The distinct nucleotide binding states of the transporter associated with antigen processing (TAP) are regulated by the nonhomologous C-terminal tails of TAP1 and TAP2.

The transporter associated with antigen processing (TAP) delivers peptides into the lumen of the endoplasmic reticulum for binding onto major histocompatibility complex class I molecules. TAP comprises two polypeptides, TAP1 and TAP2, each with an N-terminal transmembrane domain and a C-terminal cytosolic nucleotide binding domain (NBD). The two NBDs have distinct intrinsic nucleotide binding properties. In the resting state of TAP, the NBD1 has a much higher binding activity for ATP than the NBD2, while the binding of ADP to the two NBDs is equivalent. To attribute the different nucleotide binding behaviour of NBD1 and NBD2 to specific sequences, we generated chimeric TAP1 and TAP2 polypeptides in which either the nonhomologous C-terminal tails downstream of the Walker B motif, or the core NBDs which are enclosed by the conserved Walker A and B motifs, were reciprocally exchanged. Our biochemical and functional studies on the different TAP chimeras show that the distinct nucleotide binding behaviour of TAP1 and TAP2 is controlled by the nonhomologous C-terminal tails of the two TAP chains. In addition, our data suggest that the C-terminal tail of TAP2 is required for a functional transporter by regulating ATP binding. Further experiments indicate that ATP binding to NBD2 is important because it prevents simultaneous uptake of ATP by TAP1. We propose that the C-terminal tails of TAP1 and TAP2 play a crucial regulatory role in the coordination of nucleotide binding and ATP hydrolysis by TAP.     

Nucleoli and Stress Granules: Connecting Distant Relatives

Nucleoli and cytoplasmic stress granules (SGs) are subcellular compartments that modulate the response to endogenous and environmental signals to control cell survival. In our opinion, nucleoli and SGs are functionally linked; they are distant relatives that combine forces when cellular homeostasis is threatened. Several lines of evidence support this idea; nucleoli and SGs share molecular building blocks, are regulated by common signaling pathways and communicate when vital cellular functions become compromised. Together, nucleoli and SGs orchestrate physiological responses that are directly relevant to stress and human health. As both compartments have established roles in neurodegenerative diseases, cancer and virus infections, we propose that these conditions will benefit from therapeutic interventions that target simultaneously nucleoli and SGs.      

Efficient,Cyanine Dye Based Bilayer Solar Cells

Simple bilayer solar cells, using commercially available cationic cyanine dyes as donors and evaporated C₆₀ layer as an acceptor are prepared. Cyanine dyes with absorption maxima of 578, 615 and 697 nm having either perchlorate or hexafluorophosphate counter‐ions are evaluated. The perchlorate dye leads to cells with S‐shape current‐voltage curves; only the dyes with the hexafluorophosphate counter‐ions lead to efficient solar cells. When the wide bandgap dyes are employed, S‐shape current‐voltage curves are obtained when the conductive polymer PEDOT:PSS is used as hole transport layer. Substitution of PEDOT:PSS with MoO₃ leads to cells with more rectangular current–voltage curves and high fill factors. Additionally, the cells using the MoO₃ layer for hole extraction lead to high open circuit voltages of 0.9 V. In the case that a low bandgap hexafluorophosphate dye is used with the HOMO above that of the PEDOT:PSS the cell performance is independent on the type of hole transport layer employed. Using this approach, bilayer solar cells are obtained with power efficiencies ranging from 1.8 to 2.9% depending on the particular dye employed. These are impressive numbers for bilayer solar cell that are partially solution processed in ambient conditions.     

Redox-sensitive stimulation of type-1 ryanodine receptors by the scorpion toxin maurocalcine

The scorpion toxin maurocalcine acts as a high affinity agonist of the type-1 ryanodine receptor expressed in skeletal muscle. Here, we investigated the effects of the reducing agent dithiothreitol or the oxidizing reagent thimerosal on type-1 ryanodine receptor stimulation by maurocalcine. Maurocalcine addition to sarcoplasmic reticulum vesicles actively loaded with calcium elicited Ca²⁺ release from native vesicles and from vesicles pre-incubated with dithiothreitol; thimerosal addition to native vesicles after Ca²⁺ uptake completion prevented this response. Maurocalcine enhanced equilibrium [³H]-ryanodine binding to native and to dithiothreitol-treated reticulum vesicles, and increased 5-fold the apparent K_i for Mg²⁺ inhibition of [³H]-ryanodine binding to native vesicles. Single calcium release channels incorporated in planar lipid bilayers displayed a long-lived open sub-conductance state after maurocalcine addition. The fractional time spent in this sub-conductance state decreased when lowering cytoplasmic [Ca²⁺] from 10 μM to 0.1 μM or at cytoplasmic [Mg²⁺] ≥ 30 μM. At 0.1 μM [Ca²⁺], only channels that displayed poor activation by Ca²⁺ were readily activated by 5 nM maurocalcine; subsequent incubation with thimerosal abolished the sub-conductance state induced by maurocalcine. We interpret these results as an indication that maurocalcine acts as a more effective type-1 ryanodine receptor channel agonist under reducing conditions.     

