Enzyme-Induced Formation of β-Lactoglobulin Fibrils by AspN Endoproteinase

This paper describes a low temperature, enzymatic route to induce fibrillar structures in a protein solution. The route comprises two steps. First, β-lactoglobulin was hydrolyzed into peptides at pH 8 and 37 °C with the enzyme AspN endoproteinase, which resulted in the formation of random aggregates. After hydrolysis, the pH was lowered to 2. As a result, long fibrillar aggregates were formed which was observed using transmission electron microscopy and Thioflavin T fluorescence measurements.     

Methylation in methanol-water mixtures: the effect of solvent composition and high pressure

The effect of pressure (0.1 to 400 MPa) and solvent composition (methanol concentration of 5 to 30%) on the synthesis of beta-methylgalactoside was studied. Galactose was used as a reactant and the reaction was catalyzed by beta-galactosidase from Aspergillus oryzae. Under the applied conditions the enzyme was sufficiently stable and the reaction equilibrium was reached. Higher methanol concentrations obviously influenced the product yield positively due to an increase in reactant concentration but also due to a solvent effect. This solvent effect can be explained by measurement of the activities of galactose and methylgalactoside. These results may be generalized to other methylations in methanol-water systems, where methanol positively affects synthesis yields. Pressure had a small, negative effect on synthesis yields.     

The role of double covalent flavin binding in chito-oligosaccharide oxidase from Fusarium graminearum

ChitO (chito-oligosaccharide oxidase) from Fusarium graminearum catalyses the regioselective oxidation of N-acetylated oligosaccharides. The enzyme harbours an FAD cofactor that is covalently attached to His94 and Cys154. The functional role of this unusual bi-covalent flavin-protein linkage was studied by site-directed mutagenesis. The double mutant (H94A/C154A) was not expressed, which suggests that a covalent flavin-protein bond is needed for protein stability. The single mutants H94A and C154A were expressed as FAD-containing enzymes in which one of the covalent FAD-protein bonds was disrupted relative to the wild-type enzyme. Both mutants were poorly active, as the k(cat) decreased (8.3- and 3-fold respectively) and the K(m) increased drastically (34- and 75-fold respectively) when using GlcNac as the substrate. Pre-steady-state analysis revealed that the rate of reduction in the mutant enzymes is decreased by 3 orders of magnitude when compared with wild-type ChitO (k(red)=750 s(-1)) and thereby limits the turnover rate. Spectroelectrochemical titrations revealed that wild-type ChitO exhibits a relatively high redox potential (+131 mV) and the C154A mutant displays a lower potential (+70 mV), while the H94A mutant displays a relatively high potential of approximately +164 mV. The results show that a high redox potential is not the only prerequisite to ensure efficient catalysis and that removal of either of the covalent bonds may perturb the geometry of the Michaelis complex. Besides tuning the redox properties, the bi-covalent binding of the FAD cofactor in ChitO is essential for a catalytically competent conformation of the active site.     

Evolution of subterranean diving beetles (Coleoptera: Dytiscidae: Hydroporini, Bidessini) in the arid zone of Australia

Calcrete aquifers in arid inland Australia have recently been found to contain the world's most diverse assemblage of subterranean diving beetles (Coleoptera: Dytiscidae). In this study we test whether the adaptive shift hypothesis (ASH) or the climatic relict hypothesis (CRH) is the most likely mode of evolution for the Australian subterranean diving beetles by using a phylogeny based on two sequenced fragments of mitochondrial genes (CO1 and 16S-tRNA-ND1) and linearized using a relaxed molecular clock method. Most individual calcrete aquifers contain an assemblage of diving beetle species of distantly related lineages and/or a single pair of sister species that significantly differ in size and morphology. Evolutionary transitions from surface to subterranean life took place in a relatively small time frame between nine and four million years ago. Most of the variation in divergence times of the sympatric sister species is explained by the variation in latitude of the localities, which correlates with the onset of aridity from the north to the south and with an aridity maximum in the Early Pliocene (five mya). We conclude that individual calcrete aquifers were colonized by several distantly related diving beetle lineages. Several lines of evidence from molecular clock analyses support the CRH, indicating that all evolutionary transitions took place during the Late Miocene and Early Pliocene as a result of aridification.     

Functional human antigen-specific T cells produced in vitro using retroviral T cell receptor transfer into hematopoietic progenitors

In vitro production of human T cells with known Ag specificity is of major clinical interest for immunotherapy against tumors and infections. We have performed TCRalphabeta gene transfer into human hemopoietic progenitors from postnatal thymus or umbilical cord blood, and subsequently cultured these precursors on OP9 stromal cells expressing the Notch human ligand Delta-like1. We report here that fully mature, functional T cells with controlled Ag specificity are obtained from such cultures. Using vectors encoding TCRalphabeta-chains directed against melanoma (MART-1), viral (CMV), and minor histocompatibility (HA-2) Ags, we show that the obtained Ag-specific T cells exert cytolytic activity against their cognate Ag and expand in vitro upon specific TCR stimulation. Therapeutic applications may arise from these results because they provide a way to produce large numbers of autologous mature Ag-specific T cells in vitro from undifferentiated hemopoietic progenitors.     

