Primary structure of the tail sheath protein of bacteriophage T4 and its gene

Complete sequence determination of gene 18 encoding the tail sheath protein was carried out mainly by the Maxam-Gilbert method. Approximately 40 peptides contained in a tryptic digest and a lysyl endopeptidase digest of gp 18 were isolated by reversed-phase high-performance liquid chromatography. All the peptides were identified along the nucleotide sequence of gene 18 based on the amino acid compositions. These peptides cover 88% of the total primary structure. Furthermore, the amino acid sequences of 9 of the 40 peptides were determined by a gas-phase protein sequencer; one of them turned to be the N-terminal one. The C-terminal peptide in the tryptic digest was isolated from the unadsorbed fraction of affinity chromatography on immobilized anhydrotrypsin and the amino acid sequence was also determined. Thus, the complete primary structure of gp 18 was determined; it has 658 amino acid residues and a molecular weight of 71,160.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

7-O-Galloyl-(+)-catechin and 3-O-galloylprocyanidin B-3 from Sanguisorba officinalis

7-O-Galloyl-(+)-catechin and 3-O-galloylprocyanidin B-3, along with gambiriins A-1 and B-3 and four polygalloylglucoses, have been isolated fro     

AtNHX5 and AtNHX6 Are Required for the Subcellular Localization of the SNARE Complex That Mediates the Trafficking of Seed Storage Proteins in Arabidopsis

The SNARE complex composed of VAMP727, SYP22, VTI11 and SYP51 is critical for protein trafficking and PSV biogenesis in Arabidopsis. This SNARE complex directs the fusion between the prevacuolar compartment (PVC) and the vacuole, and thus mediates protein trafficking to the vacuole. In this study, we examined the role of AtNHX5 and AtNHX6 in regulating this SNARE complex and its function in protein trafficking. We found that AtNHX5 and AtNHX6 were required for seed production, protein trafficking and PSV biogenesis. We further found that the nhx5 nhx6 syp22 triple mutant showed severe defects in seedling growth and seed development. The triple mutant had short siliques and reduced seed sets, but larger seeds. In addition, the triple mutant had numerous smaller protein storage vacuoles (PSVs) and accumulated precursors of the seed storage proteins in seeds. The PVC localization of SYP22 and VAMP727 was repressed in nhx5 nhx6, while a significant amount of SYP22 and VAMP727 was trapped in the Golgi or TGN in nhx5 nhx6. AtNHX5 and AtNHX6 were co-localized with SYP22 and VAMP727. Three conserved acidic residues, D164, E188, and D193 in AtNHX5 and D165, E189, and D194 in AtNHX6, were essential for the transport of the storage proteins, indicating the importance of exchange activity in protein transport. AtNHX5 or AtNHX6 did not interact physically with the SNARE complex. Taken together, AtNHX5 and AtNHX6 are required for the PVC localization of the SNARE complex and hence its function in protein transport. AtNHX5 and AtNHX6 may regulate the subcellular localization of the SNARE complex by their transport activity.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

The sequence of histological changes in the regional lymph node and other lymphoid organs of mice injected with the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) or bacterial lipopolysaccharide (LPS) was followed. Injection of CPS-K, but not LPS, induced the following characteristic histological changes in the regional lymph node. In the early stage there was a marked decrease in the number of small lymphocytes, accompanied by the appearance of scattered fragmented nuclei and infiltration of polymorphonuclear neutrophilic leukocytes, and in the late stage there was marked proliferation of macrophage-like cells and pyroninophilic cells. Histological changes in the thymus and spleen and changes in cell populations in the bone marrow and peripheral blood after CPS-K injection were essentially the same as after LPS injection. Since CPS-K has a much stronger adjuvant action on antibody response than does LPS, it is suggested that the characteristic histological changes in the regional lymph node after injection of CPS-K are closely related to its extraordinarily strong adjuvant action.     

