Heterologous expression, characterization and site-directed mutagenesis of cutinase CUTAB1 from Alternaria brassicicola

The cutinase CUTAB1 was cloned from a cutin induced culture of Alternaria brassicicola and heterologously expressed in Pichia pastoris under the control of the methanol-inducible AOX1 promoter. From a 400-ml culture, 36 mg of purified recombinant enzyme were obtained. Biochemical characterization revealed highest catalytic activity of the enzyme at 40°C and pH 7-9 using p-nitrophenyl palmitate (p-NPP) as substrate. Among several fatty acid methyl and ethyl esters, glycerol esters and p-nitrophenyl esters tested, CUTAB1 showed highest activity towards tributyrin (3,302 ± 160 U mg⁻¹) and the activity decreased with increase in chain length of the investigated esters. Lowest activity was found for p-NPP. Replacing Leu80, Leu181 and Ile183, respectively, by the smaller alanine in the hydrophobic binding loop of CUTAB1, drastically reduced the overall activity of the enzyme. On the other hand, mutation A84F located in the small helical flap of CUTAB1 significantly increased the activity of the enzyme towards longer chain substrates like p-NPP.     

BCG strain S4-Jena: An early BCG strain is capable to reduce the proliferation of bladder cancer cells by induction of apoptosis

Background  

Intravesical immunotherapy with Mycobacterium bovis bacillus Calmette-Guérin has been established as the most effective adjuvant treatment for high risk non-muscle-invasive bladder cancer (NMIBC). We investigated the differences between the S4-Jena BCG strain and commercially available BCG strains. We tested the genotypic varieties between S4-Jena and other BCG strains and analysed the effect of the BCG strains TICE and S4-Jena on two bladder cancer cell lines.     

Analysis of photosynthetic complexes from a cyanobacterial ycf37 mutant

The Ycf37 protein has been suggested to be involved in the biogenesis and/or stability of the cyanobacterial photosystem I (PSI). With Ycf37 specific antibodies, we analyzed the localization of Ycf37 within the thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803. Inspection of a sucrose gradient profile indicated that small amounts of Ycf37 co-fractionated with monomeric photosynthetic complexes, but not with trimeric PSI. Isolating 3xFLAG epitope-tagged Ycf37 by affinity-tag purification rendered several PSI subunits that specifically co-precipitated with this protein. Blue-native PAGE newly revealed two monomeric PSI complexes (PSI and PSI*) in wild-type thylakoids. The lower amount of PsaK present in PSI* may explain its higher electrophoretic mobility. PSI* was more prominent in high-light grown cells and interestingly proved absent in the Deltaycf37 mutant. PSI* appeared again when the mutant was complemented in trans with the wild-type ycf37 gene. In the Deltaycf37 mutant the amount of trimeric PSI complexes was reduced to about 70% of the wild-type level with no significant changes in photochemical activity and subunit composition of the remaining photosystems. Our results indicate that Ycf37 plays a specific role in the preservation of PSI* and the biogenesis of PSI trimers.     

Mesodermal anatomies in cnidarian polyps and medusae

The cellular and developmental analysis of evolutionary-conserved genes directing bilaterian mesodermal and myogenic cell fate previously identified the hydromedusan entocodon and its differentiation product, the striated muscle, as mesodermal derivatives. In view of these findings we presented a hypothesis disputing the diploblast classification of cnidarians without providing further explanations for the apparent diploblasty of the polyp stage and the formation of the subepidermal striated muscle in those Medusozoa lacking the entocodon nodule (Seipel and Schmid, 2005). Hence we carried out a systematic review of the histological and experimental evidence for mesodermal differentiations in cnidarians. In anthozoan and scyphozoan but not in hydrozoan polyps the presumptive mesodermal elements include amoeboid cells, the mesentery retractor muscles and scleroblasts, all of which are embedded or deeply rooted in the extracellular matrix (mesoglea) and derive from the ectoblastemal cells invading the extracellular matrix from the gastrulation site during or shortly after endoderm formation. These data lend further support to the cnidarian mesodermate hypothesis, whereby cnidarians and bilaterians share a common triploblast ancestor, the Urtriploblast, a small, motile, possibly medusa-like organism that did not feature a sessile polyp stage in its life cycle. As a consequence the diploblasty of the hydrozoan polyps may represent a derived morphology resulting from heterochronic modulations of the gastrulation process after endoderm formation.     

