Refolding of G protein alpha subunits from inclusion bodies expressed in Escherichia coli

Heterotrimeric G proteins relay signals from G protein-coupled receptors (GPCRs) to the interior of the cell. The signaling cascades induced by G protein activation control a wide range of cellular processes. The α subunit is believed to determine which G protein couples to each GPCR, and is the primary determinant of the type of signal transmitted. Several members of the G_α family have been expressed in active form in Escherichia coli. However, production levels of these proteins are limited: in most cases only 10% of total G_α protein expressed is active; the rest accumulates in inclusion bodies. Although G_iα has been readily expressed in soluble form (to 10 mg/L), other α subunits are minimally soluble, and many are exclusively expressed to inclusion bodies. Previous efforts to solubilize and refold G_α from inclusion bodies have not been successful. Here we did a thorough study of the characteristics of G_α subunits (human G_iα(1), human G_sα(short), human G_11α and human G_tα(cone)), solubilized and purified from inclusion bodies. We find that we can obtain soluble protein both by on-column and rapid-dilution techniques. Comparison to native, soluble G_iα expressed from E. coli showed that although the refolded G_α subunits were soluble and retained partial α-helicity characteristic of the native, folded G_α subunit, they did not bind GDP or GTP as effectively as native protein. We conclude that the refolded G_iα protein has a native-like secondary structure, but is predominately in a molten globular state.     

The Pandinus imperator haemolymph lipoprotein, an unusual phosphatidylserine carrying lipoprotein

The haemolymph lipoprotein of the scorpion, Pandinus imperator was isolated and characterised. Contrary to the lipoproteins of insects and the discoidal HDL-lipoproteins of a crayfish and polychaete, the Pandinus lipoprotein consists of three instead of two apoproteins (apoPiLp I = 230 kDa, apoPiLp II = 130 kDa and apoPiLp III = 120 kDa). The apolipoproteins are arranged in varying stoichiometries as judged by cross-linking experiments. In lipoprotein samples from individual animals, the two smaller subunits occurred in a 1:1 stoichiometry, while the relative amount of the 230 kDa peptide varied. The lipoprotein is a slightly heart-shaped HDL with a diameter of 15 nm. It is present in two densities of 1100 and 1190 kg/m³, of which the latter is by far more abundant. The native molecular mass was estimated to be 500 kDa. The lipid content was determined as 33.5% and consists of 70% neutral lipids and 30% phospholipids. Strikingly, 42.5% of the phospholipids is phosphatidylserine while phosphatidylcholine and phosphatidylethanolamine account for 55.1% and 2.3%, respectively. Carbohydrate analysis suggests the presence of only high-mannose-type N-glycans. N-glycan profiling shows glycans corresponding to a size of 8.0–11.5 hexose units.     

Kringle domains and plasmin denaturation.

The rate of plasmin denaturation was in the order of Lys-plasmin greater than miniplasmin greater than microplasmin. Fibrinogen degradation products (FDP) dose dependently increased the denaturation rate of Lys-plasmin and mini-plasmin with a maximal rate constant at the FDP/plasmin ratio of about 0.5. The denaturation rate constant of microplasmin was not affected. FDP increased the rate of plasmin denaturation was in parallel with its effect on the interaction among kringle domains. Without FDP only trace amounts of plasminogen dimer could be detected by cross-linking with bis-(sulfo-succinimidyl)-suberate followed by SDS gel electrophoresis. In the low concentration of FDP significant amounts of oligomers of Glu-, mini-plasminogens, kringle 1-3 and kringle 1-5 were observed. High concentration of FDP, however, decreased plasminogen oligomer.     

