Cold-adapted enzymes: from fundamentals to biotechnology

Psychrophilic enzymes produced by cold-adapted microorganisms display a high catalytic efficiency and are most often, if not always, associated with high thermosensitivity. Using X-ray crystallography, these properties are beginning to become understood, and the rules governing their adaptation to cold appear to be relatively diverse. The application of these enzymes offers considerable potential to the biotechnology industry, for example, in the detergent and food industries, for the production of fine chemicals and in bioremediation processes.     

Tumor-derived mutated E-cadherin influences beta-catenin localization and increases susceptibility to actin cytoskeletal changes induced by pervanadate

E-cadherin participates in homophilic cell-to-cell adhesion and is localized to intercellular junctions of the adherens type. In the present study, we investigated the localization of adherens junction components in cells expressing mutant E-cadherin derivatives which had been previously cloned from diffuse-type gastric carcinoma. The mutations are in frame deletions of exons 8 or 9 and a point mutation in exon 8 and affect the extracellular domain of E-cadherin. Our findings indicate that E-cadherin mutated in exon 8 causes beta-catenin staining at lateral cell-to-cell contact sites and, in addition, abnormally located beta-catenin in the perinuclear region. Moreover, the various mutant E-cadherin derivatives increased the steady-state levels of alpha- and beta-catenin and were found in association with these catenins even after induction of tyrosine phosphorylation by pervanadate. Sustained pervanadate treatment led, however, to rounding-up of cells and induction of filopodia, changes which were first detectable in cells expressing E-cadherin mutated in exon 8. The deterioration of the cell contact was not accompanied with disassembly of the E-cadherin-catenin complex. Based on these observations, we propose a model whereby in the presence of mutant E-cadherin tyrosine phoshorylation of components of the cell adhesion complex triggers loss of cell-to-cell contact and actin cytoskeletal changes which are not caused by the disruption of the E-cadherin-catenin complex per se, but instead might be due to phosphorylation of other signaling molecules or activation of proteins involved in the regulation of the actin cytoskeleton.     

Inhibition of CIN85-Mediated Invasion by a Novel SH3 Domain Binding Motif in the Lysyl Oxidase Propeptide

The lysyl oxidase gene inhibits Ras signaling in transformed fibroblasts and breast cancer cells. Its activity was mapped to the 162 amino acid propeptide domain (LOX-PP) of the lysyl oxidase precursor protein. LOX-PP inhibited the Her-2/Ras signaling axis in breast cancer cells, and reduced the Her-2-driven breast tumor burden in a xenograft model. Since its mechanism of action is largely unknown, co-affinity-purification/mass spectrometry was performed and the “Cbl-interacting protein of 85-kDa” (CIN85) identified as an associating protein. CIN85 is an SH3-containing adapter protein that is overexpressed in invasive breast cancers. The CIN85 SH3 domains interact with c-Cbl, an E3 ubiquitin ligase, via an unconventional PxxxPR ligand sequence, with the highest affinity displayed by the SH3-B domain. Interaction with CIN85 recruits c-Cbl to the AMAP1 complex where its ubiquitination activity is necessary for cancer cells to develop an invasive phenotype and to degrade the matrix. Direct interaction of LOX-PP with CIN85 was confirmed using co-immunoprecipitation analysis of lysates from breast cancer cells and of purified expressed proteins. CIN85 interaction with c-Cbl was reduced by LOX-PP. Domain specific CIN85 regions and deletion mutants of LOX-PP were prepared and used to map the sites of interaction to the SH3-B domain of CIN85 and to an epitope encompassing amino acids 111 to 116 of LOX-PP. Specific LOX-PP point mutant proteins P111A and R116A failed to interact with CIN85 or to compete for CIN85 binding with c-Cbl. Structural modeling identified a new atypical PxpxxRh SH3-binding motif in this region of LOX-PP. The LOX-PP interaction with CIN85 was shown to reduce the invasive phenotype of breast cancer cells, including their ability to degrade the surrounding extracellular matrix and for Matrigel outgrowth. Thus, LOX-PP interacts with CIN85 via a novel SH3-binding motif and this association reduces CIN85-promoted invasion by breast cancer cells.     

