Construction of a xylanase A variant capable of polymerization

The aim of our work is to furnish enzymes with polymerization ability by creating fusion constructs with the polymerizable protein, flagellin, the main component of bacterial flagellar filaments. The D3 domain of flagellin, exposed on the surface of flagellar filaments, is formed by the hypervariable central portion of the polypeptide chain. D3 is not essential for filament formation. The concept in this project is to replace the D3 domain with suitable monomeric enzymes without adversely affecting polymerization ability, and to assemble these chimeric flagellins into tubular nanostructures. To test the feasibility of this approach, xylanase A (XynA) from B. subtilis was chosen as a model enzyme for insertion into the central part of flagellin. With the help of genetic engineering, a fusion construct was created in which the D3 domain was replaced by XynA. The flagellin-XynA chimera exhibited catalytic activity as well as polymerization ability. These results demonstrate that polymerization ability can be introduced into various proteins, and building blocks for rationally designed assembly of filamentous nanostructures can be created.     

Genetic differentiation of the Western Capercaillie highlights the importance of South-eastern Europe for understanding the species phylogeography

The Western Capercaillie (Tetrao urogallus L.) is a grouse species of open boreal or high altitude forests of Eurasia. It is endangered throughout most mountain range habitat areas in Europe. Two major genetically identifiable lineages of Western Capercaillie have been described to date: the southern lineage at the species' southernmost range of distribution in Europe, and the boreal lineage. We address the question of genetic differentiation of capercaillie populations from the Rhodope and Rila Mountains in Bulgaria, across the Dinaric Mountains to the Slovenian Alps. The two lineages' contact zone and resulting conservation strategies in this so-far understudied area of distribution have not been previously determined. The results of analysis of mitochondrial DNA control region sequences of 319 samples from the studied populations show that Alpine populations were composed exclusively of boreal lineage; Dinaric populations of both, but predominantly (96%) of boreal lineage; and Rhodope-Rila populations predominantly (>90%) of southern lineage individuals. The Bulgarian mountains were identified as the core area of the southern lineage, and the Dinaric Mountains as the western contact zone between both lineages in the Balkans. Bulgarian populations appeared genetically distinct from Alpine and Dinaric populations and exhibited characteristics of a long-term stationary population, suggesting that they should be considered as a glacial relict and probably a distinct subspecies. Although all of the studied populations suffered a decline in the past, the significantly lower level of genetic diversity when compared with the neighbouring Alpine and Bulgarian populations suggests that the isolated Dinaric capercaillie is particularly vulnerable to continuing population decline. The results are discussed in the context of conservation of the species in the Balkans, its principal threats and legal protection status. Potential conservation strategies should consider the existence of the two lineages and their vulnerable Dinaric contact zone and support the specificities of the populations.     

A full lifecycle bioenergetic model for bluefin tuna

We formulated a full lifecycle bioenergetic model for bluefin tuna relying on the principles of Dynamic Energy Budget theory. Traditional bioenergetic models in fish research deduce energy input and utilization from observed growth and reproduction. In contrast, our model predicts growth and reproduction from food availability and temperature in the environment. We calibrated the model to emulate physiological characteristics of Pacific bluefin tuna (Thunnus orientalis, hereafter PBT), a species which has received considerable scientific attention due to its high economic value. Computer simulations suggest that (i) the main cause of different growth rates between cultivated and wild PBT is the difference in average body temperature of approximately 6.5°C, (ii) a well-fed PBT individual can spawn an average number of 9 batches per spawning season, (iii) food abundance experienced by wild PBT is rather constant and sufficiently high to provide energy for yearly reproductive cycle, (iv) energy in reserve is exceptionally small, causing the weight-length relationship of cultivated and wild PBT to be practically indistinguishable and suggesting that these fish are poorly equipped to deal with starvation, (v) accelerated growth rate of PBT larvae is connected to morphological changes prior to metamorphosis, while (vi) deceleration of growth rate in the early juvenile stage is related to efficiency of internal heat production. Based on these results, we discuss a number of physiological and ecological traits of PBT, including the reasons for high Feed Conversion Ratio recorded in bluefin tuna aquaculture.     

Cryptorchidism: an indicator of testicular dysgenesis?