Historical biogeography, phylogenetic relationships and intraspecific diversity of agamid lizards in the Central Asian deserts of Kazakhstan and Uzbekistan

The Central Asian agamid lizards are ecologically and morphologically diverse, occurring across a broad range of desert environments in this biogeographically important region. It is probable that past climatic shifts have significantly influenced the diversification patterns and distributions of the agamid lizards of this region. To assess this within a phylogenetic framework we sequenced a 1200 bp region of mitochondrial DNA and a 1200 bp nuclear gene (RAG-1), incorporating both inter- and intraspecific sampling across Central Asian agamids. Our topology and divergence time estimates support an Eocene origin of the Agaminae subfamily on the Indian subcontinent, coinciding with the collision of India into Eurasia. The onset of aridification in Central Asia during the Late Oligocene, resulting from the retreat of the Paratethys Sea and the intensified uplift of the Tibetan–Himalayan complex, probably played an important role in the diversification of Phrynocephalus, one of the three genera studied. Intensification of aridity and geologic events in the Plio-Pleistocene and Quaternary glacial cycling probably had a significant influence on intraspecific diversification patterns within Phrynocephalus.     

Fine structure characterization of amylopectins from grain amaranth starch

The aim of this study was to determine the fine structure of amylopectin from grain amaranth. Amaranthus amylopectin was hydrolyzed with α-amylase, and single clusters and a group of clusters (domain) were isolated by methanol precipitation. The domain and the clusters were treated with phosphorylase a and then β-amylase to remove all external chains, whereby the internal structure was obtained. The ,β-limit dextrins were analyzed on Sepharose CL 6B. The average DP (degree of polymerization) and peak-DP values of fractions of clusters were 57 and 82, respectively; the values of the domain were 137 and 309, respectively. The unit chain length profiles were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detector (HPAEC–PAD). The results showed that the domain fraction contained 2.2 clusters, and single clusters were composed of 13 chains. The ,β-limit dextrins of the clusters were further hydrolyzed with α-amylase to characterize their building block composition. The average DP of the branched blocks was 11 and they contained on average 2.5 chains. Their average chain length, internal chain length, and degree of branching were approximately 4.3, 2.8, and 14, respectively. A cluster consisted of 6 branched blocks, and the internal chain length between the blocks was 6.8.     

Demographic and phylogeographic histories of two venomous North American snakes of the genus Agkistrodon

Many studies have revealed that lineages currently inhabiting formerly glaciated areas were pushed into southern glacial refugia and have expanded into their modern range since the last glacial maximum. There have been few studies that compare the effects of glacial cycles on lineage diversification and historical demography in closely related species with overlapping ranges. In this study we compare phylogeographic structure, historical demography, and approximate lineage age in two closely related and broadly co-occurring venomous snakes in eastern North America, the cottonmouth (Agkistrodon piscivorus) and copperhead (A. contortrix) using sequences from the mtDNA gene cytochrome b. We inferred three geographic lineages of A. contortrix and two of A. piscivorus with no common geographic or temporal pattern of lineage diversification identified for these species. Lineage diversification occurred in the Late Pliocene for A. piscivorus (2.5 mya) and in the Early Pleistocene for A. contortrix (1.5 mya). Demographic estimates revealed population expansion following the last glacial maximum (20,000 years ago) in two lineages of A. contortrix (the Central clade and Eastern clade) and one lineage of A. piscivorus (the Continental clade). The Florida clade of A. piscivorus is the only lineage for which constant population size through time was inferred, possibly due to stable populations persisting in areas unaffected by glacial advances. Our data suggest that unique habitat preferences may have shaped both the phylogeographic and demographic histories of each species.     

Isotopically labeled expression in E. coli, purification, and refolding of the full ectodomain of the influenza virus membrane fusion protein

This paper describes methods to produce an isotopically labeled 23 kDa viral membrane protein with purified yield of 20 mg/L of Escherichia coli shake flask culture. This yield is sufficient for NMR structural studies and the protein production methods are simple, straightforward, and rapid and likely applicable to other recombinant membrane proteins expressed in E. coli. The target FHA2 protein is the full ectodomain construct of the influenza virus hemagglutinin protein which catalyzes fusion between the viral and the cellular endosomal membranes during infection. The high yield of FHA2 was achieved by: (1) initial growth in rich medium to A₆₀₀  8 followed by a switch to minimal medium and induction of protein expression; and (2) obtaining protein both from purification of the detergent-soluble lysate and from solubilization, purification, and refolding of inclusion bodies. The high cell density was achieved after optimization of pH, oxygenation, and carbon source and concentration, and the refolding protocol was optimized using circular dichroism spectroscopy. For a single residue of membrane-associated FHA2 that was obtained from purification and refolding of inclusion bodies, native conformation was verified by the ¹³CO chemical shifts measured using solid-state nuclear magnetic resonance spectroscopy.     

