Engineering of Thermomyces lanuginosus lipase Lip: creation of novel biocatalyst for efficient biosynthesis of chiral intermediate of Pregabalin

Efficient and highly enantioselective hydrolysis of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE) is the most crucial step in chemoenzymatic synthesis of Pregabalin. By using site-saturation mutagenesis and high-throughput screening techniques, lipase Lip from Thermomyces lanuginosus DSM 10635 was engineered to improve its activity towards CNDE. The triple mutant, S88T/A99N/V116D exhibited a 60-fold improvement in specific activity for CNDE (2.35 U/mg) over the wild-type Lip (0.039 U/mg). Modeling and docking studies demonstrated that the mutant could more effectively stabilize oxygen anions in transition states and the lid of Lip in the open conformation. Additionally, the kinetic resolution of CNDE catalyzed by Escherichia coli cell overexpressing S88T/A99N/V116D mutant afforded (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid in 42.4 % conversion and 98 % ee within 20 h with a substrate loading of 1 M (255 g/l). These results demonstrated that a novel and promising biocatalyst was created for efficient chemoenzymatic manufacturing of Pregabalin.     

Biomedical implications from a morphoelastic continuum model for the simulation of contracture formation in skin grafts that cover excised burns

A continuum hypothesis-based model is developed for the simulation of the (long term) contraction of skin grafts that cover excised burns in order to obtain suggestions regarding the ideal length of splinting therapy and when to start with this therapy such that the therapy is effective optimally. Tissue is modeled as an isotropic, heterogeneous, morphoelastic solid. With respect to the constituents of the tissue, we selected the following constituents as primary model components: fibroblasts, myofibroblasts, collagen molecules, and a generic signaling molecule. Good agreement is demonstrated with respect to the evolution over time of the surface area of unmeshed skin grafts that cover excised burns between outcomes of computer simulations obtained in this study and scar assessment data gathered previously in a clinical study. Based on the simulation results, we suggest that the optimal point in time to start with splinting therapy is directly after placement of the skin graft on its recipient bed. Furthermore, we suggest that it is desirable to continue with splinting therapy until the concentration of the signaling molecules in the grafted area has become negligible such that the formation of contractures can be prevented. We conclude this study with a presentation of some alternative ideas on how to diminish the degree of contracture formation that are not based on a mechanical intervention, and a discussion about how the presented model can be adjusted.     

Colonization and extinction of land planarians (Platyhelminthes,Tricladida) in a Brazilian Atlantic Forest regrowth remnant

Long-term assessments of species assemblages are valuable tools for detecting species ecological preferences and their dispersal tracks, as well as for assessing the possible effects of alien species on native communities. Here we report a 50-year-long study on population dynamics of the four species of land flatworms (Platyhelminthes, Tricladida, Terricola) that have colonized or become extinct in a 70-year-old Atlantic Forest regrowth remnant through the period 1955–2006. On the one hand, the two initially most abundant species, which are native to the study site, Notogynaphallia ernesti and Geoplana multicolor have declined over decades and at present do not exist in the forest remnant. The extinction of these species is most likely related with their preference for open vegetation areas, which presently do not exist in the forest remnant. On the other hand, the neotropical Geoplaninae 1 and the exotic Endeavouria septemlineata were detected in the forest only very recently. The long-term study allowed us to conclude that Geoplaninae 1 was introduced into the study area, although it is only known from the study site. Endeavouria septemlineata, an active predator of the exotic giant African snail, is originally known from Hawaii. This land flatworm species was observed repeatedly in Brazilian anthropogenic areas, and this is the first report of the species in relatively well preserved native forest, which may be evidence of an ongoing adaptive process. Monitoring of its geographic spread and its ecological role would be a good practice for preventing potential damaging effects, since it also feeds on native mollusk fauna, as we observed in lab conditions. Júlio Pedroni: Granted by CNPQ–Brazil.     

The Effect of TCM-Induced HAMP on Key Enzymes in the Hydrolysis of AD Model Cells

Neurochemical Research - Alzheimer’s disease (AD) process is characterized classically by two hallmark pathologies: β-amyloid (Aβ) plaque deposition and neurofibrillary tangles of...     

Involvement of Protein Tyrosine Phosphatases BcPtpA and BcPtpB in Regulation of Vegetative Development,Virulence and Multi-Stress Tolerance in Botrytis cinerea