Phenotypic and Genotypic Identification of the Most Acidifiers LAB Strains Isolated from Fermented Food

Biology Bulletin - Over 18 strains of lactic acid bacteria “LAB” isolated from different traditionally fermented products mainly sourdough and fermented vegetables, have been...     

Solubilization and bio-conjugation of quantum dots and bacterial toxicity assays by growth curve and plate count

Quantum dots (QDs) are fluorescent semiconductor nanoparticles with size-dependent emission spectra that can be excited by a broad choice of wavelengths. QDs have attracted a lot of interest for imaging, diagnostics, and therapy due to their bright, stable fluorescence. QDs can be conjugated to a variety of bio-active molecules for binding to bacteria and mammalian cells. QDs are also being widely investigated as cytotoxic agents for targeted killing of bacteria. The emergence of multiply-resistant bacterial strains is rapidly becoming a public health crisis, particularly in the case of Gram negative pathogens. Because of the well-known antimicrobial effect of certain nanomaterials, especially Ag, there are hundreds of studies examining the toxicity of nanoparticles to bacteria. Bacterial studies have been performed with other types of semiconductor nanoparticles as well, especially TiO(2), but also ZnO and others including CuO. Some comparisons of bacterial strains have been performed in these studies, usually comparing a Gram negative strain with a Gram positive. With all of these particles, mechanisms of toxicity are attributed to oxidation: either the photogeneration of reactive oxygen species (ROS) by the particles or the direct release of metal ions that can cause oxidative toxicity. Even with these materials, results of different studies vary greatly. In some studies the Gram positive test strain is reportedly more sensitive than the Gram negative; in others it is the opposite. These studies have been well reviewed. In all nanoparticle studies, particle composition, size, surface chemistry, sample aging/breakdown, and wavelength, power, and duration of light exposure can all dramatically affect the results. In addition, synthesis byproducts and solvents must be considered. High-throughput screening techniques are needed to be able to develop effective new nanomedicine agents. CdTe QDs have anti-microbial effects alone or in combination with antibiotics. In a previous study, we showed that coupling of antibiotics to CdTe can increase toxicity to bacteria but decrease toxicity to mammalian cells, due to decreased production of reactive oxygen species from the conjugates. Although it is unlikely that cadmium-containing compounds will be approved for use in humans, such preparations could be used for disinfection of surfaces or sterilization of water. In this protocol, we give a straightforward approach to solubilizing CdTe QDs with mercaptopropionic acid (MPA). The QDs are ready to use within an hour. We then demonstrate coupling to an antimicrobial agent. The second part of the protocol demonstrates a 96-well bacterial inhibition assay using the conjugated and unconjugated QDs. The optical density is read over many hours, permitting the effects of QD addition and light exposure to be evaluated immediately as well as after a recovery period. We also illustrate a colony count for quantifying bacterial survival.     

sst2 Somatostatin receptor inhibits cell proliferation through Ras-, Rap1-, and B-Raf-dependent ERK2 activation

The G protein-coupled sst2 somatostatin receptor is a critical negative regulator of cell proliferation. sstII prevents growth factor-induced cell proliferation through activation of the tyrosine phosphatase SHP-1 leading to induction of the cyclin-dependent kinase inhibitor p27Kip1. Here, we investigate the signaling molecules linking sst2 to p27Kip1. In Chinese hamster ovary-DG-44 cells stably expressing sst2 (CHO/sst2), the somatostatin analogue RC-160 transiently stimulates ERK2 activity and potentiates insulin-stimulated ERK2 activity. RC-160 also stimulates ERK2 activity in pancreatic acini isolated from normal mice, which endogenously express sst2, but has no effect in pancreatic acini derived from sst2 knock-out mice. RC-160-induced p27Kip1 up-regulation and inhibition of insulin-dependent cell proliferation are both prevented by pretreatment of CHO/sst2 cells with the MEK1/2 inhibitor PD98059. In addition, using dominant negative mutants, we show that sst2-mediated ERK2 stimulation is dependent on the pertussis toxin-sensitive Gi/o protein, the tyrosine kinase Src, both small G proteins Ras and Rap1, and the MEK kinase B-Raf but is independent of Raf-1. Phosphatidylinositol 3-kinase (PI3K) and both tyrosine phosphatases, SHP-1 and SHP-2, are required upstream of Ras and Rap1. Taken together, our results identify a novel mechanism whereby a Gi/o protein-coupled receptor inhibits cell proliferation by stimulating ERK signaling via a SHP-1-SHP-2-PI3K/Ras-Rap1/B-Raf/MEK pathway.