Dynamic and static behaviors of CH4 and CO2 in small and large cavities of hydrate

We investigated the static structures and dynamic behaviors for guest molecules (CH4 and CO2) in small and large cavities which are composed of 20 and 24 water molecules, respectively, by B3LYP/6-311++G(d,p) level calculations in GAUSSIAN 09, and using quantum molecular dynamics (QMD) (NVT MD with semiempirical MO PM3 method). For the static calculations, the guest CO2 and CH4 molecules are around at the center of small and large cavities with weak H-bond formations of HOH⋯O 2C and H2O⋯H4C van der Waals interaction systems. Calculated carbon NMR chemical shifts of the CH4 in the gas-state and in the small and large cavities reflected the C-13 experimental tendency, while the calculated carbon NMR chemical shifts of the CO2 in the three states almost correspond to the experimental value in the gas-state. For QMD calculations, we used a cluster model containing 73 water molecules, and examined dynamic behavior of guest molecules in the shell cluster model of 39 water molecules which own small and large cavities. The dynamic behavior of guest molecules are simulated from the trajectory distribution of molecular center of the mass due to the translational motion, and also analyzed using librational motions of guest molecules in the cavities. © 2012 Elsevier B.V. All rights reserved

RABケツゴウインシドウテイノタメノジンソクカツカンベンナFAR-WESTERN BLOTTING

低分子量Gタンパク質Rab5は細胞膜から初期エンドソームへの小胞輸送や初期エンドソーム同士の融合に関与する因子であることが知られている。過去の報告では,GTPγSなどの加水分解耐性GTPアナログを使用しFar-western blottingを行うことによってRab5結合因子が検出されている。しかしながら,この方法ではNEおよびNS反応の際に多量の加水分解耐性GTPアナログが必要であり,実験時間やコストが非常にかかる。本論文ではFar-western blottingのプローブとして常時活性型変異体Rab5Q79Lを用いることによって,NE反応とNS反応が必要なく加水分解耐性GTPアナログを使用しない簡便且つ実験コストが低い方法について記述する。この方法を評価するため,既知のRab5結合因子であるEEA1とRab5が結合するか否か検証したところ結合が確認された。さらにLC MS/MSによってRab5結合因子の同定を試みたところactin betaが同定された。我々の方法では実験時間が3-4時間程短縮され,実験コストは90-95%削減された。我々はこの方法がRab5による細胞内小胞輸送における重要な知見をもたらすものと考える。The small GTP binding-protein Rab5 is known to be involved both in the vesicle-mediated transport from the plasma membrane to the early endosomes, and in the homotypic early endosome fusion. Previous reports showed that Rab5-binding proteins can be detected by using Far-western blotting in the presence of non-hydrolyzable GTP analogs such as GTPγS. However, this method requires significant quantities of non-hydrolyzable GTP analog and is thus time-consuming and expensive for the nucleotide exchange (NE) and nucleotide stabilization (NS) reactions. In this report we describe a faster and more cost-effective method that does not use non-hydrolyzable GTP analogs but rather, a constitutively active Rab5 mutant Q79L as a Far-western blotting probe. To validate this method, the binding of the previously characterized Rab5-binding protein EEA-1 was confirmed and actin beta was identified as a Rab5-binding protein by LC MS/MS. Our protocol can reduce the experimental time (by 3-4 h) and the cost (by 90-95%) for the experiment. We expect this method to provide fundamental insights into the molecular mechanism of intracellular transport by Rab5

D3h -Symmetric Porphyrin-Based Rigid Macrocyclic Ligands for Multicofacial Multinuclear Complexes in a One-Nanometer-Sized Cavity.

The one-step synthesis of D3h -symmetric cyclic porphyrin trimers 1 composed of three 2,2\u27-[4,4\u27-bis(methoxycarbonyl)]bipyridyl moieties and three porphyrinatozinc moieties was achieved from a nickel-mediated reductive coupling of meso-5,15-bis(6-chloro-4-methoxycarbonylpyrid-2-yl)porphyrinatozinc. Although cyclic trimers 1 were obtained as a mixture that included other cyclic and acyclic porphyrin oligomers, an extremely specific separation was observed only for cyclic trimers 1 when using columns of silica gel modified with pyrenylethyl, cyanopropyl, and other groups. Structural analysis of cyclic trimers 1 was carried out by means of NMR spectroscopy and X-ray crystallography. Treatment of an η(3) -allylpalladium complex with a cyclic trimer gave a tris(palladium) complex containing three η(3) -allylpalladium groups inside the space, which indicated that the bipyridyl moieties inside the ring could work as bidentate metalloligands.The one-step synthesis of D3h -symmetric cyclic porphyrin trimers 1 composed of three 2,2\u27-[4,4\u27-bis(methoxycarbonyl)]bipyridyl moieties and three porphyrinatozinc moieties was achieved from a nickel-mediated reductive coupling of meso-5,15-bis(6-chloro-4-methoxycarbonylpyrid-2-yl)porphyrinatozinc. Although cyclic trimers 1 were obtained as a mixture that included other cyclic and acyclic porphyrin oligomers, an extremely specific separation was observed only for cyclic trimers 1 when using columns of silica gel modified with pyrenylethyl, cyanopropyl, and other groups. Structural analysis of cyclic trimers 1 was carried out by means of NMR spectroscopy and X-ray crystallography. Treatment of an η(3) -allylpalladium complex with a cyclic trimer gave a tris(palladium) complex containing three η(3) -allylpalladium groups inside the space, which indicated that the bipyridyl moieties inside the ring could work as bidentate metalloligands

Cyclical and Patch-Like GDNF Distribution along the Basal Surface of Sertoli Cells in Mouse and Hamster Testes

BACKGROUND AND AIMS: In mammalian spermatogenesis, glial cell line-derived neurotrophic factor (GDNF) is one of the major Sertoli cell-derived factors which regulates the maintenance of undifferentiated spermatogonia including spermatogonial stem cells (SSCs) through GDNF family receptor α1 (GFRα1). It remains unclear as to when, where and how GDNF molecules are produced and exposed to the GFRα1-positive spermatogonia in vivo. METHODOLOGY AND PRINCIPAL FINDINGS: Here we show the cyclical and patch-like distribution of immunoreactive GDNF-positive signals and their close co-localization with a subpopulation of GFRα1-positive spermatogonia along the basal surface of Sertoli cells in mice and hamsters. Anti-GDNF section immunostaining revealed that GDNF-positive signals are mainly cytoplasmic and observed specifically in the Sertoli cells in a species-specific as well as a seminiferous cycle- and spermatogenic activity-dependent manner. In contrast to the ubiquitous GDNF signals in mouse testes, high levels of its signals were cyclically observed in hamster testes prior to spermiation. Whole-mount anti-GDNF staining of the seminiferous tubules successfully visualized the cyclical and patch-like extracellular distribution of GDNF-positive granular deposits along the basal surface of Sertoli cells in both species. Double-staining of GDNF and GFRα1 demonstrated the close co-localization of GDNF deposits and a subpopulation of GFRα1-positive spermatogonia. In both species, GFRα1-positive cells showed a slender bipolar shape as well as a tendency for increased cell numbers in the GDNF-enriched area, as compared with those in the GDNF-low/negative area of the seminiferous tubules. CONCLUSION/SIGNIFICANCE: Our data provide direct evidence of regionally defined patch-like GDNF-positive signal site in which GFRα1-positive spermatogonia possibly interact with GDNF in the basal compartment of the seminiferous tubules