, and the spectra obtained were in good agreement with the published spectra of naturally occurring double chain monellin.     

Protein dynamics control proton transfer from bulk solvent to protein interior: a case study with a green fluorescent protein

The kinetics of proton transfer in Green Fluorescent Protein (GFP) have been studied as a model system for characterizing the correlation between dynamics and function of proteins in general. The kinetics in EGFP (a variant of GFP) were monitored by using a laser-induced pH jump method. The pH was jumped from 8 to 5 by nanosecond flash photolysis of the "caged proton," o-nitrobenzaldehyde, and subsequent proton transfer was monitored by following the decrease in fluorescence intensity. The modulation of proton transfer kinetics by external perturbants such as viscosity, pH, and subdenaturing concentrations of GdnHCl as well as of salts was studied. The rate of proton transfer was inversely proportional to solvent viscosity, suggesting that the rate-limiting step is the transfer of protons through the protein matrix. The rate is accelerated at lower pH values, and measurements of the fluorescence properties of tryptophan 57 suggest that the enhancement in rate is associated with an enhancement in protein dynamics. The rate of proton transfer is nearly independent of temperature, unlike the rate of the reverse process. When the stability of the protein was either decreased or increased by the addition of co-solutes, including the salts KCl, KNO(3), and K(2)SO(4), a significant decrease in the rate of proton transfer was observed in all cases. The lack of correlation between the rate of proton transfer and the stability of the protein suggests that the structure is tuned to ensure maximum efficiency of the dynamics that control the proton transfer function of the protein.     

An efficient route to thioglycosides with the 2,3-anhydro-D-ribo stereochemistry

An improved route for the synthesis of p-tolyl 2,3-anhydro-5-O-benzoyl-1-thio-beta-D-ribofuranoside and its alpha anomer, which are important intermediates in the synthesis of alpha- and beta-D-arabinofuranosides, has been developed. The products are obtained in six steps from D-xylose in 39% overall yield.     

Dendrimer versus linear conjugate: Influence of polymeric architecture on the delivery and anticancer effect of paclitaxel

The relative difference in polymeric architectures of dendrimer and linear bis(poly(ethylene glycol)) (PEG) polymer in conjugation with paclitaxel has been described. Paclitaxel, a poorly soluble anticancer drug, was covalently conjugated with PAMAM G4 hydroxyl-terminated dendrimer and bis(PEG) polymer for the potential enhancement of drug solubility and cytotoxicity. Both conjugates were characterized by 1NMR, HPLC, and MALDI/TOF. In addition, molecular conformations of dendrimer, bis(PEG), paclitaxel, and its polymeric conjugates were studied by molecular modeling. Hydrolysis of the ester bond in the conjugate was analyzed by HPLC using esterase hydrolyzing enzyme. In vitro cytotoxicity of dendrimer, bis(PEG), paclitaxel, and polymeric conjugates containing paclitaxel was evaluated using A2780 human ovarian carcinoma cells. Cytotoxicity increased by 10-fold with PAMAM dendrimer-succinic acid-paclitaxel conjugate when compared with free nonconjugated drug. Data obtained indicate that the nanosized dendritic polymer conjugates can be used with good success as anticancer drug carriers.     

Activation of the NLRP3 inflammasome by intracellular poly I:C

Several RNA viruses can be detected by the inflammasome, which promotes IL-1β and IL-18 secretion, but the underlying mechanisms of detection remain unclear. Cytosolic dsRNA is a replication intermediate of many RNA viruses. We show here that transfection of the dsRNA analogue poly I:C activates the NLRP3 inflammasome via a pathway requiring endosomal acidification. This detection is independent of the other poly I:C sensors: TLR3 and MDA5. These results suggest a mechanism by which cytosolic dsRNA produced during viral infection could activate the NLRP3 inflammasome.     

Open Conformation of Ezrin Bound to Phosphatidylinositol 4,5-Bisphosphate and to F-actin Revealed by Neutron Scattering

Ezrin is a member of the ezrin-radixin-moesin family (ERM) of adapter proteins that are localized at the interface between the cell membrane and the cortical actin cytoskeleton, and they regulate a variety of cellular functions. The structure representing a dormant and closed conformation of an ERM protein has previously been determined by x-ray crystallography. Here, using contrast variation small angle neutron scattering, we reveal the structural changes of the full-length ezrin upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP₂) and to F-actin. Ezrin binding to F-actin requires the simultaneous binding of ezrin to PIP₂. Once bound to F-actin, the opened ezrin forms more extensive contacts with F-actin than generally depicted, suggesting a possible role of ezrin in regulating the interfacial structure and dynamics between the cell membrane and the underlying actin cytoskeleton. In addition, using gel filtration, we find that the conformational opening of ezrin in response to PIP₂ binding is cooperative, but the cooperativity is disrupted by a phospho-mimic mutation S249D in the 4.1-ezrin/radixin/moesin (FERM) domain of ezrin. Using surface plasmon resonance, we show that the S249D mutation weakens the binding affinity and changes the kinetics of 4.1-ERM to PIP₂ binding. The study provides the first structural view of the activated ezrin bound to PIP₂ and to F-actin.