A knowledge-based potential highlights unique features of membrane α-helical and β-barrel protein insertion and folding

Outer membrane β-barrel proteins differ from α-helical inner membrane proteins in lipid environment, secondary structure, and the proposed processes of folding and insertion. It is reasonable to expect that outer membrane proteins may contain primary sequence information specific for their folding and insertion behavior. In previous work, a depth-dependent insertion potential, E(z) , was derived for α-helical inner membrane proteins. We have generated an equivalent potential for TM β-barrel proteins. The similarities and differences between these two potentials provide insight into unique aspects of the folding and insertion of β-barrel membrane proteins. This potential can predict orientation within the membrane and identify functional residues involved in intermolecular interactions.     

Nrf2 regulates an adaptive response protecting against oxidative damage following diquat-mediated formation of superoxide anion

Mouse embryonic fibroblasts derived from Nrf2-/- mice (N0) and Nrf2+/+ mice (WT) have been used to characterize both basal and diquat (DQ)-induced oxidative stress levels and to examine Nrf2 activation during exposure to DQ-generated superoxide anion. Microarray analysis revealed that N0 cells have similar constitutive mRNA expression of genes responsible for the direct metabolism of reactive oxygen species but decreased expression of genes responsible for the production of reducing equivalents, repair of oxidized proteins and defense against lipid peroxidation, compared to WT cells. Nonetheless, the basal levels of ROS flux and oxidative damage biomarkers in WT and N0 cells were not different. Diquat dibromide (DQ), a non-electrophilic redox cycling bipyridylium herbicide, was used to generate intracellular superoxide anion. Isolated mitochondria from both cell lines exposed to DQ produced equivalent amounts of ROS, indicating a similar cellular capacity to generate ROS. However, N0 cells exposed to DQ for 24-h exhibited markedly decreased cell viability and aconitase activity as well as increased lipid peroxidation and glutathione oxidation, relative to WT cells. 2',7'-Dichlorofluorescein fluorescence was not increased in WT and N0 cells after 30-min of DQ exposure. However, increased levels of ROS were detected in N0 cells but not WT cells after 13-h of DQ treatment. Additionally, total glutathione concentrations increased in WT, but not N0 cells following a 24-h exposure to DQ. DQ exposure resulted in activation of an antioxidant response element-luciferase reporter gene, as well as induction of Nrf2-regulated genes in WT, but not N0 cells. Thus the enhanced sensitivity of N0 cells does not reflect basal differences in antioxidative capacity, but rather an impaired ability to mount an adaptive response to sustained oxidative stress.     

Stability of 100 homo and heterotypic coiled-coil a-a' pairs for ten amino acids (A, L, I, V, N, K, S, T, E, and R)

We present the thermal stability monitored by circular dichroism (CD) spectroscopy at 222 nm of 100 heterodimers that contain all possible coiled-coil a-a' pairs for 10 amino acids (I, V, L, N, A, K S, T, E, and R). This includes the stability of 36 heterodimers for 6 amino acids (I, V, L, N, A, and K) previously described and 64 new heterodimers including the 4 amino acids (S, T, E, and R). We have calculated a double mutant alanine thermodynamic cycle to determine a-a' pair coupling energies to evaluate which a-a' pairs encourage specific dimerization partners. The four new homotypic a-a' pairs (T-T, S-S, R-R, E-E) are repulsive relative to A-A and have destabilizing coupling energies. Among the 90 heterotypic a-a' pairs, the stabilizing coupling energies contain lysine or arginine paired with either an aliphatic or a polar amino acid. The range in coupling energies for each amino acid reveals its potential to regulate dimerization specificity. The a-a' pairs containing isoleucine and asparagine have the greatest range in coupling energies and thus contribute dramatically to dimerization specificity, which is to encourage homodimerization. In contrast, the a-a' pairs containing charged amino acids (K, R, and E) show the least range in coupling energies and promiscuously encourage heterodimerization.     

