Lysine residues of IGF-I are substrates for transglutaminases and modulate downstream IGF-I signalling

Numerous studies have reported associations between IGF-I and other extra cellular matrix (ECM) proteins, including fibronectin (FN), integrins, IGF-binding proteins (IGFBPs) and through IGFBPs, with vitronectin (VN). Nevertheless, the precise nature and mechanisms of these interactions are still being characterised. In this paper, we discuss transglutaminases (TGases) as a constituent of the ECM and provide evidence for the first time that IGF-I is a lysine (K)-donor substrate to TGases. When IGF-I was incubated with an alpha-2 plasmin inhibitor-derived Q peptide in the presence of tissue transglutaminase (TG2), an IGF-I:Q peptide cross-linked species was detected using Western immunoblotting and confirmed by mass spectrometry. Similar findings were observed in the presence of Factor XIIIa (FXIIIa) TGase. To identify the precise location of this K-donor TGase site/s on IGF-I, all the three IGF-I K-sites, individually and collectively (K27, K65 and K68), were substituted to arginine (R) using site-directed mutagenesis. Incubation of these K→R IGF-I analogues with Q peptide in the presence of TG2 or FXIIIa resulted in the absence of cross-linking in IGF-I analogues bearing arginine substitution at site 68. This established that K68 within the IGF-I D-domain was the principal K-donor site to TGases. We further annotated the functional significance of these K→R IGF-I analogues on IGF-I mediated actions. IGF-I analogues with K→R substitution within the D-domain at K65 and K68 hindered migration of MCF-7 breast carcinoma cells and correspondingly reduced PI3-K/AKT activation. Therefore, this study also provides first insights into a possible functional role of the previously uncharacterised IGF-I D-domain.

PEGylation of lysine residues reduces the pro-migratory activity of IGF-I

Background: The insulin-like growth factor (IGF) system is composed of ligands and receptors which regulate cell proliferation, survival, differentiation and migration. Some functions are regulated via intracellular signaling cascades, others by involvement of the extracellular milieu, including binding proteins and other extracellular matrix proteins. However, understanding of their functions and the exact nature of these interactions remains incomplete. Methods: IGF-I was PEGylated at its lysine sites - K27, K65 and K68. Binding of PEG-IGF-I to the IGFBPs was analyzed using BIAcore and its ability to activate the IGF-IR was assessed using IGF-IR phosphorylation assay. Furthermore, functional consequences of PEGylating the lysine residues of IGF-I was investigated using cell viability and cell migration assays. In addition, particular downstream signaling pathways regularly implicated in these mechanisms were also dissected using phospho-AKT and phospho-ERK1/2 assays. Results: In this study, IGF-I specifically PEGylated at lysine 27 (PEG-K27), 65 (PEG-K65) or 68 (PEG-K68) were employed. Receptor phosphorylation was only reduced by 2-fold with PEG-K65 and PEG-K68 over all the time points tested, and as observed in two cell types, 3T3 fibroblasts and MCF-7 breast cancer cells. PEGylation at K27 resulted in a much larger effect, with more than 10-fold lower activation for 3T3 fibroblasts and a ~3 fold reduced IGF-IR activation for MCF-7 breast cancer cells over 15 minutes. In addition, all PEG-IGF-I variants demonstrated a ten-fold reduction in the association rate to IGF binding proteins (IGFBPs). Functionally, all PEG variants completely lost their ability to induce cell migration in the presence of IGFBP-3/vitronectin (VN) complexes as compared to IGF-I; in contrast, cell viability was fully preserved. Further investigations into the downstream signaling pathways revealed that the PI3-K/AKT pathway was preferentially affected upon treatment with the PEG-IGF-I variants compared to the MAPK/ERK pathway. Conclusion: PEGylation of IGF-I has an impact on cell migration but not cell viability. General significance: PEG-IGF-I may differentially modulate IGF-I mediated functions that are dependent on its interaction with its receptor as well as key extracellular proteins such as VN and IGFBPs.

Regional contingencies in the relationship between aboveground biomass and litter in the world’s grasslands

Based on regional-scale studies, aboveground production and litter decomposition are thought to positively covary, because they are driven by shared biotic and climatic factors. Until now we have been unable to test whether production and decomposition are generally coupled across climatically dissimilar regions, because we lacked replicated data collected within a single vegetation type across multiple regions, obfuscating the drivers and generality of the association between production and decomposition. Furthermore, our understanding of the relationships between production and decomposition rests heavily on separate meta-analyses of each response, because no studies have simultaneously measured production and the accumulation or decomposition of litter using consistent methods at globally relevant scales. Here, we use a multi-country grassland dataset collected using a standardized protocol to show that live plant biomass (an estimate of aboveground net primary production) and litter disappearance (represented by mass loss of aboveground litter) do not strongly covary. Live biomass and litter disappearance varied at different spatial scales. There was substantial variation in live biomass among continents, sites and plots whereas among continent differences accounted for most of the variation in litter disappearance rates. Although there were strong associations among aboveground biomass, litter disappearance and climatic factors in some regions (e.g. U.S. Great Plains), these relationships were inconsistent within and among the regions represented by this study. These results highlight the importance of replication among regions and continents when characterizing the correlations between ecosystem processes and interpreting their global-scale implications for carbon flux. We must exercise caution in parameterizing litter decomposition and aboveground production in future regional and global carbon models as their relationship is complex.