Variations on the SnCl4 and CF3CO2Ag-promoted glycosidation of sugar acetates: a direct, versatile and apparently simple method with either α or β stereocontrol

Glycosidation of sugar peracetates (d-gluco, d-galacto) with SnCl₄ and CF₃CO₂Ag led to either 1,2-cis-, or 1,2-trans-glycosides, depending primarily on the alcohols used. In particular, 1,2-trans-glycosides, expected from acyl-protected glycosyl donors, were formed in high yields with alcohols sharing specific features such as bulkiness, presence of electron-withdrawing groups or polyethoxy motifs. In contrast, simple alcohols afforded 1:1 mixtures of 2,3,4,6-tetra-O-acetyl, and 3,4,6-tri-O-acetyl 1,2-cis-glycosides due to anomerization and/or acid-catalyzed fragmentation of 1,2-orthoester intermediates. After reacetylation or deacetylation, acetylated or fully deprotected 1,2-cis-glycosides (α-d-gluco, α-d-galacto) were obtained in 90% yields by a simple and direct method.     

Inoculative releases of Trichogramma ostriniae for suppression of Ostrinia nubilalis (European corn borer) in sweet corn: field biology and population dynamics

The effectiveness of inoculative releases of Trichogramma ostriniae Pang and Chen for suppression of Ostrinia nubilalis (Hübner) in sweet corn was assessed. Early-season, low-density (75,000 females ha⁻¹) releases were made, and establishment, levels of parasitism and sex ratios of emerging T. ostriniae quantified. T. ostriniae established effectively for each season that they were released, but appeared to be unable to overwinter. Parasitism levels tracked egg mass numbers closely, and T. ostriniae persisted in fields even where insecticides were applied. Parasitism by indigenous Trichogramma species was 3%. Field populations of T. ostriniae were distinctly female biased (78%), with males produced in the majority of broods. Numbers of males did not increase linearly with number of O. nubilalis eggs parasitized, but appeared to remain constant above an egg mass size of about 20 eggs. A Type-I functional response to increasing egg and egg mass density was found under field conditions, where the proportion of egg masses parasitized remained constant with increasing egg mass density. A relatively consistent percentage of eggs per egg mass was parasitized, with a linear increase in number of eggs parasitized with increasing number of eggs per egg mass. These results show that T. ostriniae established viable reproductive populations in sweet corn following inoculative release, with the potential to contribute to reduced dependence on insecticides for the control of O. nubilalis in an integrated pest management program.     

Multi-locus phylogeny of the family Acrocephalidae (Aves: Passeriformes) – The traditional taxonomy overthrown

We present the first study of the warbler family Acrocephalidae based on one mitochondrial and three nuclear DNA loci, in total 2900 bp, including most or all of the species in three (Acrocephalus, Hippolais and Chloropeta) of the four genera and one species in the fourth genus (Nesillas) in this family. All three genera were suggested to be non-monophyletic, although the non-monophyly of Acrocephalus is not fully convincingly demonstrated. Six major clades were found, which agreed largely with the results from two earlier mitochondrial studies, and for which the names Hippolais, Iduna, Acrocephalus, Calamocichla, Notiocichla and Calamodus have been used. However, the results also revealed some new constellations, due to better resolution of deeper nodes and the inclusion of more taxa. The taxonomic implications are discussed.     

