Identifying and characterizing the most significant β-glucosidase of the novel species Aspergillus saccharolyticus

The newly discovered fungal species Aspergillus saccharolyticus was found to produce a culture broth rich in β-glucosidase activity. In this present work, the main β-glucosidase of A.?saccharolyticus responsible for the efficient hydrolytic activity was identified, isolated, and characterized. Ion exchange chromatography was used to fractionate the culture broth, yielding fractions with high β-glucosidase activity and only 1 visible band on an SDS-PAGE gel. Mass spectrometry analysis of this band gave peptide matches to β-glucosidases from aspergilli. Through a polymerase chain reaction approach using degenerate primers and genome walking, a 2919 bp sequence encoding the 860 amino acid BGL1 polypeptide was determined. BGL1 of A.?saccharolyticus has 91% and 82% identity with BGL1 from Aspergillus aculeatus and BGL1 from Aspergillus niger , respectively, both belonging to Glycoside Hydrolase family 3. Homology modeling studies suggested β-glucosidase activity with preserved retaining mechanism and a wider catalytic pocket compared with other β-glucosidases. The bgl1 gene was heterologously expressed in Trichoderma reesei QM6a, purified, and characterized by enzyme kinetics studies. The enzyme can hydrolyze cellobiose, p-nitrophenyl-β-d-glucoside, and cellodextrins. The enzyme showed good thermostability, was stable at 50?°C, and at 60?°C it had a half-life of approximately 6?h.     

A genetic variant in osteoprotegerin is associated with progression of joint destruction in rheumatoid arthritis

Introduction

Progression of joint destruction in rheumatoid arthritis (RA) is partly heritably; 45 to 58% of the variance in joint destruction is estimated to be explained by genetic factors. The binding of RANKL (Receptor Activator for Nuclear Factor κ B Ligand) to RANK results in the activation of TRAF6 (tumor necrosis factor (TNF) receptor associated factor-6), and osteoclast formation ultimately leading to enhanced bone resorption. This bone resorption is inhibited by osteoprotegerin (OPG) which prevents RANKL-RANK interactions. The OPG/RANK/RANKL/TRAF6 pathway plays an important role in bone remodeling. Therefore, we investigated whether genetic variants in OPG, RANK, RANKL and TRAF6 are associated with the rate of joint destruction in RA.

Methods

1,418 patients with 4,885 X-rays of hands and feet derived from four independent data-sets were studied. In each data-set the relative increase of the progression rate per year in the presence of a genotype was assessed. First, explorative analyses were performed on 600 RA-patients from Leiden. 109 SNPs, tagging OPG, RANK, RANKL and TRAF6, were tested. Single nucleotide polymorphisms (SNPs) significantly associated in phase-1 were genotyped in data-sets from Groningen (Netherlands), Sheffield (United Kingdom) and Lund (Switzerland). Data were summarized in an inverse weighted variance meta-analysis. Bonferonni correction for multiple testing was applied.

Results

We found that 33 SNPs were significantly associated with the rate of joint destruction in phase-1. In phase-2, six SNPs in OPG and four SNPs in RANK were associated with progression of joint destruction with P-value <0.05. In the meta-analyses of all four data-sets, RA-patients with the minor allele of OPG-rs1485305 expressed higher rates of joint destruction compared to patients without these risk variants (P = 2.35x10⁻⁴). This variant was also significant after Bonferroni correction.

Conclusions

These results indicate that a genetic variant in OPG is associated with a more severe rate of joint destruction in RA.     

Multitrophic influences on egg distribution in a specialized leaf beetle at multiple spatial scales

Egg distribution in herbivorous beetles can be affected by bottom-up (host plant), and by top-down factors (parasitoids and predators), as well as by other habitat parameters. The importance of bottom-up and top-down effects may change with spatial scale.

In this study, we investigated the influence of host plant factors and habitat structure on egg distribution in the leaf beetle Cassida canaliculata Laich. (Coleoptera: Chrysomelidae), a monophagous herbivore on Salvia pratensis L. (Lamiales: Lamiaceae), on four spatial scales: individual host plant, microhabitat, macrohabitat, and landscape. At the individual host plant scale we studied the correlation between egg clutch incidence and plant size and quality. On all other scales we analyzed the relationship between the egg clutch incidence of C. canaliculata and host plant percentage cover, host plant density, and the surrounding vegetation structure. Vegetation structure was examined as herbivores might escape egg parasitism by depositing their eggs on sites with vegetation factors unfavorable for host searching parasitoids.