Photosynthetic performance and water relations in young pubescent oak (Quercus pubescens) trees during drought stress and recovery

The capability to withstand and to recover from severe summer droughts is becoming an important issue for tree species in central Europe, as dry periods are predicted to occur more frequently over the coming decades. Changes in leaf gas exchange, chlorophyll a fluorescence and leaf compounds related to photoprotection were analysed in young Quercus pubescens trees under field conditions during two summers (2004 and 2005) of progressive drought and subsequent rewatering. Photochemistry was reversibly down-regulated and dissipation of excess energy was enhanced during the stress phase, while contents of leaf pigments and antioxidants were almost unaltered. Plant water status was restored immediately after rewatering. Net photosynthesis (P(n)) measured at ambient CO2 recovered from inhibition by drought within 4 wk. P(n) measured at elevated CO2--to overcome stomatal limitations--was restored after a few days. A network of photoprotective mechanisms acted in preserving the potential functionality of the photosynthetic apparatus during severe drought, leading to a rapid recovery of photosynthetic activity after rewatering. Thus, Q. pubescens seems to be capable of withstanding and surviving extreme drought events.     

Changes of photosynthetic traits in beech saplings (Fagus sylvatica) under severe drought stress and during recovery

In the context of an increased risk of extreme drought events across Europe during the next decades, the capacity of trees to recover and survive drought periods awaits further attention. In summer 2005, 4-year-old beech (Fagus sylvatica L.) saplings were watered regularly or were kept for 4 weeks without irrigation in the field and then re-watered again. Changes of plant water status, leaf gas exchange and Chl a fluorescence parameters, as well as alterations in leaf pigment composition were followed. During the drought period, stomatal conductance (g(s)) and net photosynthesis (P(n)) decreased in parallel with increased water deficit. After 14 days without irrigation, stomata remained closed and P(n) was almost completely inhibited. Reversible downregulation of PSII photochemistry [the maximum quantum efficiency of PSII (F(v)/F(m))], enhanced thermal dissipation of excess excitation energy and an increased ratio of xanthophyll cycle pigments to chlorophylls (because of a loss of chlorophylls) contributed to an enhanced photo-protection in severely stressed plants. Leaf water potential was restored immediately after re-watering, while g(s), P(n) and F(v)/F(m) recovered only partially during the initial phase, even when high external CO(2) concentrations were applied during the measurements, indicating lasting non-stomatal limitations. Thereafter, P(n) recovered completely within 4 weeks, meanwhile g(s) remained permanently lower in stressed than in control plants, leading to an increased 'intrinsic water use efficiency' (P(n)/g(s)). In conclusion, although severe drought stress adversely affected photosynthetic performance of F. sylvatica (a rather drought-sensitive species), P(n) was completely restored after re-watering, presumably because of physiological and morphological adjustments (e.g. stomatal occlusions).     

Dynamic structure of retinylidene ligand of rhodopsin probed by molecular simulations

Rhodopsin is currently the only available atomic-resolution template for understanding biological functions of the G protein-coupled receptor (GPCR) family. The structural basis for the phenomenal dark state stability of 11-cis-retinal bound to rhodopsin and its ultrafast photoreaction are active topics of research. In particular, the beta-ionone ring of the retinylidene inverse agonist is crucial for the activation mechanism. We analyzed a total of 23 independent, 100 ns all-atom molecular dynamics simulations of rhodopsin embedded in a lipid bilayer in the microcanonical (N,V,E) ensemble. Analysis of intramolecular fluctuations predicts hydrogen-out-of-plane (HOOP) wagging modes of retinal consistent with those found in Raman vibrational spectroscopy. We show that sampling and ergodicity of the ensemble of simulations are crucial for determining the distribution of conformers of retinal bound to rhodopsin. The polyene chain is rigidly locked into a single, twisted conformation, consistent with the function of retinal as an inverse agonist in the dark state. Most surprisingly, the beta-ionone ring is mobile within its binding pocket; interactions are non-specific and the cavity is sufficiently large to enable structural heterogeneity. We find that retinal occupies two distinct conformations in the dark state, contrary to most previous assumptions. The beta-ionone ring can rotate relative to the polyene chain, thereby populating both positively and negatively twisted 6-s-cis enantiomers. This result, while unexpected, strongly agrees with experimental solid-state (2)H NMR spectra. Correlation analysis identifies the residues most critical to controlling mobility of retinal; we find that Trp265 moves away from the ionone ring prior to any conformational transition. Our findings reinforce how molecular dynamics simulations can challenge conventional assumptions for interpreting experimental data, especially where existing models neglect conformational fluctuations.     