Decomposition of four macrophytes in wetland sediments: Organic matter and nutrient decay and associated benthic processes

Decomposition rates of Phragmites australis, Carex riparia, Nuphar luteum and Salvinia natans and benthic processes were measured from December 2003 to December 2004 in a shallow wetland (Paludi di Ostiglia, Northern Italy) by means of litter bags and intact cores incubations. Decay rate was highest for N. luteum (k = 0.0152 d⁻¹), intermediate for S. natans (k = 0.0041 d⁻¹) and similar for P. australis (k = 0.0027 d⁻¹) and C. riparia (k = 0.0028 d⁻¹).Benthic metabolism followed a seasonal pattern with summer peaks of O₂ demand and TCO₂, CH₄ and NH₄⁺ efflux whilst soluble reactive phosphorus (SRP) fluxes were negligible also under hypoxic conditions, indicating that P was mainly retained by sediment. The initial C:P ratio was similar in N. luteum and S. natans (170) and significantly lower than that of P. australis and C. riparia (360). During the detritus decay P was progressively lost by N. luteum and S. natans tissues, whereas, after an initial leaching, it was probably re-used during the microbial decomposition of the more refractory P. australis and C. riparia detritus. Nuphar luteum, P. australis and S. natans had comparable initial C:N mass ratio (15), significantly lower than that of C. riparia (26). The C:N ratio was rather constant for N. luteum (12.9 ± 1.5) and S. natans (14.6 ± 0.9), decreased slightly to below 20 for C. riparia and increased up to 30 for P. australis. Overall, differences among species were likely due to the recalcitrance of decomposing detritus, whilst process rates were controlled by limitation of microbial processes by nutrients and electron acceptor availability.     

Hydrolysis of polymeric fibrin by plasmin, miniplasmin, microplasmin and trypsin]

125I-labeled polymeric fibrin hydrolyzed with plasmin, Val442-plasmin (miniplasmin, Lys530-plasmin (microplasmin) and trypsin has been studied for radioactivity of its separate electrophoretic bands. The reaction of hydrolysis was stopped at a moment of a two-fold decrease of the fibrin clot turbidity (t1/2) at the wave length 350 nm. For plasmin, miniplasmin, microplasmin and trypsin taken in the same caseinolytic activities t1/2 was 12.4, 40.0 164.1 and 76.8 min, respectively. Differences in composition of fibrin digests taken at t1/2, are demonstrated: the content of high-molecular components of digests decreases in the order of plasmin greater than miniplasmin greater than microplasmin greater than trypsin, thus showing differences in the processes of fibrin clot structure disruption by the enzymes.     

Optimization of glutaryl-7-aminocephalosporanic acid acylase expression in E. coli

A recombinant glutaryl-7-aminocephalosporanic acid acylase (GLA) from Pseudomonas N176 has been over-expressed in BL21(DE3)pLysS Escherichia coli cells. By alternating screenings of medium components and simplified factorial experimental designs, an improved microbial process was set up at shake-flask level (and then scaled up to 2L-fermentors) giving a 80- and 120-fold increase in specific and volumetric enzyme productivity, respectively. Under the best expression conditions, 1380 U/g cell and 16,100 U/L of GLA were produced versus the 18 U/g cell and the 140 U/L obtained in the initial standard conditions. Osmotic stress caused by the addition of NaCl, low cell growth rate linked to high biomass yield in the properly-designed rich medium, optimization of the time and the amount of inducer’s addition and decrease of temperature during recombinant protein production, represent the factors concurring to achieve the reported expression level. Notably, this expression level is significantly higher than any previously described production of GLAs. High volumetric production, cost reduction and the simple one-step chromatographic purification of the His-tagged recombinant enzyme, makes this GLA an economic tool to be used in the 7-ACA industrial production.     

