Heterologous protein secretion by Candida utilis

The yeast Candida utilis (also referred to as Torula) is used as a whole-cell food additive and as a recombinant host for production of intracellular molecules. Here, we report recombinant C. utilis strains secreting significant amounts of Candida antarctica lipase B (CalB). Native and heterologous secretion signals led to secretion of CalB into the growth medium; CalB was enzymatically active and it carried a short N-glycosyl chain lacking extensive mannosylation. Furthermore, CalB fusions to the C. utilis Gas1 cell wall protein led to effective surface display of enzymatically active CalB and of β-galactosidase. Secretory production in C. utilis was achieved using a novel set of expression vectors containing sat1 conferring nourseothricin resistance, which could be transformed into C. utilis, Pichia jadinii, Candida albicans, and Saccharomyces cerevisiae; C. utilis promoters including the constitutive TDH3 and the highly xylose-inducible GXS1 promoters allowed efficient gene expression. These results establish C. utilis as a promising host for the secretory production of proteins.     

Serum level of brain-derived neurotrophic factor in fibromyalgia syndrome correlates with depression but not anxiety

Brain-derived neurotrophic factor (BDNF) has been known to play a role in fibromyalgia syndrome (FMS) patients. Depression and anxiety are quite common additional symptoms in FMS. However the role of BDNF in these symptoms still needs to be elucidated. Although BDNF has been shown to be relevant in major depression, however studies could not show such differences between FMS patients with and without major depression. As mood-related symptom occurs frequently and differs in its intensity in FMS patients, BDNF level should be measured in subgroup regarding depression and anxiety scale. Therefore the aim of this study was to evaluate the correlation of BDNF in serum of FMS with intensity of depression and anxiety. Additionally, interleukin (IL)-6 was measured.     

GENETIC DIFFERENTIATION AND SELECTION AGAINST MIGRANTS IN EVOLUTIONARILY REPLICATED EXTREME ENVIRONMENTS

We investigated mechanisms of reproductive isolation in livebearing fishes (genus Poecilia) inhabiting sulfidic and nonsulfidic habitats in three replicate river drainages. Although sulfide spring fish convergently evolved divergent phenotypes, it was unclear if mechanisms of reproductive isolation also evolved convergently. Using microsatellites, we found strongly reduced gene flow between adjacent populations from different habitat types, suggesting that local adaptation to sulfidic habitats repeatedly caused the emergence of reproductive isolation. Reciprocal translocation experiments indicate strong selection against immigrants into sulfidic waters, but also variation among drainages in the strength of selection against immigrants into nonsulfidic waters. Mate choice experiments revealed the evolution of assortative mating preferences in females from nonsulfidic but not from sulfidic habitats. The inferred strength of sexual selection against immigrants (RI_s) was negatively correlated with the strength of natural selection (RI_m), a pattern that could be attributed to reinforcement, whereby natural selection strengthens behavioral isolation due to reduced hybrid fitness. Overall, reproductive isolation and genetic differentiation appear to be replicated and direct consequences of local adaptation to sulfide spring environments, but the relative contributions of different mechanisms of reproductive isolation vary across these evolutionarily independent replicates, highlighting both convergent and nonconvergent evolutionary trajectories of populations in each drainage.     

Characterization of the mouse ClC-K1/Barttin chloride channel

Several Cl⁻ channels have been described in the native renal tubule, but their correspondence with ClC-K1 and ClC-K2 channels (orthologs of human ClC-Ka and ClC-Kb), which play a major role in transcellular Cl⁻ absorption in the kidney, has yet to be established. This is partly because investigation of heterologous expression has involved rat or human ClC-K models, whereas characterization of the native renal tubule has been done in mice. Here, we investigate the electrophysiological properties of mouse ClC-K1 channels heterologously expressed in Xenopus laevis oocytes and in HEK293 cells with or without their accessory Barttin subunit. Current amplitudes and plasma membrane insertion of mouse ClC-K1 were enhanced by Barttin. External basic pH or elevated calcium stimulated currents followed the anion permeability sequence Cl⁻ > Br⁻ > NO₃⁻ > I⁻. Single-channel recordings revealed a unit conductance of ~ 40 pS. Channel activity in cell-attached patches increased with membrane depolarization (voltage for half-maximal activation: ~ − 65 mV). Insertion of the V166E mutation, which introduces a glutamate in mouse ClC-K1, which is crucial for channel gating, reduced the unit conductance to ~ 20 pS. This mutation shifted the depolarizing voltage for half-maximal channel activation to ~ + 25 mV. The unit conductance and voltage dependence of wild-type and V166E ClC-K1 were not affected by Barttin. Owing to their strikingly similar properties, we propose that the ClC-K1/Barttin complex is the molecular substrate of a chloride channel previously detected in the mouse thick ascending limb (Paulais et al., J Membr. Biol, 1990, 113:253–260).     

