Immunomodulatory activity of Bengkoang (Pachyrhizus erosus) fiber extract in vitro and in vivo

Bengkoang (Pachyrhizus erosus (L.) Urban) is one of the most popular edible root vegetables in Indonesia. Bengkoang contains fairly large amounts of carbohydrates and crude fiber. The purpose of this research is to evaluate the immunomodulatory effect of the bengkoang fiber extract (BFE) in vitro and in vivo. BFE was prepared by heating the powder of bengkoang fiber suspended in distilled water at 121 °C for 20 min. BFE facilitated IgM production by the human hybridoma cell line HB4C5 cells. In addition, production of IgM, IgG, and IgA by mouse primary splenocytes was facilitated by BFE in a dose-dependent manner. BFE also significantly facilitated production of both interleukin-5 and interleukin-10 by splenocytes. Immunoglobulin production by lymphocytes from the spleen, Peyer’s patch, and mesenteric lymph node were significantly activated by oral administration of BFE to mice for 14 days. The serum immunoglobulin levels of IgG, IgM, and IgA were also significantly enhanced. Furthermore, cytokine production by lymphocytes from the spleen, Peyer’s patch, and mesenteric lymph node were also facilitated by oral administration of BFE. These results suggest that BFE has positive effects on the immune system in vitro and in vivo.     

Crystal structure of phosphopantothenate synthetase from Thermococcus kodakarensis

Bacteria/eukaryotes share a common pathway for coenzyme A biosynthesis which involves two enzymes to convert pantoate to 4′‐phosphopantothenate. These two enzymes are absent in almost all archaea. Recently, it was reported that two novel enzymes, pantoate kinase, and phosphopantothenate synthetase (PPS), are responsible for this conversion in archaea. Here, we report the crystal structure of PPS from the hyperthermophilic archaeon, Thermococcus kodakarensis and its complexes with substrates, ATP, and ATP and 4‐phosphopantoate. PPS forms an asymmetric homodimer, in which two monomers composing a dimer, deviated from the exact twofold symmetry, displaying 4°–13° distortion. The structural features are consistent with the mutagenesis data and the results of biochemical experiments previously reported. Based on these structures, we discuss the catalytic mechanism by which PPS produces phosphopantoyl adenylate, which is thought to be a reaction intermediate. Proteins 2014; 82:1924–1936. © 2014 Wiley Periodicals, Inc.     

PCDH10 is required for the tumorigenicity of glioblastoma cells

Protocadherin10 (PCDH10)/OL-protocadherin is a cadherin-related transmembrane protein that has multiple roles in the brain, including facilitating specific cell–cell connections, cell migration and axon guidance. It has recently been reported that PCDH10 functions as a tumor suppressor and that its overexpression inhibits proliferation or invasion of multiple tumor cells. However, the function of PCDH10 in glioblastoma cells has not been elucidated. In contrast to previous reports on other tumors, we show here that suppression of the expression of PCDH10 by RNA interference (RNAi) induces the growth arrest and apoptosis of glioblastoma cells in vitro. Furthermore, we demonstrate that knockdown of PCDH10 inhibits the growth of glioblastoma cells xenografted into immunocompromised mice. These results suggest that PCDH10 is required for the proliferation and tumorigenicity of glioblastoma cells. We speculate that PCDH10 may be a promising target for the therapy of glioblastoma.     

Dual roles of a conserved pair, Arg23 and Ser20, in recognition of multiple substrates in α-aminoadipate aminotransferase from Thermus thermophilus

To clarify the mechanism for substrate recognition of α-aminoadipate aminotransferase (AAA-AT) from Thermus thermophilus, the crystal structure of AAA-AT complexed with N-(5′-phosphopyridoxyl)-l-glutamate (PPE) was determined at 1.67 Å resolution. The crystal structure revealed that PPE is recognized by amino acid residues the same as those seen in N-(5′-phosphopyridoxyl)-l-α-aminoadipate (PPA) recognition; however, to bind the γ-carboxyl group of Glu at a fixed position, the Cα atom of the Glu moiety moves 0.80 Å toward the γ-carboxyl group in the PPE complex. Markedly decreased activity for Asp can be explained by the shortness of the aspartyl side chain to be recognized by Arg23 and further dislocation of the Cα atom of bound Asp. Site-directed mutagenesis revealed that Arg23 has dual functions for reaction, (i) recognition of γ (δ)-carboxyl group of Glu (AAA) and (ii) rearrangement of α2 helix by changing the interacting partners to place the hydrophobic substrate at the suitable position.     

