Studies of lysozyme binding to histamine as a ligand for hydrophobic charge induction chromatography

Histamine was immobilized on Sepharose CL‐6B (Sepharose) for use as a ligand of hydrophobic charge induction chromatography (HCIC) of proteins. Lysozyme adsorption onto Histamine‐Sepharose (HA‐S) was studied by adsorption equilibrium and calorimetry to uncover the thermodynamic mechanism of the protein binding. In both the experiments, the influence of salt (ammonium sulfate and sodium sulfate) was examined. Adsorption isotherms showed that HA‐S exhibited a high salt tolerance in lysozyme adsorption. This property was well explained by the combined contributions of hydrophobic interaction and aromatic stacking. The isotherms were well fitted to the Langmuir equation, and the equilibrium parameters for lysozyme adsorption were obtained. In addition, thermodynamic parameters (ΔH_ads, ΔS_ads, and ΔG_ads) for the adsorption were obtained by isothermal titration calorimetry by titrating lysozyme solutions into the adsorbent suspension. Furthermore, free histamine was titrated into lysozyme solution in the same salt‐buffers. Compared with the binding of lysozyme to free histamine, lysozyme adsorption onto HA‐S was characterized by a less favorable ΔG_ads and an unfavorable ΔS_ads because histamine was covalently attached to Sepharose via a three‐carbon‐chain spacer. Consequently, the immobilized histamine could only associate with the residues on the protein surface rather than those in the hydrophobic pocket, causing a less favorable orientation between histamine and lysozyme. Further comparison of thermodynamic parameters indicated that the unfavorable ΔS_ads was offset by a favorable ΔH_ads, thus exhibiting typical enthalpy‐entropy compensation. Moreover, thermodynamic analyses indicated the importance of the dehydration of lysozyme molecule and HA‐S during the adsorption and a substantial conformational change of the protein during adsorption. The results have provided clear insights into the adsorption mechanisms of lysozyme onto the new HCIC material. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Ethanol production potential of sweet sorghum assessed using forage fiber analysis procedures

Sweet sorghum (Sorghum bicolor (L.) Moench) is widely recognized as a highly promising biomass energy crop with particular potential to complement sugarcane production in diversified cropping systems. Agronomic assessments have led to identification of four cultivars well suited for such sugarcane‐based production systems in southern Louisiana. Sweet sorghum biofuel production systems are currently being developed, and research producing large sample numbers requiring ethanol yield assessment is anticipated. Fiber analysis approaches developed for forage evaluation appear to be useful for screening such large numbers of samples for relative ethanol yield. Chemical composition, forage fiber characteristics, digestibility, and ethanol production of sweet sorghum bagasse from the four cultivars were assessed. Measures of detergent fiber, lignin, and digestibility were highly correlated with ethanol production (P < 0.01). The best linear regression models accounted for about 80% of the variation among cultivars in ethanol production. Bagasse from the cultivar Dale produced more ethanol per gram of material than any of the other cultivars. This superior ethanol production was apparently associated with less lignin in stems of Dale. Forage evaluation measures including detergent fiber analyses, in vitro digestibility, and an in vitro gas production technique successfully identified the cultivar superior in ethanol yield indicating their usefulness for screening sweet sorghum samples for potential ethanol production in research programs generating large sample numbers from evaluations of germ plasm or agronomic treatments. These screening procedures reduce time and expense of alternatives such as hexose sugar assessment for calculating theoretical ethanol yield.     

