Purification of the Chlorella HUP1 hexose—proton symporter to homogeneity and its reconstitution in vitro

A prokaryotic biotin acceptor domain was fused to the carboxy terminal end of the Chlorella hexose—proton sym- porter. The plant symporter is biotinylated in vivo when expressed in Schizosaccharomyces pombe. The extended biotinylated transport protein is fully active, catalyzes accumulation of d -glucose analogs and restores growth of a glucose-uptake-deficient yeast strain. Crude membranes were solubilized with octyl-β-d -glucoside in the presence of Escherichia colil -α-phosphatidylethanolamine. Biotinylated symporter was purified to homogeneity by biotinavidin affinity chromatography. The symporter protein was reconstituted together with cytochrome-c oxidase prepared from beef heart mitochondria into proteo-liposomes. Cytochrome-c oxidase is a redox-driven H⁺-pump generating a proton motive force (inside negative and alkaline) while transferring electrons from cytochrome-c to oxygen; this energy is used by the symporter to accumulate d -glucose at least 30-fold. In the absence of the driving force the transport protein facilitates diffusion of d -glucose until the concentration equilibrium is reached. It was shown that maximal transport activity depends highly on the amount of co-reconstituted cytochrome-c oxidase and that the symporter possesses 10% of its in vivo turnover number under optimized in vitro transport conditions.     

Shifts in bacterial community composition associated with increased carbon cycling in a mosaic of phytoplankton blooms

Marine microbes have a pivotal role in the marine biogeochemical cycle of carbon, because they regulate the turnover of dissolved organic matter (DOM), one of the largest carbon reservoirs on Earth. Microbial communities and DOM are both highly diverse components of the ocean system, yet the role of microbial diversity for carbon processing remains thus far poorly understood. We report here results from an exploration of a mosaic of phytoplankton blooms induced by large-scale natural iron fertilization in the Southern Ocean. We show that in this unique ecosystem where concentrations of DOM are lowest in the global ocean, a patchwork of blooms is associated with diverse and distinct bacterial communities. By using on-board continuous cultures, we identify preferences in the degradation of DOM of different reactivity for taxa associated with contrasting blooms. We used the spatial and temporal variability provided by this natural laboratory to demonstrate that the magnitude of bacterial production is linked to the extent of compositional changes. Our results suggest that partitioning of the DOM resource could be a mechanism that structures bacterial communities with a positive feedback on carbon cycling. Our study, focused on bacterial carbon processing, highlights the potential role of diversity as a driving force for the cycling of biogeochemical elements.     

A Combined Approach for the Assessment of Cell Viability and Cell Functionality of Human Fibrochondrocytes for Use in Tissue Engineering

Temporo-mandibular joint disc disorders are highly prevalent in adult populations. Autologous chondrocyte implantation is a well-established method for the treatment of several chondral defects. However, very few studies have been carried out using human fibrous chondrocytes from the temporo-mandibular joint (TMJ). One of the main drawbacks associated to chondrocyte cell culture is the possibility that chondrocyte cells kept in culture tend to de-differentiate and to lose cell viability under in in-vitro conditions. In this work, we have isolated human temporo-mandibular joint fibrochondrocytes (TMJF) from human disc and we have used a highly-sensitive technique to determine cell viability, cell proliferation and gene expression of nine consecutive cell passages to determine the most appropriate cell passage for use in tissue engineering and future clinical use. Our results revealed that the most potentially viable and functional cell passages were P5–P6, in which an adequate equilibrium between cell viability and the capability to synthesize all major extracellular matrix components exists. The combined action of pro-apoptotic (TRAF5, PHLDA1) and anti-apoptotic genes (SON, HTT, FAIM2) may explain the differential cell viability levels that we found in this study. These results suggest that TMJF should be used at P5–P6 for cell therapy protocols.     