The probability that egg clutches of C. canaliculata were present increased with an increasing size, percentage cover, and density of the host plant on three of the four spatial scales: individual host plant, microhabitat, and macrohabitat. There was no correlation between vegetation structure and egg clutch occurrence or parasitism on any spatial scale. A high percentage of egg clutches (38–56%) was parasitized by Foersterella reptans Nees (Hymenoptera: Tetracampidae), the only egg parasitoid, but there was no relationship between egg parasitism and the spatial distribution of egg clutches of C. canaliculata on any of the spatial scales investigated. However, we also discuss results from a further study, which revealed top-down effects on the larval stage.     

Hydrophobic plastics films synthesis by mean of agaroid acylation with lauroyl chloride in the N,N-dimethylacetamide homogeneous system

The synthesis of hydrophobic plastic films was performed by acylation of agaroids with lauroyl chloride in the N,N-dimethylacetamide homogeneous system. All the plastic films were characterized by FT-IR spectroscopy and their degrees of substitution (DS) was deduced from their ¹H-NMR spectra. In addition, thermomechanical feature of plastic films were analyzed and compared to those obtained from other kinds of hydrophobic plastic films. Latin square design of experiments helped us to determine optimized experimental conditions and identify the most important factors. Hence, mild conditions of acylation (90∘C, 1 eq/OH of lauroyl chloride, 1 eq/OH of 4-dimethylaminopyridine, 5,min) led to the production of highly substituted plastic films (DS = 3.62; maximum 4) with a high weight yield (211%) displaying mechanical properties close to polyethylene low density.     

Determination of turnover of androstenedione to estrone via aromatase in estrogen receptor positive and negative human breast cancer cell lines [Poster 48]

In one estrogen receptor (ER) negative (MDA-MB-231) and two ER positive human breast cancer cell lines (T-47-D,SK-BR-3) we measured aromatase activity by [³H]water assay and estrone (E1) production by thin-layer chromatography. Compared with ether extraction and charcoal method, lyophilization proved to be the most sensitive technique to measure the quantity of [³H]water. The extremely low contamination of the water soluble phase by [1ß-³H]androstenedione (0.02%), as well as the lack of errors due to conjugated steroids, offers the possibility to measure changes of cellular aromatase activity even at very low levels. In contrast to SK-BR-3 and MDA-MB-231 cells, we found no aromatase activity in T-47-D cells. There was no coincidence between ER status and aromatase activity. Proliferation of tumor cells was parallel with a continuous increase of aromatase activity and E1 production during mitogenic growth phase reaching highest levels at the transition from log to plateau-phase.     

Contribution of antigen-primed CD8+ T cells to the development of airway hyperresponsiveness and inflammation is associated with IL-13

The role of Th2/CD4 T cells, which secrete IL-4, IL-5, and IL-13, in allergic disease is well established; however, the role of CD8(+) T cells (allergen-induced airway hyperresponsiveness (AHR) and inflammation) is less clear. This study was conducted to define the role of Ag-primed CD8(+) T cells in the development of these allergen-induced responses. CD8-deficient (CD8(-/-)) mice and wild-type mice were sensitized to OVA by i.p. injection and then challenged with OVA via the airways. Compared with wild-type mice, CD8(-/-) mice developed significantly lower airway responsiveness to inhaled methacholine and lung eosinophilia, and exhibited decreased IL-13 production both in vivo, in the bronchoalveolar lavage (BAL) fluid, and in vitro, following Ag stimulation of peribronchial lymph node (PBLN) cells in culture. Reconstitution of sensitized and challenged CD8(-/-) mice with allergen-sensitized CD8(+) T cells fully restored the development of AHR, BAL eosinophilia, and IL-13 levels in BAL and in culture supernatants from PBLN cells. In contrast, transfer of naive CD8(+) T cells or allergen-sensitized CD8(+) T cells from IL-13-deficient donor mice failed to do so. Intracellular cytokine staining of lung as well as PBLN T cells revealed that CD8(+) T cells were a source of IL-13. These data suggest that Ag-primed CD8(+) T cells are required for the full development of AHR and airway inflammation, which appears to be associated with IL-13 production from these primed T cells.     