The catalytic machinery of chondroitinase ABC I utilizes a calcium coordination strategy to optimally process dermatan sulfate

The chondroitinases are bacterial lyases that specifically cleave chondroitin sulfate and/or dermatan sulfate glycosaminoglycans. One of these enzymes, chondroitinase ABC I from Proteus vulgaris, has the broadest substrate specificity and has been widely used to depolymerize these glycosaminoglycans. Biochemical and structural studies to investigate the active site of chondroitinase ABC I have provided important insights into the catalytic amino acids. In this study, we demonstrate that calcium, a divalent ion, preferentially increases the activity of chondroitinase ABC I toward dermatan versus chondroitin substrates in a concentration-dependent manner. Through biochemical and biophysical investigations, we have established that chondroitinase ABC I binds calcium. Experiments using terbium, a fluorescent calcium analogue, confirm the specificity of this interaction. On the basis of theoretical structural models of the enzyme-substrate complexes, specific amino acids that could potentially play a role in calcium coordination were identified. These amino acids were investigated through site-directed mutagenesis studies and kinetic assays to identify possible mechanisms for calcium-mediated processing of the dermatan substrate in the active site of the enzyme.     

Recombinant expression, purification, and biochemical characterization of chondroitinase ABC II from Proteus vulgaris

Chondroitin lyases (or chondroitinases) are a family of enzymes that depolymerize chondroitin sulfate (CS) and dermatan sulfate (DS) galactosaminoglycans, which have gained prominence as important players in central nervous system biology. Two distinct chondroitinase ABC enzymes, cABCI and cABCII, were identified in Proteus vulgaris. Recently, cABCI was cloned, recombinantly expressed, and extensively characterized structurally and biochemically. This study focuses on recombinant expression, purification, biochemical characterization, and understanding the structure-function relationship of cABCII. The biochemical parameters for optimal activity and kinetic parameters associated with processing of various CS and DS substrates were determined. The profile of products formed by action of cABCII on different substrates was compared with product profile of cABCI. A homology-based structural model of cABCII and its complexes with CS oligosaccharides was constructed. This structural model provided molecular insights into the experimentally observed differences in the product profile of cABCII as compared with that of cABCI. The critical active site residues involved in the catalytic activity of cABCII identified based on the structural model were validated using site-directed mutagenesis and kinetic characterization of the mutants. The development of such a contaminant-free cABCII enzyme provides additional tools to decode the biologically important structure-function relationship of CS and DS galactosaminoglycans and offers novel therapeutic strategies for recovery after central nervous system injury.     

Stability testing of vaccines: Developing Countries Vaccine Manufacturers' Network (DCVMN) perspective

Stability testing is an integral part of the vaccine manufacturing process and is crucial for the success of immunization programs. WHO (World Health Organization) has recently published guidelines on the stability testing of vaccines. These guidelines enlist scientific basis and principles for stability testing at various stages like development, pre-clinical, clinical, licensing, lot release and post-licensure monitoring. DCVMN (Developing Countries Vaccine Manufacturers' Network) is an international body of developing countries vaccine manufacturers and has viewpoints on technical and administrative issues in stability testing of vaccines. We here highlight viewpoints, possible roles and global expectations of DCVMN in the area of stability testing of vaccines.     

Novel In Vivo-Degradable Cellulose-Chitin Copolymer from Metabolically Engineered Gluconacetobacter xylinus