Metabolites in Blood for Prediction of Bacteremic Sepsis in the Emergency Room

A metabolomics approach for prediction of bacteremic sepsis in patients in the emergency room (ER) was investigated. In a prospective study, whole blood samples from 65 patients with bacteremic sepsis and 49 ER controls were compared. The blood samples were analyzed using gas chromatography coupled to time-of-flight mass spectrometry. Multivariate and logistic regression modeling using metabolites identified by chromatography or using conventional laboratory parameters and clinical scores of infection were employed. A predictive model of bacteremic sepsis with 107 metabolites was developed and validated. The number of metabolites was reduced stepwise until identifying a set of 6 predictive metabolites. A 6-metabolite predictive logistic regression model showed a sensitivity of 0.91(95% CI 0.69–0.99) and a specificity 0.84 (95% CI 0.58–0.94) with an AUC of 0.93 (95% CI 0.89–1.01). Myristic acid was the single most predictive metabolite, with a sensitivity of 1.00 (95% CI 0.85–1.00) and specificity of 0.95 (95% CI 0.74–0.99), and performed better than various combinations of conventional laboratory and clinical parameters. We found that a metabolomics approach for analysis of acute blood samples was useful for identification of patients with bacteremic sepsis. Metabolomics should be further evaluated as a new tool for infection diagnostics.     

Formin-like 1 (FMNL1) Is Regulated by N-terminal Myristoylation and Induces Polarized Membrane Blebbing

The formin protein formin-like 1 (FMNL1) is highly restrictedly expressed in hematopoietic lineage-derived cells and has been previously identified as a tumor-associated antigen. However, function and regulation of FMNL1 are not well defined. We have identified a novel splice variant (FMNL1γ) containing an intron retention at the C terminus affecting the diaphanous autoinhibitory domain (DAD). FMNL1γ is specifically located at the cell membrane and cortex in diverse cell lines. Similar localization of FMNL1 was observed for a mutant lacking the DAD domain (FMNL1ΔDAD), indicating that deregulation of autoinhibition is effective in FMNL1γ. Expression of both FMNL1γ and FMNL1ΔDAD induces polarized nonapoptotic blebbing that is dependent on N-terminal myristoylation of FMNL1 but independent of Src and ROCK activity. Thus, our results describe N-myristoylation as a regulative mechanism of FMNL1 responsible for membrane trafficking potentially involved in a diversity of polarized processes of hematopoietic lineage-derived cells.     

Comparison of the structures of the native form of rat liver 5S rRNA and yeast tRNAphe: small-angle and wide-angle X-ray scattering study

The small-angle and wide-angle X-ray scattering of tRNA^phe (yeast) and ribosomal 5S RNA (rat liver) in solution have been analysed and compared. tRNA^phe in solution is folded into a compact L-shaped structure similar to its structure in crystals. The geometry of the secondary structure of the double helical regions is also equivalent to the A-form in the crystalline state. Despite differences between the molar mosses of 5S rRNA (40 000 g mol^?1) and tRNA^phe (25 000 g mol^?1), and the fact that the 5S rRNA molecule is more anisometric than the tRNA^phe molecule, there are many structural similarities. The geometrical parameters of the secondary structure of double helical regions in both RNA molecules are almost identical; the mean rise per base pair is about 0.253–0.28 nm and the mean turn angle is about 32.5–33.5. Identical cross-sectional radii of gyration, R_sq,1 ≈ 1.16 nm and R_sq,2 = 0.92 nm, identical molar mass per unit length, , and a mean thickness of the molecules D ≈ 1.65 nm suggest a similar, nearly coplanar organization of isolated, double helical arms. Furthermore, there are compact regions in the central parts of both molecules, which are the sites of tertiary interactions in the tRNA^phe molecule and are a potential site of tertiary interactions in the SS rRNA molecule for stabilization of the complicated L-shape of the two molecules. Both molecules have a pseudo-twofold axis,w hich may play a role in recognition for binding of specific proteins.