Yeast water channels: an overview of orthodox aquaporins

In yeast, the presence of orthodox aquaporins has been first recognized in Saccharomyces cerevisiae, in which two genes (AQY1 and AQY2) were shown to be related to mammal and plant water channels. The present review summarizes the putative orthodox aquaporin protein sequences found in available genomes of yeast and filamentous fungi. Among the 28 yeast genomes sequenced, most species present only one orthodox aquaporin, and no aquaporins were found in eight yeast species. Alignment of amino acid sequences reveals a very diverse group. Similarity values vary from 99% among species within the Saccharomyces genus to 34% between ScAqy1 and the aquaporin from Debaryomyces hansenii. All of the fungal aquaporins possess the known characteristic sequences, and residues involved in the water channel pore are highly conserved. Advances in the establishment of the structure are reviewed in relation to the mechanisms of selectivity, conductance and gating. In particular, the involvement of the protein cytosolic N‐terminus as a channel blocker preventing water flow is addressed. Methodologies used in the evaluation of aquaporin activity frequently involve the measurement of fast volume changes. Particular attention is paid to data analysis to obtain accurate membrane water permeability parameters. Although the presence of aquaporins clearly enhances membrane water permeability, the relevance of these ubiquitous water channels in yeast performance remains obscure.     

Cooperation of Aspergillus nidulans enzymes increases plant polysaccharide saccharification

Efficient polysaccharide degradation depends on interaction between enzymes acting on the main chain and the side chains. Previous studies demonstrated cooperation between several enzymes, but not all enzyme combinations have been explored. A better understanding of enzyme cooperation would enable the design of better enzyme mixtures, optimally profiting from synergistic effects. In this study, we analyzed the cooperation of several enzymes involved in the degradation of xylan, glucan, xyloglucan and crude plant biomass from Aspergillus nidulans by single and combined incubations with their polymeric substrate. Positive effects were observed between most enzymes, although not always to the same extent. Moreover, the tailor made cocktails formulated in this study resulted in efficient release of glucose from plant biomass. This study also serves as an example for the complex cooperation that occurs between enzymes in plant biomass saccharification and how expression in easily‐accessible hosts, such as Pichia pastoris, can help in revealing these effects.     

PEGylation of lysine residues improves the proteolytic stability of fibronectin while retaining biological activity

Excessive proteolysis of fibronectin (FN) impairs tissue repair in chronic wounds. Since FN is essential in wound healing, our goal is to improve its proteolytic stability and at the same time preserve its biological activity. We have previously shown that reduced FN conjugated with polyethylene glycol (PEG) at cysteine residues is more proteolytically stable than native FN. Cysteine‐PEGylated FN supported cell adhesion and migration to the same extent as native FN. However, unlike native FN, cysteine‐PEGylated FN was not assembled into an extracellular matrix (ECM) when immobilized. Here, we present an alternative approach in which FN is preferentially PEGylated at lysine residues using different molecular weight PEGs. We show that lysine PEGylation does not perturb FN secondary structure. PEG molecular weight, from 2 to 10 kDa, positively correlates with FN–PEG proteolytic stability. Cell adhesion, cell spreading, and gelatin binding decrease with increasing molecular weight of PEG. The 2‐kDa FN–PEG conjugate shows comparable cell adhesion to native FN and binds gelatin. Moreover, immobilized FN–PEG is assembled into ECM fibrils. In summary, lysine PEGylation of FN can be used to stabilize FN against proteolytic degradation with minimal perturbation to FN structure and retained biological activity.     

Heritability and social brood effects on personality in juvenile and adult life‐history stages in a wild passerine

How has evolution led to the variation in behavioural phenotypes (personalities) in a population? Knowledge of whether personality is heritable, and to what degree it is influenced by the social environment, is crucial to understanding its evolutionary significance, yet few estimates are available from natural populations. We tracked three behavioural traits during different life‐history stages in a pedigreed population of wild house sparrows. Using a quantitative genetic approach, we demonstrated heritability in adult exploration, and in nestling activity after accounting for fixed effects, but not in adult boldness. We did not detect maternal effects on any traits, but we did detect a social brood effect on nestling activity. Boldness, exploration and nestling activity in this population did not form a behavioural syndrome, suggesting that selection could act independently on these behavioural traits in this species, although we found no consistent support for phenotypic selection on these traits. Our work shows that repeatable behaviours can vary in their heritability and that social context influences personality traits. Future efforts could separate whether personality traits differ in heritability because they have served specific functional roles in the evolution of the phenotype or because our concept of personality and the stability of behaviour needs to be revised.     

