Cellulolytic enzymes for the hydrolysis of leached beet cosette

Summary Cellulolytic fungi were isolated from rotting leaves and tested for extra-cellular cellulase activities (CMCase, avicelase, cellobiase and xylanase). The effect of the proportion of the enzyme activities on the rate of degradation of leached beet cosette was observed using a range of supernatant fluids in appropriate combinations. At low cosette concentrations (1.5–3.0 g/l), avicelase and cellobiase were the rate limiting enzymes; avicelase in the initial stages of reaction and cellobiase after 6–8 hours, when cellobiose inhibition becomes important. A ratio of celiobiase to avicelase of approx 2.0 was established as appropriate. At higher substrate concentrations (10 g/l, 40 g/l) the best cellobiase to avicelase ratio was maintained and up to 40% hydrolysis was obtained in the 10 g/l incubation with 10 Uav/l and 20 Ucellob/l. At 100 g/l cosette concentration, substrate inhibition was observed.     

Denitrification is a common feature among members of the genus Bacillus

Although several Gram-positive denitrifiers have been characterized in the past, there is still uncertainty about the occurrence of the denitrification trait among these bacteria. In an isolation campaign on luvisol soil, Bacillus spp. were among the most abundant retrieved cultured denitrifiers next to members of Rhizobiaceae family and genus Cupriavidus. Subsequent screening of 180 representatives of the genus Bacillus (encompassing more than half of the current validly described species diversity in Bacillus) was performed and demonstrated the potential for dissimilatory reduction of nitrogen compounds in 45 of the 87 investigated species, with 19 species containing denitrifying members. The influence of several electron donors and acceptors was tested. The use of more than one electron acceptor, e.g. both nitrate and nitrite, was crucial to detect the denitrification potential of reference strains. Complex electron donors, most suitable for aerobic growth, were ideal for denitrification testing, while retrieval of denitrifiers from the environment was facilitated by the use of defined electron donors, due to less interference of other anaerobic growers. The outcome of the isolation campaign and screening of reference strain set suggest that bacilli may be potential contributors to denitrification in terrestrial and possibly other ecosystems.     

Analysis of epithelial and mesenchymal markers in ovarian cancer reveals phenotypic heterogeneity and plasticity

In our studies of ovarian cancer cells we have identified subpopulations of cells that are in a transitory E/M hybrid stage, i.e. cells that simultaneously express epithelial and mesenchymal markers. E/M cells are not homogenous but, in vitro and in vivo, contain subsets that can be distinguished based on a number of phenotypic features, including the subcellular localization of E-cadherin, and the expression levels of Tie2, CD133, and CD44. A cellular subset (E/M-MP) (membrane E-cadherin(low)/cytoplasmic E-cadherin(high)/CD133(high), CD44(high), Tie2(low)) is highly enriched for tumor-forming cells and displays features which are generally associated with cancer stem cells. Our data suggest that E/M-MP cells are able to differentiate into different lineages under certain conditions, and have the capacity for self-renewal, i.e. to maintain a subset of undifferentiated E/M-MP cells during differentiation. Trans-differentiation of E/M-MP cells into mesenchymal or epithelial cells is associated with a loss of stem cell markers and tumorigenicity. In vivo xenograft tumor growth is driven by E/M-MP cells, which give rise to epithelial ovarian cancer cells. In contrast, in vitro, we found that E/M-MP cells differentiate into mesenchymal cells, in a process that involves pathways associated with an epithelial-to-mesenchymal transition. We also detected phenotypic plasticity that was dependent on external factors such as stress created by starvation or contact with either epithelial or mesenchymal cells in co-cultures. Our study provides a better understanding of the phenotypic complexity of ovarian cancer and has implications for ovarian cancer therapy.     

Development of substrate analogue inhibitors for the human airway trypsin-like protease HAT

A series of substrate analogue inhibitors of the serine protease HAT, containing a 4-amidinobenzylamide moiety as the P1 residue, was prepared. The most potent compounds possess a basic amino acid in the d-configuration as P3 residue. Whereas inhibitor 4 (K_i 13 nM) containing proline as the P2 residue completely lacks selectivity, incorporation of norvaline leads to a potent inhibitor (15, K_i 15 nM) with improved selectivity for HAT in comparison to the coagulation proteases thrombin and factor Xa or the fibrinolytic plasmin. Selected inhibitors were able to suppress influenza virus replication in a HAT-expressing MDCK cell model.     

Phosphopeptides with improved cellular uptake properties as ligands for the polo-box domain of polo-like kinase 1

Human polo-like kinase 1 (Plk1) is involved in cell proliferation and overexpressed in a broad variety of different cancer types. Due to its crucial role in cancerogenesis Plk1 is a potential target for diagnostic and therapeutic applications. Peptidic ligands can specifically interact with the polo-box domain (PBD) of Plk1, a C-terminal located phosphoepitope binding motif. Recently, phosphopeptide MQSpTPL has been identified as ligand with high binding affinity. However, a radiolabeled version of this peptide showed only insufficient cellular uptake. The present study investigated peptide dimers consisting of PBD-targeting phosphopeptide MQSpTPL and a cell-penetrating peptide (CPP) moiety. The new constructs demonstrate superior uptake in different cancer cell-lines compared to the phosphopeptide alone. Furthermore, we could demonstrate binding of phosphopeptide-CPP dimers to PBD of Plk1 making the compounds interesting leads for the development of molecular probes for imaging Plk1 in cancer.     