Tyrosine phosphorylation and dephosphorylation have emerged as fundamentally important mechanisms of signal transduction and regulation in eukaryotic cells, governing many processes, but little has been known about their functions in filamentous fungi. In this study, we deleted two putative protein tyrosine phosphatase (PTP) genes (BcPTPA and BcPTPB) in Botrytis cinerea, encoding the orthologs of Saccharomyces cerevisiae Ptp2 and Ptp3, respectively. Although BcPtpA and BcPtpB have opposite functions in conidiation, they are essential for sclerotial formation in B. cinerea. BcPTPA and BcPTPB deletion mutants ΔBcPtpA-10 and ΔBcPtpB-4 showed significantly increased sensitivity to osmotic and oxidative stresses, and to cell wall damaging agents. Inoculation tests showed that both mutants exhibited dramatically decreased virulence on tomato leaves, apples and grapes. In S. cerevisiae, it has been shown that Ptp2 and Ptp3 negatively regulate the high-osmolarity glycerol (HOG) pathway and the cell wall integrity (CWI) pathway. Although both BcPtpA and BcPtpB were able to inactive Hog1 and Mpk1 in S. cerevisiae, in contrast to S. cerevisiae, they positively regulate phosphorylation of BcSak1 (the homologue of Hog1) and BcBmp3 (the homologue of Mpk1) in B. cinerea under stress conditions. These results demonstrated that functions of PTPs in B. cinerea are different from those in S. cerevisiae, and BcPtpA and BcPtpB play important roles in regulation of vegetative development, virulence and in adaptation to oxidative, osmotic and cell-wall damage stresses in B. cinerea.     

Oligomeric State and Thermal Stability of Apo- and Holo- Human Ornithine δ-Aminotransferase

Human ornithine δ-aminotransferase (hOAT) (EC 2.6.1.13) is a mitochondrial pyridoxal 5′-phosphate (PLP)-dependent aminotransferase whose deficit is associated with gyrate atrophy, a rare autosomal recessive disorder causing progressive blindness and chorioretinal degeneration. Here, both the apo- and holo-form of recombinant hOAT were characterized by means of spectroscopic, kinetic, chromatographic and computational techniques. The results indicate that apo and holo-hOAT (a) show a similar tertiary structure, even if apo displays a more pronounced exposure of hydrophobic patches, (b) exhibit a tetrameric structure with a tetramer-dimer equilibrium dissociation constant about fivefold higher for the apoform with respect to the holoform, and (c) have apparent T_m values of 46 and 67?°C, respectively. Moreover, unlike holo-hOAT, apo-hOAT is prone to unfolding and aggregation under physiological conditions. We also identified Arg217 as an important hot-spot at the dimer–dimer interface of hOAT and demonstrated that the artificial dimeric variant R217A exhibits spectroscopic properties, T_m values and catalytic features similar to those of the tetrameric species. This finding indicates that the catalytic unit of hOAT is the dimer. However, under physiological conditions the apo-tetramer is slightly less prone to unfolding and aggregation than the apo-dimer. The possible implications of the data for the intracellular stability and regulation of hOAT are discussed.     

Population dynamics of two small pelagic fish in the central-south area off Chile: delayed density-dependence and biological interaction

It is widely believed that environmental variability is the main cause for fluctuations in commercially exploited small pelagic fish populations around the world. Nevertheless, density-dependent factors also can drive population dynamics. In this paper, we analyzed thirteen years of a relative abundance index of two clupeoids fish populations coexisting in the central-south area off Chile, namely the common sardine, Strangomera bentincki, and anchovy, Engraulis ringens. We applied the classical diagnostic tools of time series analysis to the observed time-series. Also, the realized per capita population growth rate was studied with the aim of detecting the feedback structure that is characterizing the population dynamics of the two species. The analysis suggests that population fluctuations of the two species have an important density-dependent component, displaying first-order (direct density-dependent) and second-order (delayed density-dependent) simultaneously. The density-dependent component explained 70.5 and 55.6 % of the realized per capita population growth rate of common sardine and anchovy, respectively. The deterministic skeleton model showed an asymptotic convergence to equilibrium density. In presence of a stochastic environment, fluctuations were reproduced for the species showing a component of fluctuation with a period of 4 year. The intrinsic dynamics of each species is typical of interacting species resulting from trophic interactions. It is postulated that the second-order dynamics of S. bentincki and E. ringens in central-south Chile, may be the result from interactions with a specialist predator (the fishing fleet), interacting with exogenous environmental factors.     

Do terrestrial gastropods use olfactory cues to locate and select food actively?

Having been investigated for over 40 years, some aspects of the biology of terrestrial gastropod’s olfactory system have been challenging and highly contentious, while others still remain unresolved. For example, a number of terrestrial gastropod species can track the odor of food, while others have no strong preferences toward food odor; rather they find it by random encounter. Here, while assessing the most recent findings and comparing them with earlier studies, the aspects of the food selection based on olfactory cues are examined critically to highlight the speculations and controversies that have arisen. We analyzed and compared the potential role of airborne odors in the feeding behavior of several terrestrial gastropod species. The available results indicate that in the foraging of most of the terrestrial gastropod species odor cues contribute substantially to food finding and selection. The results also suggest, however, that what they will actually consume largely depends on where they live and the species of gastropod that they are. Due to the voluminous literature relevant to this object, this review is not intended to be exhaustive. Instead, I selected what I consider to be the most important or critical in studies regarding the role of the olfaction in feeding of terrestrial gastropods.