Identification of a novel sex determining chromosome in cichlid fishes that acts as XY or ZW in different lineages

Sex determination systems are highly conserved among most vertebrates with genetic sex determination, but can be variable and evolve rapidly in some. Here, we study sex determination in a clade with exceptionally high sex chromosome turnover rates. We identify the sex determining chromosomes in three interspecific crosses of haplochromine cichlid fishes from Lakes Victoria and Malawi. We find evidence for different sex determiners in each cross. In the Malawi cross and one Victoria cross the same chromosome is sex-linked but while females are the heterogametic sex in the Malawi species, males are the heterogametic sex in the Victoria species. This chromosome has not previously been reported to be sex determining in cichlids, increasing the number of different chromosomes shown to be sex determining in cichlids to 12. All Lake Victoria species of our crosses are less than 15,000 years divergent, and we identified different sex determiners among them. Our study provides further evidence for the diversity and evolutionary flexibility of sex determination in cichlids, factors which might contribute to their rapid adaptive radiations.

     

Incidence of Second Malignancies for Prostate Cancer

Introduction

There is a need to assess risk of second primary cancers in prostate cancer (PCa) patients, especially since PCa treatment may be associated with increased risk of second primary tumours.

Methods

We calculated standardized incidence ratios (SIRs) for second primary tumours comparing men diagnosed with PCa between 1980 and 2010 in the Canton of Zurich, Switzerland (n = 20,559), and the general male population in the Canton.

Results

A total of 1,718 men developed a second primary tumour after PCa diagnosis, with lung and colon cancer being the most common (15 and 13% respectively). The SIR for overall second primary cancer was 1.11 (95%CI: 1.06–1.17). Site-specific SIRs varied from 1.19 (1.05–1.34) to 2.89 (2.62–4.77) for lung and thyroid cancer, respectively. When stratified by treatment, the highest SIR was observed for thyroid cancer (3.57 (1.30–7.76)) when undergoing surgery, whereas liver cancer was common when treated with radiotherapy (3.21 (1.54–5.90)) and kidney bladder was most prevalent for those on hormonal treatment (3.15 (1.93–4.87)). Stratification by time since PCa diagnosis showed a lower risk of cancer for men with PCa compared to the general population for the first four years, but then a steep increase in risk was observed.

Conclusion

In the Canton of Zurich, there was an increased risk of second primary cancers among men with PCa compared to the general population. Increased diagnostic activity after PCa diagnosis may partly explain increased risks within the first years of diagnosis, but time-stratified analyses indicated that increased risks remained and even increased over time.     

Phosphorus and Nitrogen Regulate Arbuscular Mycorrhizal Symbiosis in Petunia hybrida

Phosphorus and nitrogen are essential nutrient elements that are needed by plants in large amounts. The arbuscular mycorrhizal symbiosis between plants and soil fungi improves phosphorus and nitrogen acquisition under limiting conditions. On the other hand, these nutrients influence root colonization by mycorrhizal fungi and symbiotic functioning. This represents a feedback mechanism that allows plants to control the fungal symbiont depending on nutrient requirements and supply. Elevated phosphorus supply has previously been shown to exert strong inhibition of arbuscular mycorrhizal development. Here, we address to what extent inhibition by phosphorus is influenced by other nutritional pathways in the interaction between Petunia hybrida and R. irregularis. We show that phosphorus and nitrogen are the major nutritional determinants of the interaction. Interestingly, the symbiosis-promoting effect of nitrogen starvation dominantly overruled the suppressive effect of high phosphorus nutrition onto arbuscular mycorrhiza, suggesting that plants promote the symbiosis as long as they are limited by one of the two major nutrients. Our results also show that in a given pair of symbiotic partners (Petunia hybrida and R. irregularis), the entire range from mutually symbiotic to parasitic can be observed depending on the nutritional conditions. Taken together, these results reveal complex nutritional feedback mechanisms in the control of root colonization by arbuscular mycorrhizal fungi.