Water and salt movement in soil driven by crop roots: a controlled column study

Water deficit and salt accumulation in soil presents serious problems to crop production in semi-arid regions. These problems depend on the active transpiration stream and the selective absorption of ions by crop roots. In this study, a large sized soil column system was used to examine the dynamics of water and ion transport and salt accumulation in soil layers. Special reference was placed on the effects of the active and selective absorption by roots of different crops (i.e., corn plants, sunflower plants and no plants). The column system was equipped with on-line systems for the control of groundwater level. Soil water content sensors enabled time-course evaluations of the volumetric water content and hence upward flux of the groundwater in the soils at different depths. Furthermore, the distribution and accumulation of ions in soil layers, plant organs and xylem sap were analyzed using ion chromatography. In this column experiment, diurnal and longer term changes in water movement and ion accumulation in soil, affected by root absorption characteristics of plants, were evaluated quantitatively. The results demonstrated that the column system was applicable for the quantitative analysis of the effects of root absorption by different crops on water deficit and salinization in soils.     

A synthetic biology approach to the construction of membrane proteins in semi-synthetic minimal cells

Synthetic biology is an emerging field that aims at constructing artificial biological systems by combining engineering and molecular biology approaches. One of the most ambitious research line concerns the so-called semi-synthetic minimal cells, which are liposome-based system capable of synthesizing the lipids within the liposome surface. This goal can be reached by reconstituting membrane proteins within liposomes and allow them to synthesize lipids. This approach, that can be defined as biochemical, was already reported by us (Schmidli et al. J. Am. Chem. Soc. 113, 8127-8130, 1991). In more advanced models, however, a full reconstruction of the biochemical pathway requires (1) the synthesis of functional membrane enzymes inside liposomes, and (2) the local synthesis of lipids as catalyzed by the in situ synthesized enzymes. Here we show the synthesis and the activity - inside liposomes - of two membrane proteins involved in phospholipids biosynthesis pathway. The proteins, sn-glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LPAAT), have been synthesized by using a totally reconstructed cell-free system (PURE system) encapsulated in liposomes. The activities of internally synthesized GPAT and LPAAT were confirmed by detecting the produced lysophosphatidic acid and phosphatidic acid, respectively. Through this procedure, we have implemented the first phase of a design aimed at synthesizing phospholipid membrane from liposome within from within — which corresponds to the autopoietic growth mechanism.     

Phosphorylation of Tyr-398 and Tyr-402 in Occludin Prevents Its Interaction with ZO-1 and Destabilizes Its Assembly at the Tight Junctions