Cyclical mechanical stretch enhances degranulation and IL‐4 secretion in RBL‐2H3 mast cells

Mast cells are widely distributed in the body and affect their surrounding environment through degranulation and secretion of cytokines. Conversely, mast cells are influenced by environmental stimuli such as cyclical mechanical stretch (CMS), such as that induced by heartbeat and respiration. Peripherally distributed mast cells are surrounded by extracellular matrix, where they bind IgE on their surface by expressing the high‐affinity Fc receptor for IgE (FcεRI), and they release mediators after cross‐linking of surface‐bound IgE by allergen. To analyse how CMS affects mast cell responses, we examined the effect of applying CMS on the behaviour of IgE‐bound mast cells (RBL‐2H3 cell line) adhering to fibronectin as a substitute for extracellular matrix. We found that CMS enhanced FcεRI‐mediated secretion in the presence of antigen (2,4‐dinitrophenol–bovine serum albumin). CMS increased expression of IL‐4 mRNA and secretion of IL‐4 protein. Western blot analysis showed that CMS changes the signal transduction in mitogen‐activated protein kinases and AKT, which in turn alters the regulation of IL‐4 and increases the secretion of IL‐4. These results suggest that CMS modulates the effect of mast cells on inflammation and resultant tissue remodelling. Understanding how CMS affects mast cell responses is crucial for developing therapies to treat mast cell‐related diseases. Copyright © 2013 John Wiley & Sons, Ltd.     

A Theoretical Model of Jigsaw-Puzzle Pattern Formation by Plant Leaf Epidermal Cells

Plant leaf epidermal cells exhibit a jigsaw puzzle–like pattern that is generated by interdigitation of the cell wall during leaf development. The contribution of two ROP GTPases, ROP2 and ROP6, to the cytoskeletal dynamics that regulate epidermal cell wall interdigitation has already been examined; however, how interactions between these molecules result in pattern formation remains to be elucidated. Here, we propose a simple interface equation model that incorporates both the cell wall remodeling activity of ROP GTPases and the diffusible signaling molecules by which they are regulated. This model successfully reproduces pattern formation observed in vivo, and explains the counterintuitive experimental results of decreased cellulose production and increased thickness. Our model also reproduces the dynamics of three-way cell wall junctions. Therefore, this model provides a possible mechanism for cell wall interdigitation formation in vivo.     

Monitoring Autophagy Flux and Activity: Principles and Applications

Macroautophagy is a major degradation mechanism of cell components via the lysosome. Macroautophagy greatly contributes to not only cell homeostasis but also the prevention of various diseases. Because macroautophagy proceeds through multi-step reactions, researchers often face a persistent question of how macroautophagic activity can be measured correctly. To make a straightforward determination of macroautophagic activity, diverse monitoring assays have been developed. Direct measurement of lysosome-dependent degradation of radioisotopically labeled cell proteins has long been applied. Meanwhile, indirect monitoring procedures have been developed. In these assays, autophagosome marker proteins, microtubule-associated proteins 1A/1B light chain 3B-II (LC3B-II) and gamma-aminobutyric acid receptor-associated protein-II (GABARAP-II) have been analyzed and the validity of the assays strongly depends on appropriate assessment of the fluctuation of LC3-II and/or GABARAP-II levels in the presence or absence of lysosomal inhibitors. This article describes these monitoring methods, paying special attention to the principles and characteristics of each procedure.     

Embigin/basigin subgroup of the immunoglobulin superfamily: Different modes of expression during mouse embryogenesis and correlated expression with carbohydrate antigenic markers

Embigin and basigin are highly glycosylated transmembrane glycoproteins with two immunoglobulin domains and form a subgroup in the immunoglobulin superfamily. Previous studies have demonstrated the functional significance of these molecules. In the present study, in situ hybridization analysis of their expression was performed during mouse embryogenesis. Embigin was strongly expressed in the endoderm during early postimplantation embryogenesis, and in the somite stage in the gut and visceral endoderm. Embryonic ectoderm and its derivative tissues weakly to moderately expressed this molecule. From day 10 to 15 of gestation, no embigin signal was detected. Basigin was more broadly expressed. During the organogenesis period, basigin was expressed in various epithelial tissues, brain ventricles, the spinal cord and dorsal root ganglion. The modes of expression of these two proteins throughout the egg cylinder stage correlated with the expression of the carbohydrate markers that they carry; embigin with Dolichos biflorus agglutinin binding sites and basigin with Le^x antigen and more closely with fucosyltransferase IV, which forms the antigenic epitope. These findings imply that proteins with specific carbohydrate epitopes play roles in early postimplantation embryogenesis.