Generalization of the QST framework in hierarchically structured populations: Impacts of inbreeding and dominance

Q_ST is a differentiation parameter based on the decomposition of the genetic variance of a trait. In the case of additive inheritance and absence of selection, it is analogous to the genic differentiation measured on individual loci, F_ST. Thus, Q_ST?F_ST comparison is used to infer selection: selective divergence when Q_ST > F_ST, or convergence when Q_ST < F_ST. The definition of Q‐statistics was extended to two‐level hierarchical population structures with Hardy–Weinberg equilibrium. Here, we generalize the Q‐statistics framework to any hierarchical population structure. First, we developed the analytical definition of hierarchical Q‐statistics for populations not at Hardy–Weinberg equilibrium. We show that the Q‐statistics values obtained with the Hardy–Weinberg definition are lower than their corresponding F‐statistics when F_IS > 0 (higher when F_IS < 0). Then, we used an island model simulation approach to investigate the impact of inbreeding and dominance on the Q_ST?F_ST framework in a hierarchical population structure. We show that, while differentiation at the lower hierarchical level (Q_SR) is a monotonic function of migration, differentiation at the upper level (Q_RT) is not. In the case of additive inheritance, we show that inbreeding inflates the variance of Q_RT, which can increase the frequency of Q_RT > F_RT cases. We also show that dominance drastically reduces Q‐statistics below F‐statistics for any level of the hierarchy. Therefore, high values of Q‐statistics are good indicators of selection, but low values are not in the case of dominance.     

Diterpenoids from Wedelia prostrata and Their Derivatives and Cytotoxic Activities

One new ent‐kaurane diterpenoid, 11β,16α‐dihydroxy‐ent‐kauran‐19‐oic acid ( 1 ), together with eight known analogues 2 – 9 were isolated from the aerial parts of Wedelia prostrata. One of the acidic diterpenoids, kaurenoic acid ( 3 ), was converted to seven derivatives, 10 – 16 . All compounds were evaluated for their cytotoxic activity in vitro against human leukemia (K562), liver (HepG‐2), and stomach (SGC‐7901) cancer cell lines. Only four kaurenoic acid derivatives, 13 – 16 , with 15‐keto and substitutions at C(19) position, exhibited notable cytotoxic activities on these tumor cell lines with IC₅₀ value ranging from 0.05 to 3.71 μm . Compounds 10 – 12 , with oxime on C(15) showed moderate inhibitory effects and compounds 1 – 9 showed no cytotoxicities on them. Structure–activity relationships were also discussed based on the experimental data obtained. The known derivative, 15‐oxokaurenoic acid 4‐piperdin‐1‐ylbutyl ester ( 17 ), induced typical apoptotic cell death in colon SW480 cells upon evaluation of the apoptosis‐inducing activity by flow‐cytometric analysis.     

Detection of antimicrobial substances from larvae of the black soldier fly,Hermetia illucens (Diptera: Stratiomyidae)

Maggots have become highly successful in the treatment of non‐healing wounds and multidrug‐resistant pathogen infections. The main objective of this study was to extract antibacterial substances from larvae of the black soldier fly, Hermetia illucens. To induce immune responses, we septically injured the larvae with a contaminated needle. Lyophilized H. illucens larvae were homogenized and extracted with acidic methanol. We examined the antifungal and antibacterial effects of the low molecular weight antimicrobial factors within the larval extract on the growth of a broad range of microorganisms, including Gram‐positive Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA), and Gram‐negative Pseudomonas aeruginosa. Furthermore, we isolated the anti‐MRSA substances from the larval extract using high performance liquid chromatography. These investigations revealed that the larval extract possessed a broad‐spectrum of antibacterial activity, demonstrating that secretions of H. illucens larvae prove useful in the fight against MRSA and can potentially be a source of novel antibiotic‐like compounds for infection control.