Occludin is phosphorylated on tyrosine residues during the oxidative stress-induced disruption of tight junction, and in vitro phosphorylation of occludin by c-Src attenuates its binding to ZO-1. In the present study mass spectrometric analyses of C-terminal domain of occludin identified Tyr-379 and Tyr-383 in chicken occludin as the phosphorylation sites, which are located in a highly conserved sequence of occludin, YETDYTT; Tyr-398 and Tyr-402 are the corresponding residues in human occludin. Deletion of YETDYTT motif abolished the c-Src-mediated phosphorylation of occludin and the regulation of ZO-1 binding. Y398A and Y402A mutations in human occludin also abolished the c-Src-mediated phosphorylation and regulation of ZO-1 binding. Y398D/Y402D mutation resulted in a dramatic reduction in ZO-1 binding even in the absence of c-Src. Similar to wild type occludin, its Y398A/Y402A mutant was localized at the plasma membrane and cell-cell contact sites in Rat-1 cells. However, Y398D/Y402D mutants of occludin failed to localize at the cell-cell contacts. Calcium-induced reassembly of Y398D/Y402D mutant occludin in Madin-Darby canine kidney cells was significantly delayed compared with that of wild type occludin or its T398A/T402A mutant. Furthermore, expression of Y398D/Y402D mutant of occludin sensitized MDCK cells for hydrogen peroxide-induced barrier disruption. This study reveals a unique motif in the occludin sequence that is involved in the regulation of ZO-1 binding by reversible phosphorylation of specific Tyr residues.Epithelial tight junctions (TJs)² form a selective barrier to the diffusion of toxins, allergens, and pathogens from the external environment into the tissues in the gastrointestinal tract, lung, liver, and kidney (1). Disruption of TJs is associated with the gastrointestinal diseases such as inflammatory bowel disease, celiac disease, infectious enterocolitis, and colon cancer (2–4) as well as in diseases of lung and kidney (5, 6). Numerous inflammatory mediators such as tumor necrosis factor α, interferon γ, and oxidative stress (7–12) are known to disrupt the epithelial TJs and the barrier function. Several studies have indicated that hydrogen peroxide disrupts the TJs in intestinal epithelium by a tyrosine kinase-dependent mechanism (11, 12).Four types of integral proteins, occludin, claudins, junctional adhesion molecules, and tricellulin are associated with TJs. Occludin, claudins, and tricellulin are tetraspan proteins, and their extracellular domains interact with homotypic domains of the adjacent cells (1, 2, 13). The intracellular domains of these proteins interact with a variety of soluble proteins such as ZO-1, ZO-2, ZO-3, 7H6, cingulin, and symplekin (14–23); this protein complex interacts with the perijunctional actomyosin ring. The interactions among TJ proteins are essential for the assembly and the maintenance of TJs. Therefore, regulation of the interactions among TJ proteins may regulate the TJ integrity. A significant body of evidence indicates that numerous signaling molecules are associated with the TJs. Protein kinases and protein phosphatases such as protein kinase Cζ (PKCζ), PKCι/λ (24), c-Src (25), c-Yes (26, 27), mitogen-activated protein kinase (28), PP2A, and PP1 (29) interact with TJs, indicating that TJs are dynamically regulated by intracellular signal transduction involving protein phosphorylation. Additionally, other signaling molecules such as calcium (30), phosphatidylinositol 3-kinase (31), Rho (32), and Rac (33) are involved in the regulation of TJs.Occludin, a ∼65-kDa protein, has been well characterized to be assembled into the TJs. Although occludin knock-out mice showed the formation of intact TJs in different epithelia (34), numerous studies have emphasized that it plays an important role in the regulation of TJ integrity. Occludin spans the membrane four times to form two extracellular loops and one intracellular loop, and the N-terminal and C-terminal domains hang into the intracellular compartment (35–37). In epithelium with intact TJs, occludin is highly phosphorylated on Ser and Thr residues (38), whereas Tyr phosphorylation is undetectable. However, the disruption of TJs in Caco-2 cell monolayers by oxidative stress and acetaldehyde leads to Tyr phosphorylation of occludin; the tyrosine kinase inhibitors attenuate the disruption of TJs (39, 40). Furthermore, a previous in vitro study demonstrated that Tyr phosphorylation of the C-terminal domain of occludin leads to the loss of its interaction with ZO-1 and ZO-3 (25).In the present study we identified the Tyr residues in occludin that are phosphorylated by c-Src and determined their role in regulated interaction between occludin and ZO-1 and its assembly into the TJs. Results show that 1) Tyr-379 and Tyr-383 in chicken occludin and Tyr-398 and Tyr-402 in human occludin are the exclusive sites of phosphorylation by c-Src, and these Tyr residues are located in a highly conserved sequence of occludin, YET-DYTT, 2) deletion of YEDTYTT or point mutation of Tyr-398 and Tyr-402 in human occludin attenuates the phosphorylation-dependent regulation of ZO-1 binding, 3) Y398D/Y402D mutation of human occludin leads to loss of ZO-1 binding and prevents its translocation to the plasma membrane and cell-cell contact sites in Rat-1 cells, 4) Y398D/Y402D mutation of occludin delays its assembly into the intercellular junctions during the calcium-induced assembly of TJs, and 5) expression of Y398D/Y402D mutant occludin sensitizes cell monolayers for hydrogen peroxide-induced disruption of barrier function.