Self-assemblies of Rab- and Arf-family small GTPases on lipid bilayers in membrane tethering

Small GTPases of the Ras superfamily, which include Ras-, Rho-, Rab-, Arf-, and Ran-family isoforms, are generally known to function as a nucleotide-dependent molecular switch in eukaryotic cells. In the GTP-loaded forms, they selectively recruit their cognate interacting proteins or protein complexes, termed “effectors,” to the cytoplasmic face of subcellular membrane compartments, thereby switching on the downstream effector functions, which are vital for fundamental cellular events, such as cell proliferation, cytoskeletal organization, and intracellular membrane trafficking. Nevertheless, in addition to acting as the classic nucleotide-dependent switches for the effectors, recent studies have uncovered that small GTPases themselves can be self-assembled specifically into homo-dimers or higher-order oligomers on membranes, and these assembly processes are likely responsible for their physiological functions. This Review focuses particularly on the self-assembly processes of Rab- and Arf-family isoforms during membrane tethering, the most critical step to ensure the fidelity of membrane trafficking. A summary of the current experimental evidence for self-assemblies of Rab and Arf small GTPases on lipid bilayers in chemically defined reconstitution system is provided     

Synergistic role of c-Myc and ERK1/2 in the mitogenic response to TGFβ-1 in cultured rat nucleus pulposus cells

Introduction  

Although transforming growth factor β1 (TGFβ1) is known to be a potent inhibitor of proliferation in most cell types, it accelerates proliferation in certain mesenchymal cells, such as articular chondrocytes and nucleus pulposus cells. The low ability for self-renewal of nucleus pulposus cells is one obstacle in developing new therapeutic options for intervertebral disc diseases, and utilizing cytokines is one of the strategies to regulate nucleus pulposus cell proliferation. However, the precise cell cycle progression and molecular mechanisms by which TGFβ1 stimulates cell growth remain unclear. The aim of this study was to elucidate a mechanism that enables cell proliferation with TGFβ1 stimulation.     

A New Natural Quinone, 2-Methyl-3-II,III,VIII,IX-octahydromultiprenyl9-1,4-naphthoquinone Isolated from Streptomyces albus

Purification of the precipitates obtained from the juice oil of Citrus hassaku by chromatography afforded 7-geranyloxycoumarin (aurapten) and two compounds (mp 43~45°C and 122~124°C), whose structures were determined to be epoxyaurapten and marmin on the basis of spectral evidence. These compounds were isolated from Citrus hassaku for the first time. The spasmolytic activity was tested of aurapten, epoxyaurapten, marmin and their related compounds, which were synthesized from aurapten and marmin, on the small intestine removed from a male guinea pig. Epoxyaurapten exhibited the highest activity among them against the small intestine’s contraction induced by BaCl₂.     

Structure of the carboxypeptidase Y inhibitor IC in complex with the cognate proteinase reveals a novel mode of the proteinase-protein inhibitor interaction

Carboxypeptidase Y (CPY) inhibitor, IC, shows no homology to any other known proteinase inhibitors and rather belongs to the phosphatidylethanolamine-binding protein (PEBP) family. We report here on the crystal structure of the IC-CPY complex at 2.7 A resolution. The structure of IC in the complex with CPY consists of one major beta-type domain and a N-terminal helical segment. The structure of the complex contains two binding sites of IC toward CPY, the N-terminal inhibitory reactive site (the primary CPY-binding site) and the secondary CPY-binding site, which interact with the S1 substrate-binding site of CPY and the hydrophobic surface flanked by the active site of the enzyme, respectively. It was also revealed that IC had the ligand-binding site, which is conserved among PEBPs and the putative binding site of the polar head group of phospholipid. The complex structure and analyses of IC mutants for inhibitory activity and the binding to CPY demonstrate that the N-terminal inhibitory reactive site is essential both for inhibitory function and the complex formation with CPY and that the binding of IC to CPY constitutes a novel mode of the proteinase-protein inhibitor interaction. The unique binding mode of IC toward the cognate proteinase provides insights into the inhibitory mechanism of PEBPs toward serine proteinases and into the specific biological functions of IC belonging to the PEBP family as well.     