Pronase digestion of brush border membrane-bound Cry1Aa shows that almost the whole activated Cry1Aa molecule penetrates into the membrane

Bacillus thuringiensis insecticidal proteins, Cry toxins, following ingestion by insect larvae, induce insecticidal effect by penetrating the brush border membranes (BBM) of midgut epithelial cells. Purified, activated B. thuringiensis Cry1Aa bound to Bombyx mori BBMV or unbound Cry1Aa were vigorously digested with Pronase. Both digests were compared by Western blotting. Free Cry1Aa was digested to α-helix and/or to amino acids at 1 mg Pronase/mL within 2.4 h at 37 °C. Whereas, BBMV-bound Cry1Aa was very resistant to Pronase digestion and even at 2 mg for 24 h, 7.5 kDa and 30 kDa peptide were detected by α-2,3 antiserum, and α-4,5 and α-6,7 antisera, respectively. Another 30 kDa peptide was also detected by β-6-11 and domain III antisera. These fragments are believed either to be embedded in or to strongly interact with the BBMV. The 7.5 and former 30 kDa peptides are thought to be derived from α-2,3 helix and stretch of α-4 to α-7 helices. Furthermore the latter 30 kDa was thought to include the stretch of β-6 to domain III. Moreover, the embedded Cry1Aa molecule appears to be segregated in some areas of β-1-5 sheets, resulting in the above two 30 kDa peptides. From these digestion patterns, we proposed new membrane insertion model for single Cry1Aa molecule. On the other hand, in digestion of BBMV-bound Cry1Aa, 15 kDa peptide which was recognized only by α-4,5 antiserum was observed. This fragment must be dimeric α-4,5 helices and we discussed the origin of this peptide.     

Use of RNA interference to dissect the roles of trans-acting factors in alternative pre-mRNA splicing

RNA interference (RNAi) is becoming a popular method for analyzing gene function in a variety of biological processes. We have used RNAi in cultured Drosophila cells to identify trans-acting factors that regulate the alternative splicing of endogenously transcribed pre-mRNAs. We have generated a dsRNA library comprising 70% of the Drosophila genes encoding RNA binding proteins and assessed the function of each protein in the regulation of alternative splicing. This approach not only identifies trans-acting factors regulating specific alternative splicing events, but also can provide insight into the alternative splicing regulatory networks of Drosophila. Here, we describe this RNAi approach to identify alternative splicing regulatory proteins in detail.     

Phylogeography of the frog Leptodactylus validus (Amphibia: Anura): Patterns and timing of colonization events in the Lesser Antilles

The frog Leptodactylus validus occurs in northern South America, Trinidad and Tobago, and the southern Lesser Antilles (Grenada and St. Vincent). Mitochondrial DNA sequences were used to perform a nested clade phylogeographic analysis (NCPA), to date colonization events, and to analyze colonization patterns using on a relaxed molecular clock and coalescent simulations. L. validus originated on the mainland and first colonized Trinidad with subsequent independent colonizations of Tobago and the Lesser Antilles from Trinidad. The NCPA suggests a historical vicariant event between populations in Trinidad and Tobago from those in the Lesser Antilles. The colonization of Trinidad occurred 1 million years ago (mya) and the colonization of the Lesser Antillean islands occurred 0.4 mya. The coalescent approach supported the scenario where L. validus dispersed from Trinidad to St. Vincent and from there to Grenada, a dispersal event that could have been mediated by human introduction as recent as 1600 years ago.     

Adult Brugia malayi 34 kDa (BMT-5) antigen offers Th1 mediated significant protection against infective larval challenge in Mastomys coucha

We earlier reported a sizeable protection conferred by ‘mitochondria rich’ (MT) fraction of adult B. malayi and the present study was planned to locate the candidate protective molecule/s in the active fraction. The MT fraction was subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and the antigen bands showing strong immune-reactivity with the resistant mastomys sera were assayed for their lymphoproliferative response using splenocytes of protected animals. Of the eight such protein bands, one sub fraction with a molecular weight of  34 kDa (BMT-5) produced utmost cellular proliferation and was therefore exploited for vaccination study. BMT-5 emulsified in Freund's adjuvant produced discernible protection causing 69 and 67% reductions in microfilaraemia and adult worm burden respectively along with sterilization of 68% of the recovered female parasites. Significant levels of filaria-specific and non-specific lymphoproliferation along with enhanced release of Th1 cytokines (TNF-α, IFN-γ and IL-2) by splenocytes were observed in the vaccinated mastomys in addition to elevated levels of antigen-specific IgG, IgG2a, IgG2b and IgA. The peritoneal macrophages of immunized animals also revealed enhanced nitric oxide production in the presence of BMT-5. The findings suggest that  34 kDa (BMT-5) molecules present in the MT fraction of adult B. malayi provided sizeable protection against infective larval challenge by generating a Th1 biased milieu in the host.     