Cryptorchidism is a common ailment of new-born boys, affecting 1–9% of full term boys at birth. Cryptorchidism has been associated with an increased risk of testicular cancer and reduced fertility. Aetiology of cryptorchidism remains obscure in most cases. Familial occurrence suggests a heritable susceptibility to cryptorchidism; however, seasonal variation in the incidence of cryptorchidism suggests that environmental factors also contribute. Testicular descent is characterised by androgen-dependent regression of cranial suspensory ligament and androgen + insulin-like hormone 3 (Insl3)-dependent gubernacular outgrowth. Even though hormonal defects are rarely detected in patients, both hypo-and hypergonadotropic hormonal patterns have been associated with cryptorchidism. Moreover, cryptorchid boys have significantly reduced serum androgen bioactivity at 3 months of age when normal boys have a strong surge of reproductive hormones. Defects in Insl3 action cause cryptorchidism in male mice, and over-expression in female mice causes ovarian descent. Defects in leucine-rich repeat-containing G-protein-coupled receptor 8/G-protein-coupled receptor affecting testis descent (LGR8/GREAT), the receptor for Insl3, manifest the same phenotype as Insl3 knockout mutants. Even though mutations found in Insl3 and LGR8/GREAT genes are not a common cause of cryptorchidism in patients, it remains to be resolved whether low Insl3 levels during development are associated with cryptorchidism. Cryptorchidism may reflect foetal testicular dysgenesis that may later manifest as subfertility or testicular cancer.This work was supported by the Turku University Central Hospital, the Academy of Finland and the European Commission (contracts BMH4-CT96-0314, QLK4-CT1999-01422, QLK4-CT2001-00269 and QLK4-CT2002-00603).     

Functional coupling of adenine nucleotide translocase and mitochondrial creatine kinase is enhanced after exercise training in lung transplant skeletal muscle

Mechanisms responsible for limitation of exercise capacity in lung transplant recipients (LR) and benefits gained by exercise training were studied. Mitochondrial respiration parameters, energy transfer, and cell structure were assessed in vastus lateralis biopsies using the permeabilized fiber technique with histochemical and morphometric measurements. Twelve male controls (C) and 12 LR performed exercise training over 12 wk. Before exercise training, there were strong correlations between exercise capacity (maximal O(2) consumption and endurance time at 70% maximal power output) and cellular events, as assessed by percentage of type I fibers and apparent K(m) for exogenous ADP. Anticalcineurins were not involved in LR exercise limitation, since there were no differences in maximal mitochondrial rate of respiration before exercise training and no abnormalities in respiratory chain complexes compared with C. Training resulted in a significant increase in physiological parameters both at the cellular (apparent K(m) for exogenous ADP and stimulating effect of creatine) and integrated (maximal O(2) consumption, power output at ventilatory threshold, maximal power output, and endurance time at 70% maximal power output) levels in LR and C. After the training period, improvements in maximal O(2) consumption and in maximal mitochondrial rate of respiration were noted, as well as changes in endurance time and percentage of type I fibers. Because there were no changes in diameters and fiber types, baseline alteration of apparent K(m) for exogenous ADP and its improvement after training might be related to changes within the intracellular energetic units. After the training period, intracellular energetic units exhibited a higher control of mitochondrial respiration by creatine linked to a more efficient functional coupling adenine nucleotide translocase-mitochondrial creatine kinase, resulting in better exercise performances in C and LR.     

A modular design for the clathrin- and actin-mediated endocytosis machinery

Endocytosis depends on an extensive network of interacting proteins that execute a series of distinct subprocesses. Previously, we used live-cell imaging of six budding-yeast proteins to define a pathway for association of receptors, adaptors, and actin during endocytic internalization. Here, we analyzed the effects of 61 deletion mutants on the dynamics of this pathway, revealing functions for 15 proteins, and we analyzed the dynamics of 8 of these proteins. Our studies provide evidence for four protein modules that cooperate to drive coat formation, membrane invagination, actin-meshwork assembly, and vesicle scission during clathrin/actin-mediated endocytosis. We found that clathrin facilitates the initiation of endocytic-site assembly but is not needed for membrane invagination or vesicle formation. Finally, we present evidence that the actin-meshwork assembly that drives membrane invagination is nucleated proximally to the plasma membrane, opposite to the orientation observed for previously studied actin-assembly-driven motility processes.     

