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.     

Impact of H216 on the DNA Binding and Catalytic Activities of the HIV Restriction Factor APOBEC3G

APOBEC3G has an important role in human defense against retroviral pathogens, including HIV-1. Its single-stranded DNA cytosine deaminase activity, located in its C-terminal domain (A3Gctd), can mutate viral cDNA and restrict infectivity. We used time-resolved nuclear magnetic resonance (NMR) spectroscopy to determine kinetic parameters of A3Gctd''s deamination reactions within a 5′-CCC hot spot sequence. A3Gctd exhibited a 45-fold preference for 5′-CCC substrate over 5′-CCU substrate, which explains why A3G displays almost no processivity within a 5′-CCC motif. In addition, A3Gctd''s shortest substrate sequence was found to be a pentanucleotide containing 5′-CCC flanked on both sides by a single nucleotide. A3Gctd as well as full-length A3G showed peak deamination velocities at pH 5.5. We found that H216 is responsible for this pH dependence, suggesting that protonation of H216 could play a key role in substrate binding. Protonation of H216 appeared important for HIV-1 restriction activity as well, since substitutions of H216 resulted in lower restriction in vivo.     

Effects of Detergents on Lysis of Micrococcus lysodeikticus by Lysozyme

The effects of detergents on the lysozyme-catalyzed hydrolysis of Micrococcus lysodeikticus cells were investigated by changing the concentration of Na-phosphate buffer and pH in the presence or absence of sucrose. Also, a parallel study of the hydrolysis of glycolchitin by lysozyme was conducted and compared to the lytic reaction. Electron microscopy was utilized to follow the changes in cell morphology during the various treatments.

None of the detergents changed turbidity of the cell suspension. However, they did affect the change in turbidity during lysis in unique ways. SDS, which is an anionic detergent, inhibited lysozyme activity and its addition to the reaction mixture caused a rapid and large decrease in the turbidity. Brij 35 and Triton X-100, which are non-ionic detergents, did not inhibit lysozyme activity, but their presence in the reaction mixture changed the rate of turbidity change. Apparently non-ionic detergents disrupt only the protoplast, while anionic detergents disrupt both the protoplast and the damaged cell. The lytic mechanism of M. lysodeikticus by lysozyme was discussed in detail.     

The Biosynthetic Pathway of (Z)-11-Hexadecenal,the Sex Pheromone Component of the Rice Stem Borer,Chilo sappressalis WALKER (Lepidoptera: Pyralidae)

Lipoxygenase-3, the major component of the enzyme in rice grain, was purified 2980-fold with a yield of 7% from embryos. The purified enzyme had a specific activity of 280 μmol O₂ formed/min per mg protein. This enzyme was inactivated by SH compounds, such as cysteine and glutathione. The inactivation was prevented by the addition of catalase or replacement of the air by N₂ gas. These two treatments were also effective for the stable storage of the purified enzyme. The molecular weights measured by sodium dodecyl sulfate gel and gradient gel electrophoresis were 93,000 and 89,000, respectively, indicating that the enzyme is a single polypeptide chain. The purified enzyme contained 0.73 Fe atom per molecule. The absorption spectrum suggested that the enzyme is a non-heme iron protein. Some similarities in amino-acid composition were observed between rice, soybean, and pea lipoxygenases. The purified enzyme specifically produced 9-d-hydroperoxy-10,12(E,Z)-octadecadienoic acid when linoleic acid was used as a substrate.     

Chitin Coated Cellulose as an Adsorbent of lysozyme like Enzymes Preparation and Properties

As a new adsorbent of lysozyme-like enzymes, chitin coated (CC-)cellulose was prepared. CC-cellulose was stable and had good flow properties for use in column chromatography. Affinity chromatography with CC-cellulose showed that 3~5 mg of lysozyme/ml resin was adsorbed specifically and desorbed quantitatively under mild conditions. The utilities of the method of affinity chromatography with CC-cellulose are discussed.     

Preparation of Optically Active Secondary Alcohols Related to Synthetic Pyrethroids by Microbial Asymmetric Hydrolysis of the Corresponding Acetates

Asymmetric hydrolysis of the acetates of racemic secondary alcohols related to synthetic pyrethroids by Bacillus subtilis var. niger (IFO 3108) yielded optically active acetates and alcohols of varying optical purities.     

Method for Determination and Productivity of Sclerothionine in Sclerotinia Fungi

For determining sclerothionine (STH, S-2-hydroxyethylergothioneine), the method of P. C. Jocelyn which determined ergothioneine in blood was applied, and it was shown that STH was necessary to be degraded with 70% KOH for 1 hr at 100°C, in order to get a theoretical amount of trimethylamine. Trimethylamine produced was trapped by picric acid and spectrophotometrically measured as picrate at 410 mμ. By this method and using paper chromatography, STH in Sclerotinia culture could be determined successfully, and it was found that, among Sclerotinia fungi, a strain of Sclerotinia libertiana which can form sclerotium normally only produced STH, and other various strains of the same genus produced ergothioneine. The cultural condition for production of STH by the Sclerotinia libertiana strain was investigated. As a result, in the shaking liquid culture containing wheat bran, 1.0%; glucose, 1.0%; Polypepton, 0.6%; KH₂PO₄, 0.05%; MgCl₂, 0.05% and cystine-HCl, 0.003% as nutrient, the addition of methionine at a later period in about 0.01% concentration was found to stimulate the accumulation of STH in mycelium.