Identification of a new membrane-permeable inhibitor against inositol-1,4,5-trisphosphate-3-kinase A

Ectopic expression of the neuron-specific inositol-1,4,5-trisphosphate-3-kinase A (ITPKA) in lung cancer cells increases their metastatic potential because the protein exhibits two actin regulating activities; it bundles actin filaments and regulates inositol-1,4,5-trisphosphate (InsP₃)-mediated calcium signals by phosphorylating InsP₃. Thus, in order to inhibit the metastasis-promoting activity of ITPKA, both its actin bundling and its InsP₃kinase activity has to be blocked. In this study, we performed a high throughput screen in order to identify specific and membrane-permeable substances against the InsP₃kinase activity. Among 341,44 small molecules, 237 compounds (0.7%) were identified as potential InsP₃kinase inhibitors. After determination of IC₅₀-values, the three compounds with highest specificity and highest hydrophobicity (EPPC-3, BAMB-4, MEPTT-3) were further characterized. Only BAMB-4 was nearly completely taken up by H1299 cells and remained stable after cellular uptake, thus exhibiting a robust stability and a high membrane permeability. Determination of the inhibitor type revealed that BAMB-4 belongs to the group of mixed type inhibitors. Taken together, for the first time we identified a highly membrane-permeable inhibitor against the InsP₃kinase activity of ITPKA providing the possibility to partly inhibit the metastasis-promoting effect of ITPKA in lung tumor cells.     

Visualization of cyclooxygenase-2 using a 2,3-diarylsubstituted indole-based inhibitor and confocal laser induced cryofluorescence microscopy at 20 K in melanoma cells in vitro

This study aimed at visualization of cyclooxygenase-2 (COX-2) protein expression in melanoma cells by confocal laser induced cryofluorescence microscopy using 4-(3-(4-methoxyphenyl)-1H-indol-2-yl)benzene-sulfonamide (C1) representative for a novel class of autofluorescent 2,3-diarylsubstituted indole-based selective COX-2 inhibitors.COX-2 expression was measured in human melanoma cell lines A2058 and MelJuso by immunocytochemistry and immunoblotting. Cellular uptake experiments using varying C1 concentrations down to 0.1 nM (with/without molar excess of celecoxib as control) were performed at 37 °C. Cryofluorescence microscopy was conducted at 20 K.COX-2 protein expression was successfully visualized by C1 in A2058 cells. COX-2-negative MelJuso cells showed no specific accumulation of C1. Control experiments using celecoxib and, additionally, implemented fluorescence spectroscopy confirmed specificity of both cellular uptake and intracellular association of C1.Cryofluorescence microscopy in combination with spectroscopy allowed for visualization of COX-2 protein expression in melanoma cells in vitro using a selective COX-2 inhibitor at very low concentrations.     

‘Real time’ genetic manipulation: a new tool for ecological field studies

Field experiments with transgenic plants often reveal the functional significance of genetic traits that are important for the performance of the plants in their natural environments. Until now, only constitutive overexpression, ectopic expression and gene silencing methods have been used to analyze gene‐related phenotypes in natural habitats. These methods do not allow sufficient control over gene expression for the study of ecological interactions in real time, of genetic traits that play essential roles in development, or of dose‐dependent effects. We applied the sensitive dexamethasone (DEX)‐inducible pOp6/LhGR expression system to the ecological model plant Nicotiana attenuata and established a lanolin‐based DEX application method to facilitate ectopic gene expression and RNA interference‐mediated gene silencing in the field and under challenging conditions (e.g. high temperature, wind and UV radiation). Fully established field‐grown plants were used to silence phytoene desaturase and thereby cause photobleaching only in specific plant sectors, and to activate expression of the cytokinin (CK) biosynthesis gene isopentenyl transferase (ipt). We used ipt expression to analyze the role of CKs in both the glasshouse and the field to understand resistance to the native herbivore Tupiocoris notatus, which attacks plants at small spatial scales. By spatially restricting ipt expression and elevating CK levels in single leaves, damage by T. notatus increased, demonstrating the role of CKs in this plant–herbivore interaction at a small scale. As the arena of most ecological interactions is highly constrained in time and space, these tools will advance the genetic analysis of dynamic traits that matter for plant performance in nature.     

Synthesis and evaluation of carbamoylmethylene linked prodrugs of BMS-582949, a clinical p38α inhibitor

A series of carbamoylmethylene linked prodrugs of 1 (BMS-582949), a clinical p38α inhibitor, were synthesized and evaluated. Though the phosphoryloxymethylene carbamates (3, 4, and 5) and α-aminoacyloxymethylene carbamates (22, 23, and 26) were found unstable at neutral pH values, fumaric acid derived acyloxymethylene carbamates (2, 28, and 31) were highly stable under both acidic and neutral conditions. Prodrugs 2 and 31 were also highly soluble at both acidic and neutral pH values. At a solution dose of 14.2 mpk (equivalent to 10 mpk of 1), 2 gave essentially the same exposure of 1 compared to dosing 10 mpk of 1 itself. At a suspension dose of 142 mpk (equivalent to 100 mpk of 1), 2 demonstrated that it could overcome the solubility issue associated with 1 and provide a much higher exposure of 1. To our knowledge, the unique type of prodrugs like 2, 28, and 31 was not reported in the past and could represent a novel prodrug approach for secondary amides, a class of molecules frequently identified as drug candidates.