Initial Contact of Glioblastoma Cells with Existing Normal Brain Endothelial Cells Strengthen the Barrier Function via Fibroblast Growth Factor 2 Secretion: A New In Vitro Blood–Brain Barrier Model

Glioblastoma multiforme (GBM) cells invade along the existing normal capillaries in brain. Normal capillary endothelial cells function as the blood–brain barrier (BBB) that limits permeability of chemicals into the brain. To investigate whether GBM cells modulate the BBB function of normal endothelial cells, we developed a new in vitro BBB model with primary cultures of rat brain endothelial cells (RBECs), pericytes, and astrocytes. Cells were plated on a membrane with 8 μm pores, either as a monolayer or as a BBB model with triple layer culture. The BBB model consisted of RBEC on the luminal side as a bottom, and pericytes and astrocytes on the abluminal side as a top of the chamber. Human GBM cell line, LN-18 cells, or lung cancer cell line, NCI-H1299 cells, placed on either the RBEC monolayer or the BBB model increased the transendothelial electrical resistance (TEER) values against the model, which peaked within 72 h after the tumor cell application. The TEER value gradually returned to baseline with LN-18 cells, whereas the value quickly dropped to the baseline in 24 h with NCI-H1299 cells. NCI-H1299 cells invaded into the RBEC layer through the membrane, but LN-18 cells did not. Fibroblast growth factor 2 (FGF-2) strengthens the endothelial cell BBB function by increased occludin and ZO-1 expression. In our model, LN-18 and NCI-H1299 cells secreted FGF-2, and a neutralization antibody to FGF-2 inhibited LN-18 cells enhanced BBB function. These results suggest that FGF-2 would be a novel therapeutic target for GBM in the perivascular invasive front.     

Substrate‐shielding and hydrolytic reaction in hydrolases

In general, transferases undergo large structural changes and sequester substrate molecules, to shield them from water. By contrast, hydrolases exhibit only small structural changes, and expose substrate molecules to water. However, some hydrolases deeply bury their substrates within the proteins. To clarify the relationship between substrate‐shielding and enzymatic functions, we investigated 70 representative hydrolase structures, and examined the relative accessible surface areas of their substrates. As compared to the hydrolases employing the single displacement reaction, the hydrolases employing the double displacement reaction bury the substrate within the proteins. The exo hydrolases display significantly more substrate‐shielding from water than the endo hydrolases. It suggests that the substrate‐shielding is related to the chemical reaction mechanism of the hydrolases and the substrate specificity. Proteins 2013; © 2012 Wiley Periodicals, Inc.     

Identification of a Novel Subgroup of Koala Retrovirus from Koalas in Japanese Zoos

We identified a new subgroup of koala retrovirus (KoRV), named KoRV-J, which utilizes thiamine transport protein 1 as a receptor instead of the Pit-1 receptor used by KoRV (KoRV-A). By subgroup-specific PCR, KoRV-J and KoRV-A were detected in 67.5 and 100% of koalas originating from koalas from northern Australia, respectively. Altogether, our results indicate that the invasion of the koala population by KoRV-J may have occurred more recently than invasion by KoRV-A.     