Cold winter effects on benthic macrofauna communities in near- and offshore regions of the North Sea

In order to investigate the effect of severe winters on the long-term variability of benthic macrofauna communities four stations from the inner German Bight towards the Dogger Bank (North Sea) were sampled in March 1990 and from May 1995 to 2002. The stations were chosen to reflect a gradient in the hydrographic regime, temperature and organic matter supply. We initially hypothesized that the effect of severe winters on benthic communities will be more pronounced in the offshore more stable environment than in the nearshore regions. Our results clearly showed the opposite pattern. The benthic communities at the nearshore stations in the German Bight changed dramatically in species abundance and community structure after the severe winter 1995/1996. But the community structure in the German Bight returned to the previous stage within 2 years. In contrast, the offshore stations in the Oyster Ground and at the Dogger Bank remained rather unaffected by the severe winter and changed gradually during the study period. The results are discussed concerning the ecological importance of severe winters for benthic communities and the utility of our results for interpreting long-term changes.     

The impacts of different management strategies and environmental forcing in ecological communities

Understanding the effects of population management on the community a target species belongs to is of key importance for successful management. It is known that the removal or extinction of a single species in a community may lead to extinctions of other community members. In our study, we assess the impacts of population management on competitive communities, studying the response of both locally stable and unstable communities of varying size (between four and 10 species) to three different management strategies; harvesting of a target species, harvesting with non-targeted catch, and stocking of the target species. We also studied the consequences of selecting target species with different relative abundances, as well as the effects of varying environmental conditions.We show here how the effects of management in competitive communities extend far beyond the target population. A crucial role is played by the underlying stability properties of the community under management. In general, locally unstable communities are more vulnerable to perturbation through management. Furthermore, the community response is shown to be sensitive to the relative density of the target species. Of considerable interest is the result that even a small (2.5%) increase in the population size of the target species through stocking may lead to extinction of other community members. These results emphasize the importance of considering and understanding multi-species interactions in population management.     

Design, synthesis and structure-activity relationships of new phosphinate inhibitors of MurD

A series of new phosphinate compounds were designed and synthesized as inhibitors of the d-glutamic acid-adding enzyme (MurD) involved in peptidoglycan biosynthesis. They were tested against the MurD enzyme from Escherichia coli, allowing initial structure-activity relationships to be deduced. Two compounds had IC(50) values near 100 microM and constitute a promising starting point for further development.     

Flavonoids and cinnamic acid esters as inhibitors of fungal 17beta-hydroxysteroid dehydrogenase: a synthesis, QSAR and modelling study

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) modulate the biological potency of estrogens and androgens by interconversion of inactive 17-keto-steroids and their active 17beta-hydroxy- counterparts. We have shown previously that flavonoids are potentially useful lead compounds for developing inhibitors of 17beta-HSDs. In this paper, we describe the synthesis and biochemical evaluation of structurally analogous inhibitors, the trans-cinnamic acid esters and related compounds. Additionally, quantitative structure-activity relationship (QSAR) and modelling studies were performed to rationalize the results and to suggest further optimization. The results stress the importance of a hydrogen bond with Asn154 and hydrophobic interactions with the aromatic side chain of Tyr212 for optimal molecular recognition.     

Characterization of 37 breed-specific single-nucleotide polymorphisms in sheep

We identified 37 single-nucleotide polymorphisms (SNPs) in sheep and screened 16 individuals from 8 different sheep breeds selected throughout Europe. Population genetic measures based on the genotyping of about 30 sheep from the same 8 breeds are reported. To date, there are no sheep SNPs documented in the National Center for Biotechnology Information dbSNP database. Therefore, the markers presented here contribute significantly to those currently available.