Identification and Characterization of a High-Affinity Choline Uptake System of Brucella abortus

Phosphatidylcholine (PC), a common phospholipid of the eukaryotic cell membrane, is present in the cell envelope of the intracellular pathogen Brucella abortus, the etiological agent of bovine brucellosis. In this pathogen, the biosynthesis of PC proceeds mainly through the phosphatidylcholine synthase pathway; hence, it relies on the presence of choline in the milieu. These observations imply that B. abortus encodes an as-yet-unknown choline uptake system. Taking advantage of the requirement of choline uptake for PC synthesis, we devised a method that allowed us to identify a homologue of ChoX, the high-affinity periplasmic binding protein of the ABC transporter ChoXWV. Disruption of the choX gene completely abrogated PC synthesis at low choline concentrations in the medium, thus indicating that it is a high-affinity transporter needed for PC synthesis via the PC synthase (PCS) pathway. However, the synthesis of PC was restored when the mutant was incubated in media with higher choline concentrations, suggesting the presence of an alternative low-affinity choline uptake activity. By means of a fluorescence-based equilibrium-binding assay and using the kinetics of radiolabeled choline uptake, we show that ChoX binds choline with an extremely high affinity, and we also demonstrate that its activity is inhibited by increasing choline concentrations. Cell infection assays indicate that ChoX activity is required during the first phase of B. abortus intracellular traffic, suggesting that choline concentrations in the early and intermediate Brucella-containing vacuoles are limited. Altogether, these results suggest that choline transport and PC synthesis are strictly regulated in B. abortus.     

Prognostic factors and survival in metastatic breast cancer: A single institution experience

Background

The current retrospective study aims to identify some determinants of survival in metastatic breast cancer.

Methods

The study concerned 332 patients with synchronous (SM) or metachronous (MM) metastatic breast cancer treated between January 2000 and December 2007. Statistical comparison between subgroups of patients concerning survival was carried out employing log-rank test for the invariable analysis and Cox model for the multivariable analysis. Factors included: age group (≤50 years vs. >50; ≤70 years vs. >70; ≤35 years vs. >35), menopausal status, presentation of metastatic disease (SM vs. MM), disease free interval (DFI) (≤24 months vs. >24 months; ≤60 months vs. >60 months), performance status at diagnosis of metastatic disease (PS) (0–1 vs. >1), hormone receptors (HR), number of metastatic sites (1 site vs. >1), nature of the metastatic site (visceral vs. non visceral), first line therapy, surgery of the primary tumor (SPT), locoregional radiotherapy (LRRT) and use or not of bisphosphonates.

Results

Overall survival at 5 years was 12%. Positive prognostic factors in univariate analysis were: age ≤ 70 years, hormono-dependence of the tumor, good PS (PS 0–1), less than two metastatic sites, no visceral metastases, DFI ≥ 24 months, SPT or LRRT. In multivariate analysis, favorable independent prognostic factors included: good PS (PS 0–1), absence of visceral metastases (liver, lung, brain) and age ≤ 70 years.

Conclusion

Many of the prognostic factors in metastatic breast cancer found in our study are known in the literature but some of them, like the application of locoregional treatment (radiotherapy or surgery) and the use of bisphosphonates, need to be further investigated in randomized clinical trials.     

Tau,XMAP215/Msps and Eb1 co-operate interdependently to regulate microtubule polymerisation and bundle formation in axons

The formation and maintenance of microtubules requires their polymerisation, but little is known about how this polymerisation is regulated in cells. Focussing on the essential microtubule bundles in axons of Drosophila and Xenopus neurons, we show that the plus-end scaffold Eb1, the polymerase XMAP215/Msps and the lattice-binder Tau co-operate interdependently to promote microtubule polymerisation and bundle organisation during axon development and maintenance. Eb1 and XMAP215/Msps promote each other’s localisation at polymerising microtubule plus-ends. Tau outcompetes Eb1-binding along microtubule lattices, thus preventing depletion of Eb1 tip pools. The three factors genetically interact and show shared mutant phenotypes: reductions in axon growth, comet sizes, comet numbers and comet velocities, as well as prominent deterioration of parallel microtubule bundles into disorganised curled conformations. This microtubule curling is caused by Eb1 plus-end depletion which impairs spectraplakin-mediated guidance of extending microtubules into parallel bundles. Our demonstration that Eb1, XMAP215/Msps and Tau co-operate during the regulation of microtubule polymerisation and bundle organisation, offers new conceptual explanations for developmental and degenerative axon pathologies.