The First Identification of Carbohydrate Binding Modules Specific to Chitosan

Two carbohydrate binding modules (DD1 and DD2) belonging to CBM32 are located at the C terminus of a chitosanase from Paenibacillus sp. IK-5. We produced three proteins, DD1, DD2, and tandem DD1/DD2 (DD1+DD2), and characterized their binding ability. Transition temperature of thermal unfolding (T_m) of each protein was elevated by the addition of cello-, laminari-, chitin-, or chitosan-hexamer (GlcN)₆. The T_m elevation (ΔT_m) in DD1 was the highest (10.3 °C) upon the addition of (GlcN)₆ and was markedly higher than that in DD2 (1.0 °C). A synergistic effect was observed (ΔT_m = 13.6 °C), when (GlcN)₆ was added to DD1+DD2. From isothermal titration calorimetry experiments, affinities to DD1 were not clearly dependent upon chain length of (GlcN)_n; ΔG_r° values were −7.8 (n = 6), −7.6 (n = 5), −7.6 (n = 4), −7.6 (n = 3), and −7.1 (n = 2) kcal/mol, and the value was not obtained for GlcN due to the lowest affinity. DD2 bound (GlcN)_n with the lower affinities (ΔG_r° = −5.0 (n = 3) ∼ −5.2 (n = 6) kcal/mol). Isothermal titration calorimetry profiles obtained for DD1+DD2 exhibited a better fit when the two-site model was used for analysis and provided greater affinities to (GlcN)₆ for individual DD1 and DD2 sites (ΔG_r° = −8.6 and −6.4 kcal/mol, respectively). From NMR titration experiments, (GlcN)_n (n = 2∼6) were found to bind to loops extruded from the core β-sandwich of individual DD1 and DD2, and the interaction sites were similar to each other. Taken together, DD1+DD2 is specific to chitosan, and individual modules synergistically interact with at least two GlcN units, facilitating chitosan hydrolysis.     

Distinct tRNA modifications in the thermo-acidophilic archaeon, Thermoplasma acidophilum

Thermoplasma acidophilum is a thermo-acidophilic archaeon. We purified tRNA^Leu (UAG) from T. acidophilum using a solid-phase DNA probe method and determined the RNA sequence after determining via nucleoside analysis and m⁷G-specific aniline cleavage because it has been reported that T. acidophilum tRNA contains m⁷G, which is generally not found in archaeal tRNAs. RNA sequencing and liquid chromatography–mass spectrometry revealed that the m⁷G modification exists at a novel position 49. Furthermore, we found several distinct modifications, which have not previously been found in archaeal tRNA, such as 4-thiouridine9, archaeosine13 and 5-carbamoylmethyuridine34. The related tRNA modification enzymes and their genes are discussed.     

Flagella proteins contribute to the production of outer membrane vesicles from Escherichia coli W3110

Gram-negative bacteria, including Escherichia coli, release outer membrane vesicles (OMVs) that are derived from the bacterial outer membrane. OMVs contribute to bacterial cell–cell communications and host–microbe interactions by delivering components to locations outside the bacterial cell. In order to explore the molecular machinery involved in OMV biogenesis, the role of a major OMV protein was examined in the production of OMVs from E. coli W3110, which is a widely used standard E. coli K-12 strain. In addition to OmpC and OmpA, which are used as marker proteins for OMVs, an analysis of E. coli W3110 OMVs revealed that they also contain abundant levels of FliC, which is also known as flagellin. A membrane-impermeable biotin-labeling reagent did not label FliC in intact OMVs, but labeled FliC in sonically disrupted OMVs, suggesting that FliC is localized in the lumen of OMV. Compared to the parental strain expressing wild-type fliC, an E. coli strain with a fliC-null mutation produced reduced amounts of OMVs based on both protein and phosphate levels. In addition, an E. coli W3110-derived strain with a null-mutation in flgK, which encodes flagellar hook-associated protein that is essential along with FliC for flagella synthesis, also produced fewer OMVs than the parental strain. Taken together, these results indicate that the ability to form flagella, including the synthesis of flagella proteins, affects the production of E. coli W3110 OMVs.     

Identification of a novel benzimidazole derivative as a highly potent NPY Y5 receptor antagonist with an anti-obesity profile

Optimization of HTS hit 1 for NPY Y5 receptor binding affinity, CYP450 inhibition, solubility and metabolic stability led to the identification of some orally available oxygen-linker derivatives for in vivo study. Among them, derivative 4i inhibited food intake induced by the NPY Y5 selective agonist, and chronic oral administration of 4i in DIO mice caused a dose-dependent reduction of body weight gain.