Despite excellent biocompatibility and mechanical properties, the poor in vitro and in vivo degradability of cellulose has limited its biomedical and biomass conversion applications. To address this issue, we report a metabolic engineering-based approach to the rational redesign of cellular metabolites to introduce N-acetylglucosamine (GlcNAc) residues into cellulosic biopolymers during de novo synthesis from Gluconacetobacter xylinus. The cellulose produced from these engineered cells (modified bacterial cellulose [MBC]) was evaluated and compared with cellulose produced from normal cells (bacterial cellulose [BC]). High GlcNAc content and lower crystallinity in MBC compared to BC make this a multifunctional bioengineered polymer susceptible to lysozyme, an enzyme widespread in the human body, and to rapid hydrolysis by cellulase, an enzyme commonly used in biomass conversion. Degradability in vivo was demonstrated in subcutaneous implants in mice, where modified cellulose was completely degraded within 20 days. We provide a new route toward the production of a family of tailorable modified cellulosic biopolymers that overcome the longstanding limitation associated with the poor degradability of cellulose for a wide range of potential applications.A variety of biopolymers, such as polysaccharides, polyesters, and polyamides, have been produced by bacteria. Due to less complexity, genetic manipulation in these microbes opens up an enormous potential to tailor biopolymers for high-value medical applications and drug delivery systems (32). Cellulose is the most abundant biopolymer on Earth, recognized as the major component of plant biomass but also as a representative of microbial extracellular polymers. Bacterial cellulose (BC), produced by Gluconacetobacter xylinus (formerly Acetobacter xylinum) is extruded as fibrils that accumulate to form microfibrils. These microfibrils then aggregate into ribbon structures, which can be disrupted by incorporation of various compounds during de novo synthesis (34). BC has been demonstrated to be a remarkably versatile biomaterial and can be used in a wide variety of applied endeavors, such as paper products, electronics, acoustics, and biomedical devices (4). Due to its unique nanostructure and properties that closely resemble the structure of native extracellular matrices, BC has been considered for numerous medical and tissue-engineering applications, such as wound healing, skin replacement, blood vessel replacement, cartilage engineering, and guided tissue regeneration (GTR) (4, 5, 7, 17, 33).Despite the excellent biocompatibility and mechanical properties of BC, the lack of cellulose-hydrolyzing enzymes in the human body and the high crystallinity restrict its utility (10). Therefore, cellulose with low crystallinity and more facile degradability could be an important polymer for biomass conversion and tissue engineering applications. While in biofuel refineries plant cellulose is the main source for cellulosic biomass, this source is still not efficient due to crystallinity, inhibited access to enzymes, and poor purity of plant cellulose (24).Advances in metabolic engineering over the last 2 decades have led to high-level production of specific metabolites at commercially viable levels. More recently, advancements in genetic engineering combined with those in metabolic engineering have led to the generation of microbes that express heterologous pathways, resulting in the production of natural products beyond the genetic confines of the natural host (29, 39). Examples of this include the production of biofuels (ethanol), adhesives like xanthan gum, and biodegradable plastics, such as poly(3-hydroxybutyric acid) (PHA) (9, 22, 26, 43). In the case of ethanol production, metabolic alterations have been carried out to expand the substrate range utilized by a microbe that naturally ferments sugars into alcohol and thus have improved the overall biomass conversion yield. Another metabolic engineering strategy successfully produced a fast-growing and technically amenable Escherichia coli strain which normally does not produce alcohols at high yields (6).To address the challenges associated with BC, prior efforts were made to modify the polymer based on chemical modifications or on feeding strategies, with limited success (16, 27). It is our hypothesis that the most likely cause of the limitations to these studies was related to insufficient levels of UDP-charged nonglucose monomers available to the cellulose synthase. While these studies proved a limited ability to incorporate nonglucose monomers, they did demonstrate that the cellulose synthase (glycosyltransferase) of G. xylinus can utilize both UDP-glucose and UDP-N-acetylglucosamine (UDP-GlcNAc) as substrates. Therefore, in the present work, an attempt was made to engineer G. xylinus to rationally redesign the flow of cellular metabolites to incorporate GlcNAc sugar residues into cellulose during de novo synthesis. The goal was to generate modified cellulose with more facile enzyme degradation and improved degradability in vivo. To achieve this goal, an operon containing three genes from Candida albicans (Table ​(Table1)1) for UDP-GlcNAc synthesis was expressed in G. xylinus to produce activated cytoplasmic UDP-GlcNAc monomers accessible to cellulose synthase to produce a chimeric polymer comprising both glucose and GlcNAc (see Fig. S1 in the supplemental material). The presence of GlcNAc not only enables BC to be susceptible to lysozyme but also disrupts the highly ordered cellulose crystalline structure. Moreover, in contrast to plant-derived cellulose, the coupling between biosynthesis and material processing in this bacterium offers an experimentally accessible system (1).

TABLE 1.

Candida albicans genes constructed into a heterologous operon for GlcNac incorporation into cellulose fibrils

Preliminary estimates of the population parameters of major fish species in Lake Ayamé I (Bia basin; Côte d'Ivoire)

Length frequency data collected from artisanal fisheries in Lake Ayamé I (Côte d'Ivoire) from August 2004 to 2005 were analysed with F isat software using the E lefan package to estimate the population parameters of 11 fish species. Asymptotic values for total length ( L∞ ) ranged from 20.5 cm for Brycinus imberi to 78 cm for Mormyrops anguilloides . Growth rates ( k ) varied from 0.24 year⁻¹ for Chrysichthys nigrodigitatus to 0.57 year⁻¹ for Hemichromis fasciatus . The growth performance estimates were close to the values found by others authors and reported in FishBase 2008. Fishing mortality ( F ) and exploitation rate ( E ) were found to be below optimum levels of exploitation for most fish species. Recruitment was noted as year–round and bimodal for most studied populations. The data sets were limited in most cases, thus this study provides preliminary population parameters only, but for species for which information is scarce. For application in stock assessment, the growth parameters and especially the natural mortality data require further confirmation.     

Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples

Background  

Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals.