CD45<Superscript>−</Superscript>CD14<Superscript>+</Superscript>CD34<Superscript>+</Superscript> murine bone marrow low-adherent mesenchymal primitive cells preserve multilineage differentiation potential in long-term <Emphasis Type="Italic">in vitro</Emphasis> culture

Bone marrow-derived cells have been postulated as a source of multipotent mesenchymal stem cells (MSC). However, the whole fraction of MSC remains heterogeneous and the expansion of primitive subset of these cells is still not well established. Here, we optimized the protocol for propagating the low-adherent subfraction of MSC which results in long-term expansion of population characterized by CD45⁻CD14⁺CD34⁺ phenotype along with expression of common MSC markers. We established that the expanded MSC are capable of differentiating into endothelial cells highly expressing angiogenic markers and exhibiting functional properties of endothelium. Moreover, we found these cells to be multipotent and capable of giving rise into cells from neuronal lineages. Interestingly, the expanded MSC form characteristic cellular spheres in vitro indicating primitive features of these cells. In sum, we isolated the novel multipotent subpopulation of CD45⁻CD14⁺ CD34⁺ bone marrow-derived cells that could be maintained in long-term culture without losing this potential.     

The ATPase activity of the <Emphasis Type="Italic">G2alt</Emphasis> gene encoding an aluminium tolerance protein from <Emphasis Type="Italic">Anoxybacillus gonensis</Emphasis> G2

The G2ALT gene was cloned and sequenced from the thermophilic bacterium Anoxybacillus gonensis G2. The gene is 666 bp long and encodes a protein 221 amino acids in length. The gene was overexpressed in E. coli and purified to homogeneity and biochemically characterized. The enzyme has a molecular mass of 24.5 kDa and it could be classified as a member of the family of bacterial aluminium resistance proteins based on homology searches. When this fragment was expressed in E. coli, it endowed E. coli with Al tolerance to 500 μM. The purified G2ALT protein is active at a broad pH range (pH 4.0–10.0) and temperature range (25°C–80°C) with optima of 6.0 and the apparent optimal temperature of 73°C respectively. Under optimal conditions, G2ALT exhibited a low ATPase activity with K _m ⁻ and V _max ⁻ values of 10±0.55 μM and 26.81±0.13 mg Pi released/min/mg enzyme, respectively. The ATPase activity of G2ALT requires Mg²⁺ and Na⁺ ions, while Zn²⁺ and Al³⁺ stimulate the activity. Cd²⁺ and Ag⁺ reduced the activity and Li⁺, Cu²⁺, and Co²⁺ inhibited the activity. Known inhibitors of most ATPases, like such as β-mercaptoethanol and ouabain, also inhibited the activity of the G2ALT. These biochemical characterizations suggested that G2ALT belongs to the PP-loop ATPase superfamily and it can be responsible for aluminium tolerance in A. gonensis G2.     

Disassembly and reassembly of amyloid fibrils in water-ethanol mixtures

This work presents the structural analysis of amyloid-like β-lactoglobulin fibrils incubated in ethanol-water mixtures after their formation in water. We observe for the first time the disassembly of semiflexible heat-denatured β-lactoglobulin fibrils and reassembly into highly flexible wormlike fibrils in ethanol-water solutions. Tapping mode atomic force microscopy is performed to follow structural changes. Our results show that in addition to their growth in length, there is a continuous nucleation process of new wormlike objects with time at the expense of the original β-lactoglobulin fibrils. The persistence length of wormlike fibrils (29.43 nm in the presence of 50% ethanol), indicative of their degree of flexibility, differs by 2 orders of magnitude from that of untreated β-lactoglobulin fibrils (2368.75 nm in pure water). Interestingly, wormlike fibrils do not exhibit a multiple strands nature like the pristine fibrils, as revealed by the lower maximum height and the lack of clear height periodicity along their contour length profile. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrates that the set of polypeptides obtained by ethanol degradation differs in some fractions from that present in pristine β-lactoglobulin fibrils. ATR-FTIR (attenuated total reflectance-Fourier transform infrared) spectroscopy also supports a different composition of the secondary structure of wormlike fibrils with a decreased amount of α-helix and increased random coils and turns content. These findings can contribute to deciphering the molecular mechanisms of protein aggregation into amyloid fibrils and their disassembly as well as enabling tailor-made production of protein fibrils.     