The Akt/GSK-3beta axis as a new signaling pathway of the histamine H(3) receptor

Drugs targeting the histamine H(3) receptor (H(3)R) are suggested to be beneficial for the treatment of neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. The H(3)R activates G(i/o)-proteins to inhibit adenylyl cyclase activity and modulates phospholipase A(2) and MAPK activity. Herein we show that, in transfected SK-N-MC cells, the H(3)R modulates the activity of the Akt/Glycogen synthase kinase 3beta (GSK-3beta) axis both in a constitutive and agonist-dependent fashion. H(3)R stimulation with the H(3)R agonist immepip induces the phosphorylation of both Ser473 and Thr308 on Akt, a serine/threonine kinase that is important for neuronal development and function. The H(3)R-mediated activation of Akt can be inhibited by the H(3)R inverse agonist thioperamide, and by Wortmannin, LY294002 and PTX, suggesting the observed Akt activation occurs via a G(i/o)-mediated activation of phosphoinositide-3-kinase. H(3)R activation also results in the phosphorylation of Ser9 on GSK-3beta, which acts downstream of Akt and has a prominent role in brain function. In addition, we show the H(3)R-mediated phosphorylation of Akt at Ser473 to occur in primary rat cortical neurons and in rat brain slices. The discovery of this signaling property of the H(3)R adds new understanding to the roles of histamine and the H(3)R in brain function and pathology.     

Heterologous Protection against Malaria after Immunization with Plasmodium falciparum Sporozoites

Background

Sterile protection in >90% of volunteers against homologous Plasmodium falciparum infection has been achieved only using the controlled human malaria infection (CHMI) model. This efficient model involves whole parasite immunizations under chloroquine prophylaxis (CPS-immunization), requiring only 30–45 mosquitoes bites infected with P. falciparum-sporozoites. Given the large diversity of P. falciparum parasites, it is essential to assess protection against heterologous parasite strains.

Methods

In an open-label follow-up study, 16 volunteers previously CPS-immunized and challenged with P. falciparum NF54 (West-Africa) in a dose de-escalation and challenge trial were re-challenged with clone NF135.C10 (Cambodia) at 14 months after the last immunization (NCT01660854).

Results

Two out of thirteen NF54 protected volunteers previously fully protected against NF54 were also fully protected against NF135.C10, while 11/13 showed a delayed patency (median prepatent period of 10.5 days (range 9.0–15.5) versus 8.5 days in 5 malaria-naïve controls (p = 0.0005). Analysis of patency by qPCR indicated a 91 to >99% estimated reduction of liver parasite load in 7/11 partially protected subjects. Three volunteers previously not protected against NF54, were also not protected against NF135.C10.

Conclusion

This study shows that CPS-immunization can induce heterologous protection for a period of more than one year, which is a further impetus for clinical development of whole parasite vaccines.

Trial Registration

Clinicaltrials.gov NCT01660854     

Plantecophys - An R Package for Analysing and Modelling Leaf Gas Exchange Data

Here I present the R package ''plantecophys'', a toolkit to analyse and model leaf gas exchange data. Measurements of leaf photosynthesis and transpiration are routinely collected with portable gas exchange instruments, and analysed with a few key models. These models include the Farquhar-von Caemmerer-Berry (FvCB) model of leaf photosynthesis, the Ball-Berry models of stomatal conductance, and the coupled leaf gas exchange model which combines the supply and demand functions for CO₂ in the leaf. The ''plantecophys'' R package includes functions for fitting these models to measurements, as well as simulating from the fitted models to aid in interpreting experimental data. Here I describe the functionality and implementation of the new package, and give some examples of its use. I briefly describe functions for fitting the FvCB model of photosynthesis to measurements of photosynthesis-CO₂ response curves (''A-C_i curves''), fitting Ball-Berry type models, modelling C3 photosynthesis with the coupled photosynthesis-stomatal conductance model, modelling C4 photosynthesis, numerical solution of optimal stomatal behaviour, and energy balance calculations using the Penman-Monteith equation. This open-source package makes technically challenging calculations easily accessible for many users and is freely available on CRAN.     

Plant evolution: AGC kinases tell the auxin tale

The signaling molecule auxin is a central regulator of plant development, which instructs tissue and organ patterning, and couples environmental stimuli to developmental responses. Here, we discuss the function of PINOID (PID) and the phototropins, members of the plant specific AGCVIII protein kinases, and their role in triggering and regulating development by controlling PIN-FORMED (PIN) auxin transporter-generated auxin gradients and maxima. We propose that the AGCVIII kinase gene family evolved from an ancestral phototropin gene, and that the co-evolution of PID-like and PIN gene families marks the transition of plants from water to land. We hypothesize that the PID-like kinases function in parallel to, or downstream of, the phototropins to orient plant development by establishing the direction of polar auxin transport.