Characterization of 1-(2-[18F] fluoro-3-pyridyl)-4-(2-isopropyl-1-oxo- isoindoline-5-yl)-5-methyl-1H-1,2,3-triazole, a PET ligand for imaging the metabotropic glutamate receptor type 1 in rat and monkey brains

Neurovascular degeneration contributes to the pathogenesis of Alzheimer's disease (AD). Because erythropoietin (EPO) promotes endothelial regeneration, we investigated the therapeutic effects of EPO in animal models of AD. In aged Tg2576 mice, EPO receptors (EPORs) were expressed in the cortex and hippocampus. Tg2576 mice were treated with daily injection of EPO (5000 IU/kg/day) for 5 days. At 14 days, EPO improved contextual memory as measured by fear-conditioning test. EPO enhanced endothelial proliferation and the level of synaptophysin expression in the brain. EPO also increased capillary density, and decreased the level of the receptor for advanced glycation endproducts (RAGE) in the brain, while decreasing in the amount of amyloid plaque and amyloid-β (Aβ). In cultured human endothelial cells, EPO enhanced angiogenesis and suppressed the expression of the RAGE. These results show that EPO improves memory and ameliorates endothelial degeneration induced by Aβ in AD models. This pre-clinical evidence suggests that EPO may be useful for the treatment of AD.     

A Neural Network Model for K(λ) Retrieval and Application to Global K par Monitoring

Accurate estimation of diffuse attenuation coefficients in the visible wavelengths K _d(λ) from remotely sensed data is particularly challenging in global oceanic and coastal waters. The objectives of the present study are to evaluate the applicability of a semi-analytical K _d(λ) retrieval model (SAKM) and Jamet’s neural network model (JNNM), and then develop a new neural network K _d(λ) retrieval model (NNKM). Based on the comparison of K _d(λ) predicted by these models with in situ measurements taken from the global oceanic and coastal waters, all of the NNKM, SAKM, and JNNM models work well in K _d(λ) retrievals, but the NNKM model works more stable and accurate than both SAKM and JNNM models. The near-infrared band-based and shortwave infrared band-based combined model is used to remove the atmospheric effects on MODIS data. The K _d(λ) data was determined from the atmospheric corrected MODIS data using the NNKM, JNNM, and SAKM models. The results show that the NNKM model produces <30% uncertainty in deriving K _d(λ) from global oceanic and coastal waters, which is 4.88-17.18% more accurate than SAKM and JNNM models. Furthermore, we employ an empirical approach to calculate K _par from the NNKM model-derived diffuse attenuation coefficient at visible bands (443, 488, 555, and 667 nm). The results show that our model presents a satisfactory performance in deriving K _par from the global oceanic and coastal waters with 20.2% uncertainty. The K _par are quantified from MODIS data atmospheric correction using our model. Comparing with field measurements, our model produces ~31.0% uncertainty in deriving K _par from Bohai Sea. Finally, the applicability of our model for general oceanographic studies is briefly illuminated by applying it to climatological monthly mean remote sensing reflectance for time ranging from July, 2002- July 2014 at the global scale. The results indicate that the high K _d(λ) and K _par values are usually found around the coastal zones in the high latitude regions, while low K _d(λ) and K _par values are usually found in the open oceans around the low-latitude regions. These results could improve our knowledge about the light field under waters at either the global or basin scales, and be potentially used into general circulation models to estimate the heat flux between atmosphere and ocean.     