Effects of dissolved oxygen availability and culture biomass at induction upon the intracellular expression of monoamine oxidase by recombinant E. coli in fed batch bioprocesses

The effects of oxygen availability and induction culture biomass upon production of an industrially important monoamine oxidase (MAO) were investigated in fed-batch cultures of a recombinant E. coli. For each induction cell biomass 2 different oxygenation methods were used, aeration and oxygen enriched air. Induction at higher biomass levels increased the culture demand for oxygen, leading to fermentative metabolism and accumulation of high levels of acetate in the aerated cultures. Paradoxically, despite an almost eight fold increase in acetate accumulation to levels widely reported to be highly detrimental to protein production, when induction wet cell weight (WCW) rose from 100% to 137.5%, MAO specific activity in these aerated processes showed a 3 fold increase. By contrast, for oxygenated cultures induced at WCW's 100% and 137.5% specific activity levels were broadly similar, but fell rapidly after the maxima were reached. Induction at high biomass levels (WCW 175%) led to very low levels of specific MAO activity relative to induction at lower WCW's in both aerated and oxygenated cultures. Oxygen enrichment of these cultures was a useful strategy for boosting specific growth rates, but did not have positive effects upon specific enzyme activity. Based upon our findings, consideration of the amino acid composition of MAO and previous studies on related enzymes, we propose that this effect is due to oxidative damage to the MAO enzyme itself during these highly aerobic processes. Thus, the optimal process for MAO production is aerated, not oxygenated, and induced at moderate cell density, and clearly represents a compromise between oxygen supply effects on specific growth rate/induction cell density, acetate accumulation, and high specific MAO activity. This work shows that the negative effects of oxygen previously reported in free enzyme preparations, are not limited to these acellular environments but are also discernible in the sheltered environment of the cytosol of E. coli cells.

Mechanisms of mono- and poly-ubiquitination : ubiquitination specificity depends on compatibility between the E2 catalytic core and amino acid residues proximal to the lysine

Ubiquitination involves the attachment of ubiquitin to lysine residues on substrate proteins or itself, which can result in protein monoubiquitination or polyubiquitination. Ubiquitin attachment to different lysine residues can generate diverse substrate-ubiquitin structures, targeting proteins to different fates. The mechanisms of lysine selection are not well understood. Ubiquitination by the largest group of E3 ligases, the RING-family E3 s, is catalyzed through co-operation between the non-catalytic ubiquitin-ligase (E3) and the ubiquitin-conjugating enzyme (E2), where the RING E3 binds the substrate and the E2 catalyzes ubiquitin transfer. Previous studies suggest that ubiquitination sites are selected by E3-mediated positioning of the lysine toward the E2 active site. Ultimately, at a catalytic level, ubiquitination of lysine residues within the substrate or ubiquitin occurs by nucleophilic attack of the lysine residue on the thioester bond linking the E2 catalytic cysteine to ubiquitin. One of the best studied RING E3/ E2 complexes is the Skp1/Cul1/F box protein complex, SCFCdc4, and its cognate E2, Cdc34, which target the CDK inhibitor Sic1 for K48-linked polyubiquitination, leading to its proteasomal degradation. Our recent studies of this model system demonstrated that residues surrounding Sic1 lysines or lysine 48 in ubiquitin are critical for ubiquitination. This sequence-dependence is linked to evolutionarily conserved key residues in the catalytic region of Cdc34 and can determine if Sic1 is mono- or poly-ubiquitinated. Our studies indicate that amino acid determinants in the Cdc34 catalytic region and their compatibility to those surrounding acceptor lysine residues play important roles in lysine selection. This may represent a general mechanism in directing the mode of ubiquitination in E2 s.