Proteomics on brefeldin A-treated Arabidopsis roots reveals profilin 2 as a new protein involved in the cross-talk between vesicular trafficking and the actin cytoskeleton

The growing importance of vesicular trafficking and cytoskeleton dynamic reorganization during plant development requires the exploitation of novel experimental approaches. Several genetic and cell biological studies have used diverse pharmaceutical drugs that inhibit vesicular trafficking and secretion to study these phenomena. Here, proteomic and cell biology approaches were applied to study effects of brefeldin A (BFA), an inhibitor of vesicle recycling and secretion, in Arabidopsis roots. The main aim of this study was to obtain an overview of proteins affected by BFA, but especially to identify new proteins involved in the vesicular trafficking and its cross-talk to the actin cytoskeleton. The results showed that BFA altered vesicular trafficking and caused the formation of BFA-compartments which was accompanied by differential expression of several proteins in root cells. Some of the BFA-up-regulated proteins belong to the class of the vesicular trafficking proteins, such as V-ATPase and reversibly glycosylated polypeptide, while others, such as profilin 2 and elongation factor 1 alpha, are rather involved in the remodeling of the actin cytoskeleton. Upregulation of profilin 2 by BFA was verified by immunoblot and live imaging at subcellular level. The latter approach also revealed that profilin 2 accumulated in BFA-compartments which was accompanied by remodeling of the actin cytoskeleton in BFA-treated root cells. Thus, profilin 2 seems to be involved in the cross-talk between vesicular trafficking and the actin cytoskeleton, in a BFA-dependent manner.     

A tritrophic approach to the preference–performance hypothesis involving an exotic and a native plant

Exotic plants often generate physical and chemical changes in native plant communities where they become established. A major challenge is to understand how novel plants may affect trophic interactions in their new habitats, and how native herbivores and their natural enemies might respond to them. We compared the oviposition preference and offspring performance of the crucifer specialist, Pieris brassicae, on an exotic plant, Bunias orientalis, and on a related native plant, Sinapis arvensis. Additionally, we studied the response of the parasitoid, Cotesia glomerata to herbivore-induced plant volatiles (HIPV) and determined the volatile blend composition to elucidate which compound(s) might be involved in parasitoid attraction. On both host plants we also compared the parasitism rate of P. brassicae by C. glomerata. Female butterflies preferred to oviposit on the native plant and their offspring survival and performance was higher on the native plant compared to the exotic. Although, headspace analysis revealed qualitative and quantitative differences in the volatile blends of both plant species, C. glomerata did not discriminate between the HIPV blends in flight-tent bioassays. Nevertheless, parasitism rate of P. brassicae larvae was higher on the native plant under semi-field conditions. Overall, P. brassicae oviposition preference may be more influenced by bottom-up effects of the host plant on larval performance than by top-down pressure exerted by its parasitoid. The potential for dietary breadth expansion of P. brassicae to include the exotic B. orientalis and the role of top-down processes played by parasitoids in shaping herbivore host shifts are further discussed.     

Synthesis and Biological Evaluation of 4-Carbamoyl-2-β-D-Ribofuranosyl-Pyridine

Abstract

D-Allo/D-altro 2-(2,4:3, 5-di-O-benzylidenepentitol-1-y1)-4-(4,4-dimethyloxazolin-2-y1)pyridine was synthesized from 2-lithio-4-(4,4-dimethyloxazolin-2-y1)pyridine and 2, 4:3,5-di-O-benzylidenealdehydo-D-ribose. After mesylation and subsequent treatment of the adduct with CF₃COOH/H₂O and then ammonia, 4-carbamoyl-2-D-ribofuranosylpyridine was formed. The α- and β-anomers were separated by semipreparative hplc on a LICHROSORB 10 DIOL column. The β-anomer had no antiviral activity, but it had modest cytostatic activity against tumor cells.     

Ubiquitin C-Terminal Hydrolase-L1 Potentiates Cancer Chemosensitivity by Stabilizing NOXA

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Highlights? Proteasomal degradation of NOXA is enhanced in chemoresistant tumor samples ? UCH-L1 is the specific deubiquitylating enzyme of NOXA ? Lack of UCH-L1 results in inefficient DNA damage-induced apoptosis in cancer ? Knockdown of UCH-L1 homolog in C. elegans results in reduced apoptosis     

Copper Directs ATP7B to the Apical Domain of Hepatic Cells via Basolateral Endosomes

Physiologic Cu levels regulate the intracellular location of the Cu ATPase ATP7B. Here, we determined the routes of Cu‐directed trafficking of endogenous ATP7B in the polarized hepatic cell line WIF‐B and in the liver in vivo. Copper (10 µm ) caused ATP7B to exit the trans‐Golgi network (TGN) in vesicles, which trafficked via large basolateral endosomes to the apical domain within 1 h. Although perturbants of luminal acidification had little effect on the TGN localization of ATP7B in low Cu, they blocked delivery to the apical membrane in elevated Cu. If the vesicular proton‐pump inhibitor bafilomycin‐A1 (Baf) was present with Cu, ATP7B still exited the TGN, but accumulated in large endosomes located near the coverslip, in the basolateral region. Baf washout restored ATP7B trafficking to the apical domain. If ATP7B was staged apically in high Cu, Baf addition promoted the accumulation of ATP7B in subapical endosomes, indicating a blockade of apical recycling, with concomitant loss of ATP7B at the apical membrane. The retrograde pathway to the TGN, induced by Cu removal, was far less affected by Baf than the anterograde (Cu‐stimulated) case. Overall, loss of acidification‐impaired Cu‐regulated trafficking of ATP7B at two main sites: (i) sorting and exit from large basolateral endosomes and (ii) recycling via endosomes near the apical membrane.