Antigen-dependent and -independent mechanisms of T and B cell hyperactivation during chronic HIV-1 infection

Continuous loss of CD4(+) T lymphocytes and systemic immune activation are hallmarks of untreated chronic HIV-1 infection. Chronic immune activation during HIV-1 infection is characterized by increased expression of activation markers on T cells, elevated levels of proinflammatory cytokines, and B cell hyperactivation together with hypergammaglobulinemia. Importantly, hyperactivation of T cells is one of the best predictive markers for progression toward AIDS, and it is closely linked to CD4(+) T cell depletion and sustained viral replication. Aberrant activation of T cells is observed mainly for memory CD4(+) and CD8(+) T cells and is documented, in addition to increased expression of surface activation markers, by increased cell cycling and apoptosis. Notably, the majority of these activated T cells are neither HIV specific nor HIV infected, and the antigen specificities of hyperactivated T cells are largely unknown, as are the exact mechanisms driving their activation. B cells are also severely affected by HIV-1 infection, which is manifested by major changes in B cell subpopulations, B cell hyperactivation, and hypergammaglobulinemia. Similar to those of T cells, the mechanisms underlying this aberrant B cell activation remain largely unknown. In this review, we summarized current knowledge about proposed antigen-dependent and -independent mechanisms leading to lymphocyte hyperactivation in the context of HIV-1 infection.     

N-cadherin specifies first asymmetry in developing neurons

The precise polarization and orientation of developing neurons is essential for the correct wiring of the brain. In pyramidal excitatory neurons, polarization begins with the sprouting of opposite neurites, which later define directed migration and axo-dendritic domains. We here show that endogenous N-cadherin concentrates at one pole of the newborn neuron, from where the first neurite subsequently emerges. Ectopic N-cadherin is sufficient to favour the place of appearance of the first neurite. The Golgi and centrosome move towards this newly formed morphological pole in a second step, which is regulated by PI3K and the actin/microtubule cytoskeleton. Moreover, loss of function experiments in vivo showed that developing neurons with a non-functional N-cadherin misorient their cell axis. These results show that polarization of N-cadherin in the immediate post-mitotic stage is an early and crucial mechanism in neuronal polarity.     

Incorporation of mineral nitrogen into the soil food web as affected by plant community composition

Although nitrogen (N) deposition is increasing globally, N availability still limits many organisms, such as microorganisms and mesofauna. However, little is known to which extent soil organisms rely on mineral‐derived N and whether plant community composition modifies its incorporation into soil food webs. More diverse plant communities more effectively compete with microorganisms for mineral N likely reducing the incorporation of mineral‐derived N into soil food webs. We set up a field experiment in experimental grasslands with different levels of plant species and functional group richness. We labeled soil with ¹⁵NH₄ ¹⁵NO₃ and analyzed the incorporation of mineral‐derived ¹⁵N into soil microorganisms and mesofauna over 3 months. Mineral‐derived N incorporation decreased over time in all investigated organisms. Plant species richness and presence of legumes reduced the uptake of mineral‐derived N into microorganisms. In parallel, the incorporation of mineral‐derived ¹⁵N into mesofauna species declined with time and decreased with increasing plant species richness in the secondary decomposer springtail Ceratophysella sp. Effects of both plant species richness and functional group richness on other mesofauna species varied with time. The presence of grasses increased the ¹⁵N incorporation into Ceratophysella sp., but decreased it in the primary decomposer oribatid mite Tectocepheus velatus sarekensis. The results highlight that mineral N is quickly channeled into soil animal food webs via microorganisms irrespective of plant diversity. The amount of mineral‐derived N incorporated into soil animals, and the plant community properties affecting this incorporation, differed markedly between soil animal taxa, reflecting species‐specific use of food resources. Our results highlight that plant diversity and community composition alter the competition for N in soil and change the transfer of N across trophic levels in soil food webs, potentially leading to changes in soil animal population dynamics and community composition. Sustaining high plant diversity may buffer detrimental effects of elevated N deposition on soil biota.     

Interleukin-7 inactivates the pro-apoptotic protein Bad promoting T cell survival

Interleukin-7 (IL-7) is a cytokine that is required for T cell development and survival. The anti-apoptotic function of IL-7 is partly through induction of Bcl-2 synthesis and cytosolic retention of Bax. Here we show that the Bcl-2 homology 3 domain-only protein, Bad, is involved in cell death following IL-7 withdrawal from D1 cells, an IL-7-dependent murine thymocyte cell line. IL-7 stimulation resulted in the inactivation of Bad by phosphorylation at Ser-112, -136, and -155. The phosphoinositide 3-kinase (PI3K)/Akt pathway has been implicated previously in Bad phosphorylation. In response to IL-7, the PI3K/Akt pathway induced phosphorylation at Ser-136 and -155, but Ser-112 was partly independent of the PI3K/Akt pathway, indicating an as yet unknown pathway in this response. Following IL-7 withdrawal, dephosphorylated Bad translocated from cytosol to mitochondria, bound to Bcl-2, and accelerated cell death. Thus, the inactivation of Bad contributes to the survival function of IL-7.