A study of the effects metal residues in poly(9,9-dioctylfluorene) have on field-effect transistor device characteristics

The amount of metal residues from organometallic reagents used in preparation of poly(9,9-dioctylfluorene) by palladium catalysed Suzuki and nickel-induced Yamamoto polycondensations have been determined, and their effect upon the behaviour of the polymer in field-effect transistors (FETs) has been measured. The metal levels from material polymerised by Suzuki method were found to be much higher than from that made by the Yamamoto procedure. Simple treatment of the polymers with suitable metal trapping reagents lowered the metal levels significantly, with EDTA giving best results for nickel and triphenylphosphine for palladium. Comparison of the behaviour of FETs using polyfluorenes with varying levels of metal contamination, showed that the metal residues have little effect upon the mobility values, but often affect the degree of hysteresis, possibly acting as charge traps. Satisfactory device performances were obtained from polymer with palladium levels of 2000 μg/g suggesting that complete removal of metal residues may not be necessary for satisfactory device performance.

Microalgal biomass as a fermentation feedstock for bioethanol production

BACKGROUND The increasing cost of fossil fuels as well as the escalating social and industrial awareness of the environmental impacts associated with the use of fossil fuels has created the need for more sustainable fuel options. Bioethanol, produced from renewable biomass such as sugar and starch materials, is believed to be one of these options, and it is currently being harnessed extensively. However, the utilization of sugar and starch materials as feedstocks for bioethanol production creates a major competition with the food market in terms of land for cultivation, and this makes bioethanol from these sources economically less attractive. RESULT This study explores the suitability of microalgae (Chlorococum sp.) as a substrate for bioethanol production via yeast (Saccharomycesbayanus)under different fermentation conditions. Results show a maximum ethanol concentration of 3.83 g L -1 obtained from 10 g L-1 of lipid-extracted microalgae debris. CONCLUSION This productivity level (∼38% w/w), which is in keeping with that of current production systems endorses microalgae as a promising substrate for bioethanol production.

The combination of plant-expressed cellobiohydrolase and low dosages of cellulases for the hydrolysis of sugar cane bagasse

Background The expression of biomass-degrading enzymes (such as cellobiohydrolases) in transgenic plants has the potential to reduce the costs of biomass saccharification by providing a source of enzymes to supplement commercial cellulase mixtures. Cellobiohydrolases are the main enzymes in commercial cellulase mixtures. In the present study, a cellobiohydrolase was expressed in transgenic corn stover leaf and assessed as an additive for two commercial cellulase mixtures for the saccharification of pretreated sugar cane bagasse obtained by different processes. Results Recombinant cellobiohydrolase in the senescent leaves of transgenic corn was extracted using a simple buffer with no concentration step. The extract significantly enhanced the performance of Celluclast 1.5 L (a commercial cellulase mixture) by up to fourfold on sugar cane bagasse pretreated at the pilot scale using a dilute sulfuric acid steam explosion process compared to the commercial cellulase mixture on its own. Also, the extracts were able to enhance the performance of Cellic CTec2 (a commercial cellulase mixture) up to fourfold on a range of residues from sugar cane bagasse pretreated at the laboratory (using acidified ethylene carbonate/ethylene glycol, 1-butyl-3-methylimidazolium chloride, and ball-milling) and pilot (dilute sodium hydroxide and glycerol/hydrochloric acid steam explosion) scales. We have demonstrated using tap water as a solvent (under conditions that mimic an industrial process) extraction of about 90% recombinant cellobiohydrolase from senescent, transgenic corn stover leaf that had minimal tissue disruption. Conclusions The accumulation of recombinant cellobiohydrolase in senescent, transgenic corn stover leaf is a viable strategy to reduce the saccharification cost associated with the production of fermentable sugars from pretreated biomass. We envisage an industrial-scale process in which transgenic plants provide both fibre and biomass-degrading enzymes for pretreatment and enzymatic hydrolysis, respectively.

Measuring the regional availability of biomass for biofuels and the potential for microalgae

Biomass is an important energy resource for producing bioenergy and growing the global economy whilst minimising greenhouse gas emissions. Many countries, like Australia have a huge amount of biomass with the potential for bioenergy, but non-edible feedstock resources are significantly under-exploited. Hence it is essential to map the availability of these feedstocks to identify the most appropriate bioenergy solution for each region and develop supply chains for biorefineries. Using Australia as a case study,we present the spatial availability and opportunities for second and third generation feedstocks. Considerations included current land use, the presence of existing biomass industries and climatic conditions. Detailed information on the regional availability of biomass was collected from government statistics, technical reports and energy assessments as well as from academic literature. Second generation biofuels have the largest opportunity in New South Wales, Queensland and Victoria (NSW, QLD and VIC) and the regions with the highest potential for microalgae are Western Australia and Northern Territory (WA, NT), based on land use opportunity cost and climate. The approach can be used in other countries with a similar climate. More research is needed to overcome key technical and economic hurdles.