Enhancement of the endotoxin recognition pathway by ventilation with a large tidal volume in rabbits

Ventilation with a small tidal volume (V(t)) is associated with better clinical outcomes than with a large V(t), particularly in critical settings, including acute lung injury. To determine whether V(t) influences the lipopolysaccaharide (LPS) recognition pathway, we studied CD14 expression in rabbit lungs and the release of TNF-alpha by cultured alveolar macrophages after 240 min of ventilation with a large (20 ml/kg) vs. a small (5 ml/kg) V(t). We also applied small or large V(t) to lungs instilled with 50 microg/kg of LPS. The alveolar macrophages collected after large V(t) ventilation revealed a 20-fold increase in LPS-induced TNF-alpha release compared with those collected after small V(t) ventilation, whereas TNF-alpha was undetectable without LPS stimulation. In animals ventilated with a large V(t), the expression of CD14 mRNA in whole lung homogenates and the expression of CD14 protein on alveolar macrophages, assessed by immunohistochemistry, were both significantly increased in the absence of LPS stimulation. A large V(t) applied to LPS-instilled lungs increased the pulmonary albumin permeability and TNF-alpha release into the plasma. These results suggest that mechanical stress caused by a large V(t) sensitizes the lungs to endotoxin, a phenomenon that may occur partially via the upregulation of CD14.     

Protein anatomy: structure and function of peptide fragments corresponding to the secondary structure units of barnase

Globular proteins can be decomposed into several modules or secondary structure units. It is useful to investigate the functions of such structural units in order to understand the folding units of proteins. In our previous work, barnase was divided into six peptide fragments corresponding to modules, and some of them were shown to have RNA-binding and RNase activity [Yanagawa, et al. (1993) J. Biol. Chem. 268, 5861-5865]. Barnase mutant proteins obtained by permutation of the structural units also had RNase activity [Tsuji, T. et al. (1999) J. Mol. Biol. 286, 1581-1596]. Here we investigated the structure and function of peptide fragments corresponding to secondary structure units of barnase. The results of circular dichroism spectroscopy indicated that some of the peptide fragments form helical structures in aqueous solutions containing over 30% 2,2,2-trifluoroethanol, and the S6 (94-110) peptide fragment is induced to form a beta-sheet structure in the presence of RNA. The S6 peptide fragment forms aggregate complexes with RNA. Electron microscopic analysis showed that the aggregate complexes were comprised of filaments. These results indicate that not only modules but also secondary structure units dissected from a globular protein have functional and structure-forming capabilities.     

Human bronchial smooth muscle cell proliferation via thromboxane A2 receptor

Thromboxane A2 receptor (TP) mediates bronchial smooth muscle cell (BSMC) contraction, airway hyperresponsiveness, and airway inflammation in patients with asthma. In the present study, a pathogenic role of TP activation in airway remodeling was examined using primary cultures of human BSMC. A TP agonist, I-BOP, concentration-dependently enhanced not only bromodeoxyuridine (BrdU) uptake but also cell proliferation of BSMC. A TP-selective antagonist, AA-2414, blocked the effects of I-BOP on both BrdU uptake and cell proliferation. I-BOP-induced BrdU uptake was significantly blocked by two non-selective tyrosine kinase inhibitors, genistein and herbimycin A, or a Src family tyrosine kinase inhibitor, PP2, but not by an inhibitor of epidermal growth factor (EGF) receptor-associated tyrosine kinase, AG1478. In conclusion, TP receptor activation causes DNA synthesis and cell proliferation of human BSMC by activating tyrosine kinases including Src, but not by EGF receptor transactivation.     

Expression of a sweet cherry DREB1/CBF ortholog in Arabidopsis confers salt and freezing tolerance

Dehydration responsive element binding protein 1 (DREB1)/C-repeat binding factor (CBF) induces the expression of many stress-inducible genes in Arabidopsis. We have previously reported the identification of three DREB1/ICBF homologs from sweet cherry (Prunus avium). To identify the function of these homologs, one of the genes, CIG-B, was transformed into Arabidopsis. In one of the transgenic plant lines, the DREB1/CBF target gene cor15a was induced in the absence of stress treatment. The cor15a-overexpressing transgenic plant exhibited mild growth retardation and had greater salt and freezing tolerance than did the wild-type and the transgenic lines in which cor15a was not induced. These results suggest that this sweet cherry DREB1/CBF homolog has a function similar to that of DREB1/CBF.