Comparison of two management strategies for Helicobacter pylori treatment: clinical study and cost-effectiveness analysis

BACKGROUND: First-line proton pump inhibitor-based triple and quadruple therapies for Helicobacter pylori eradication present similar levels of efficacy. Cross-over treatment (quadruple following triple failure, and triple following quadruple failure) seems the most sensible approach to treatment failures, but the two strategies -'quadruple first' versus 'triple first'- have not been previously compared. The aims of our study were to assess the usefulness and the cost-effectiveness of the two treatment strategies. MATERIAL AND METHODS: Forty-nine out of 344 patients included in a previous study comparing triple therapy - 7 days of omeprazole, amoxicillin and clarithromycin twice a day - with quadruple therapy - 7 days of omeprazole twice a day, plus tetracycline, metronidazole and bismuth subcitrate three times a day - failed initial treatment and were assigned to cross-over therapy. Cure was determined by urea breath test. A decision analysis was performed to compare the two eradication strategies. RESULTS: Intention to treat cure rates were 46% (10/22 patients; 95% CI 24-68%) for second-line triple therapy and 63% (17/27 patients; 95% CI 42-81%) for second-line quadruple therapy. Per protocol cure rates were 71% and 85%, respectively. Intention to treat cure rates were 87% (95% CI 81-92%) for the 'triple first' versus 86% (95% CI 80-91%) for the 'quadruple first' strategy (p = .87). The 'quadruple first' strategy was more cost-effective. The incremental cost of 'triple first' strategy per person was 19 in the low-cost area and 65 US dollars in the high-cost area. CONCLUSIONS: The effectiveness of 'triple first' and 'quadruple first' strategies is similar, although the latter seems slightly more cost-effective.     

In silico fragment-based discovery of indolin-2-one analogues as potent DNA gyrase inhibitors

We describe here the fragment-based design of potent DNA gyrase inhibitors. Using the tools of virtual screening and NMR spectroscopy we identified the binding of two low-molecular weight fragments (2-aminobenzimidazole and indolin-2-one) to the 24kDa N-terminal fragment of DNA gyrase B. Further in silico optimization of indolin-2-one led to the discovery of potent DNA gyrase inhibitors.     

An intraspecific comparative analysis of character divergence between sympatric species

Although sympatric character divergence between closely related species has been described in a wide variety of taxa, the evolutionary processes responsible for generating these patterns are difficult to identify. One hypothesis that can explain sympatric differences is ecological character displacement: the sympatric origin of morphologically divergent phenotypes in response to selection caused by interspecific competition. Alternatively, populations may adapt to different conditions in allopatry, with sympatric distributions evolving through selective colonization and proliferation of ecologically compatible phenotypes. In this study, I characterize geographic variation within two sibling species of rocky-shore gastropods that have partially overlapping distributions in central California. In sympatry, both Nucella emarginata and N. ostrina show significant differences in shell shape and shell ornamentation that together suggest that where the two species co-exist, divergent phenotypes arose as an evolutionary consequence of competition. To examine the evolutionary origins of divergent characters in sympatry, I used a comparative method based on spatial autocorrelation to remove the portion of the phenotypic variance among populations that is explained by genetic distance (using mitochondrial DNA sequences and allozyme frequency data). Because the remaining portion of the phenotypic variance represents the independent divergence of individual populations, a significant sympatric difference in the corrected dataset provides evidence of true character displacement: significant sympatric character evolution that is independent of population history. After removal of genetic distance effects in Nucella, shell shape differences remain statistically significant in N. emarginata, providing evidence of significant sympatric character divergence. However, for external shell ornamentation in both species and shell shape in N. ostrina, the significance of sympatric differences is lost in the corrected dataset, indicating that colonization events and gene flow have played important roles in the evolutionary history of character divergence in sympatry. Although the absence of a widely dispersing planktonic larva in the life cycle of Nucella will promote local adaptation, the results here indicate that once advantageous traits arise, demographic processes, such as recurrent gene flow between established populations and extinction and recolonization, are important factors contributing to the geographic pattern of sympatric character divergence.     

Dynamic imaging of protease activity with fluorescently quenched activity-based probes

Protease activity is tightly regulated in both normal and disease conditions. However, it is often difficult to monitor the dynamic nature of this regulation in the context of a live cell or whole organism. To address this limitation, we developed a series of quenched activity-based probes (qABPs) that become fluorescent upon activity-dependent covalent modification of a protease target. These reagents freely penetrate cells and allow direct imaging of protease activity in living cells. Targeted proteases are directly identified and monitored biochemically by virtue of the resulting covalent tag, thereby allowing unambiguous assignment of protease activities observed in imaging studies. We report here the design and synthesis of a selective, cell-permeable qABP for the study of papain-family cysteine proteases. This probe is used to monitor real-time protease activity in live human cells with fluorescence microscopy techniques as well as standard biochemical methods.