Microbial oil production from palm oil empty fruit bunch hydrolysates

This study explores the potential use of empty fruit bunch (EFB) residues from palm oil processing residues, as an alternative feedstock for microbial oil production. EFB is a readily available, lignocellulosic biomass that provides cheaper substrates for oil production in comparison to the use of pure sugars. In this study, potential oleaginous microorganisms were selected based on a multi-criteria analysis (MCA) framework which utilised Analytical Hierarchy Process (AHP) with Preference Ranking Organization Method for Enrichment Evaluation (PROMETHEE) aided by Geometrical Analysis for Interactive Aid (GAIA). The MCA framework was used to evaluate several strains of microalgae (Chlorella protothecoides and Chlorella zofingiensis), yeasts (Cryptococcus albidus and Rhodotorula mucilaginosa) and fungi (Aspergillus oryzae and Mucor plumbeus) on glucose, xylose and glycerol. Based on the results of PROMETHEE rankings and GAIA plane, fungal strains A. oryzae and M. plumbeus and yeast strain R. mucilaginosa showed great promise for oil production from lignocellulosic hydrolysates. The study further cultivated A. oryzae, M. plumbeus and R. mucilaginosa on EFB hydrolysates for oil production. EFB was pretreated with dilute sulfuric acid, followed by enzymatic saccharification of solid residue. Hydrolysates tested in this study are detoxified liquid hydrolysates (LH) and enzymatic hydrolysate (EH).

Effects of sewer conditions on the degradation of selected illicit drug residues in wastewater

The stability of five illicit drug markers in wastewater was tested under different sewer conditions using laboratory-scale sewer reactors. Wastewater was spiked with deuterium labelled isotopes of cocaine, benzoyl ecgonine, methamphetamine, MDMA and 6-acetyl morphine to avoid interference from the native isotopes already present in the wastewater matrix. The sewer reactors were operated at 20 °C and pH 7.5, and wastewater was sampled at 0, 0.25, 0.5, 1, 2, 3, 6, 9 and 12 h to measure the transformation/degradation of these marker compounds. The results showed that while methamphetamine, MDMA and benzoyl ecgonine were stable in the sewer reactors, cocaine and 6-acetyl morphine degraded quickly. Their degradation rates are significantly higher than the values reportedly measured in wastewater alone (without biofilms). All the degradation processes followed first order kinetics. Benzoyl ecgonine and morphine were also formed from the degradation of cocaine and 6-acetyl morphine, respectively, with stable formation rates throughout the test. These findings suggest that, in sewage epidemiology, it is essential to have relevant information of the sewer system (i.e. type of sewer, hydraulic retention time) in order to accurately back-estimate the consumption of illicit drugs. More research is required to look into detailed sewer conditions (e.g. temperature, pH and ratio of biofilm area to wastewater volume among others) to identify their effects on the fate of illicit drug markers in sewer systems.

Biosorption of metals using nonliving biomass - A review

A review of published literature on the biosorption of metals using nonliving biomass is presented. Factors such as pH, temperature, initial metal concentration, biomass loaning, the presence of co-ions and the pretreatment of biomass influence the metal uptake by biomass. Although few generalizations are possible, unified theor ies regarding the mechanism of uptake are not available. Therefore, the above aspects of metal biosorption have to be defined individually for each biomass and metal-ion pair.

Conformation-activity correlations for chemotactic tripeptide analogs incorporating dialkyl residues with linear and cyclic alkyl sidechains at position 2

Five stereochemically constrained analogs of the chemotactic tripeptide incorporating 1-aminocycloalkane-1-carboxylic acid (Ac(n)c) and alpha,alpha-dialkylglycines (Deg, diethylglycine; Dpg, n,n-dipropylglycine and Dbg, n,n-dibutylglycine) at position 2 have been synthesized. NMR studies of peptides For-Met-Xxx-Phe-OMe (Xxx = Ac(7)c, I; Ac(8)c, II; Deg, III; Dpg, IV and Dbg, V; For, formyl) establish that peptides with cycloalkyl residues, I and II, adopt folded beta-turn conformations in CDCl3 and (CD3)(2)SO. In contrast, analogs with linear alkyl sidechains, III-V, favour fully extended (C-5) conformations in solution. Peptides I-V exhibit high activity in inducing beta-glucosaminidase release from rabbit neutrophils, with ED(50) values ranging from 1.4-8.0 x 10(-11)M. In human neutrophils the Dxg peptides III-V have ED(50) values ranging from 2.3 x 10(-8) to 5.9 x 10(-10) M, with the activity order being V > IV > III. While peptides I-IV are less active than the parent. For-Met-Leu-Phe-OH, in stimulating histamine release from human basophils, the Dbg peptide V is appreciably more potent, suggesting its potential utility as a probe for formyl peptide receptors.