Growth phase-dependant enzyme profile of pyruvate catabolism and end-product formation in Clostridium thermocellum ATCC 27405

End-product synthesis and enzyme activities involved in pyruvate catabolism, H₂ synthesis, and ethanol production in mid-log (OD₆₀₀  0.25), early stationary (OD₆₀₀  0.5), and stationary phase (OD₆₀₀  0.7) cell extracts were determined in Clostridium thermocellum ATCC 27405 grown in batch cultures on cellobiose. Carbon dioxide, hydrogen, ethanol, acetate and formate were major end-products and their production paralleled growth and cellobiose consumption. Lactate dehydrogenase, pyruvate:formate lyase, pyruvate:ferredoxin oxidoreductase, methyl viologen-dependant hydrogenase, ferredoxin-dependant hydrogenase, NADH-dependant hydrogenase, NADPH-dependant hydrogenase, NADH-dependant acetaldehyde dehydrogenase, NADH-dependant alcohol dehydogenase, and NADPH-dependant alcohol dehydrogenase activities were detected in all extracts, while pyruate dehydrogenase and formate dehydrogenase activities were not detected. All hydrogenase activities decreased (2–12-fold) as growth progressed from early exponential to stationary phase. Alcohol dehydrogenase activities fluctuated only marginally (<45%), while lactate dehydrogenase, pyruvate:formate lyase, and pyruvate:ferredoxin oxidoreductase remained constant in all cell extracts. We have proposed a pathway involved in pyruvate catabolism and end-product formation based on enzyme activity profiles in conjunction with bioinformatics analysis.     

Propionic acid production from glycerol by metabolically engineered Propionibacterium acidipropionici

Large amounts of crude glycerol produced in the biodiesel industry can be used as a low-cost renewable feedstock to produce chemicals and fuels. Compared to sugars (sucrose, glucose, xylose, etc.), glycerol has a lower reducing level, which is of benefit to the production of reduced chemicals. In this work, glycerol as the sole carbon source in propionic acid fermentation by metabolically engineered Propionibacterium acidipropionici (ACK-Tet) was studied. It was found that the adapted ACK-Tet mutant could use glycerol for its growth and produced propionic acid at a high yield of 0.54–0.71 g/g, which was much higher than that from glucose (0.35 g/g). In addition, the production of acetic acid in glycerol fermentation was much less than that from glucose. Thus, glycerol fermentation produced a high purity propionic acid with a high propionic acid to acetic acid ratio of 22.4 (vs. 5 for glucose fermentation), facilitating the recovery and purification of propionic acid from the fermentation broth. The highest propionic acid concentration obtained from glycerol fermentation was 106 g/L, which was 2.5 times of the highest concentration (42 g/L) previously reported in the literature.     

Phylogeography of Indonesian and Sino-Himalayan region bush warblers (Cettia, Aves)

We present a hypothesis for the phylogeny and phylogeography of a group of bush warblers in the genus Cettia, based on parts of the mitochondrial Cytochrome b gene and the nuclear myoglobin intron II (in all 1.7 kb). Ancestral areas were reconstructed by dispersal-vicariance analysis and constrained Bayesian inference. The results suggest that the insular taxa in the Cettia vulcania group are most closely related to Cettia flavolivacea, and originated from a dispersal by an ancestral population in the Himalayas towards the south, to the Sunda region. From this population, a second dispersal along a different route colonized China and northern Vietnam. Hence, the Chinese taxon intricata and Vietnamese oblita, currently allocated to C. flavolivacea, are more closely related to the vulcania group than to the other taxa in the flavolivacea group, and we propose that they be treated as conspecific with C. vulcania, restricting C. flavolivacea to Myanmar and the Himalayas.