Liposome Vector Containing Biosurfactant-Complexed DNA as Herpes Simplex Virus Thymidine Kinase Gene Delivery System

For injectable-sized liposome complexed with DNA (lipoplexes) with high transfection efficiency of genes, we initially prepared small-sized liposomes by addition of biosurfactant. For selectivity of gene expression, the thymidine kinase (MK-tk) gene controlled by midkine was used for herpes simplex virus thymidine kinase (HSV-tk) gene therapy. Liposomes composed of 3([N-(N′,N′–dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), L-dioleoylphosphatidylethanolamine (DOPE), and a biosurfactant, such as β-sitosterol β-D-glucoside (Sit-G) for Sit-G-liposomes and mannosylerythrytol lipid A (MEL) for MEL-liposomes, produced about 300-nm-sized lipoplexes. Sit-G- and MEL-liposomes showed higher transfection efficiency of the luciferase marker gene and thymidine kinase activity in the presence of serum in the cells. The treatment with transfection of MK-tk gene by Sit-G-liposome and injection of ganciclovir significantly reduced tumor growth in a solid tumor model, compared with that by Sit-G-liposome alone. This finding suggested that Sit-G-liposome is a potential vector for HSV-tk gene therapy.     

Inhibition of HIV-1 replication by a tricyclic coumarin GUT-70 in acutely and chronically infected cells

The anti-HIV-1 activity of GUT-70, a natural product derived from the stem bark of Chlophyllum brasiliense, was evaluated. GUT-70 inhibited HIV-1 replication in both acutely and chronically infected cells through suppression of NF-κB. Our results strengthen the idea that NF-κB pathway is one of the potential targets to control HIV-1 replication and that GUT-70 could serve as a lead compound to develop novel therapeutic agents against HIV-1 infection.     

Conformational restriction approach to BACE1 inhibitors II: SAR study of the isocytosine derivatives fixed with a cis-cyclopropane ring

To improve the efficacy of the conformationally restricted BACE1 inhibitors, structural modifications were investigated using two strategies: (a) modification of the terminal aromatic ring and (b) insertion of a spacer between the aromatic rings. In the latter approach, another type of inhibitor 17 bearing an ethylene spacer between two aromatic rings was found to exhibit good BACE1 inhibitory activity, while the corresponding conformationally unrestricted compound 25 showed no activity. This result revealed an interesting effect of a conformational restriction with a cyclopropane ring.     

Studies on Amylolytic Enzymes Produced by Endornyces sp

Various saccharides were hydrolyzed with the purified amyloglucosidase of Endornyces sp. IFO 0111.

Glucose was the only reducing product in the digest of soluble starch. The amyloglucosidase could hydrolyze starch and amylose only incompletely though it had the ability to split α-d-(1→6) bonds and hydrolyzed amylopectin and glycogen to high extents.

It hydrolyzed maito-oligosaccharides by stepwise removal of glucose units from the nonreducing end of the molecules.     

Assay and Inhibitors of Spinach Superoxide Dismutase

Allosamidin, a product of Streptomyces sp. No 1713, inhibited Bombyx mori chitinase specifically in a competitive way with a Ki o f about 0.1 μm. The effect of allosamidin on chitinases from r Streptomyces griseus and Serratia marcescens was weaker, about 1/500 that on B. mori chitinase. Allosamidin did not inhibit yam chitinase, lysozymes of hen egg-white or human urine, or B. mori α-N-acetyl-d-glucosaminidase. The results suggest that allosamidin is a specific inhibitor of the insect chitinase.     

Microbial Production of d-Arabitol by n-Alkane-grown Candida tropicalis

In the course of study on citric acid fermentation by Candida tropicalis KY6224, in which n-alkane mixture (C–12 to C–15) was used as the sole source of carbon, we found that a arabitollike substance was accumulated when the medium-pH was controlled at low level (3.0 to 4.0). This substance was isolated in crystalline forms and identified as d-arabitol.

d-Arabitol production was also observed with ethanol, acetic acid and glucose as the sole source of carbon. Important factors for efficient production of d-arabitol were keeping the medium-pH at low-level (3.0 to 4.0) and the concentration of NaCl or KCl at high level (1 to 5%). This strain produced 75 mg/ml of d-arabitol in 120 hr incubation under optimal culture conditions; this corresponds to 50 % of n-alkane consumed.     

Isolation of a Novel Auxin,Methyl 4-Chloroindoleacetate from Immature Seeds of Pisum sativum

Human casein was separated by gel filtration on a column of Sephadex G–200 with 0.1 m Tris buffer (pH 8.5) containing 1.0 m NaCl. The effluent which increased in turbidity at 25°C was centrifuged at 25,000 × g for 30 min and the precipitate was obtained as Fraction 6. After centrifugation, the effluent was separated into 5 elution fractions.

Disc gel electrophoretic patterns of each fraction showed occurrence of secondary bands other than major bands especially in Fractions 3, 4 and 5. The casein solutions unheated and heated at 100°C for 5 and 10 min were kept at 5°C for 5 days. No marked changes of electrophoretic pattern were observed among these casein solutions. However, when a casein solution heated at 100°C for 5 min was chroma to graphed under the same condition, secondary bands also appeared.     

Overexpression of the NADP+-specific isocitrate dehydrogenase gene (icdA) in citric acid-producing Aspergillus niger WU-2223L

In the tricarboxylic acid (TCA) cycle, NADP⁺-specific isocitrate dehydrogenase (NADP⁺-ICDH) catalyzes oxidative decarboxylation of isocitric acid to form α-ketoglutaric acid with NADP⁺ as a cofactor. We constructed an NADP⁺-ICDH gene (icdA)-overexpressing strain (OPI-1) using Aspergillus niger WU-2223L as a host and examined the effects of increase in NADP⁺-ICDH activity on citric acid production. Under citric acid-producing conditions with glucose as the carbon source, the amounts of citric acid produced and glucose consumed by OPI-1 for the 12-d cultivation period decreased by 18.7 and 10.5%, respectively, compared with those by WU-2223L. These results indicate that the amount of citric acid produced by A. niger can be altered with the NADP⁺-ICDH activity. Therefore, NADP⁺-ICDH is an important regulator of citric acid production in the TCA cycle of A. niger. Thus, we propose that the icdA gene is a potentially valuable tool for modulating citric acid production by metabolic engineering.     

Production of Erythritol by n-Alkane-grown Yeasts

In the course of study on citric acid fermentation by Candida zeylanoides, in which n-alkane (a mixture of C–12 to C–15) was used as the sole source of carbon, we found that a polyol-like substance was accumulated when the medium-pH fell down to below 4.0. This was isolated in crystalline forms and identified as meso-erythritol. Comparing erythritol production among fifty yeast strains, Candida zeylanoides, particularly its glycerol-requiring mutant KY 6166, was found to be an excellent producer.

Erythritol production was also observed with ethanol or acetic acid as the sole carbon source but not with glucose. An efficient condition for large production of erythritol was to keep the medium-pH at low level (2.5 to 4.0) and the concentration of NaCl or KCl at high level (1 to 3%). Under conditions established in this work, more than 55 mg/ml of erythritol was successfully produced in 120 hr incubation in 300-ml flasks, which corresponded to 55% of the alkane used.     

Lactobacillus helveticus SBT2171 alleviates allergic symptoms in a murine model for pollen allergy

ABSTRACT

Lactic acid bacteria are known to have various health-promoting effects and are highly expected to find applications in anti-allergic food materials. In this study, we focused on Lactobacillus helveticus SBT2171 (LH2171), which reportedly modifies some unique immune responses and ameliorated symptoms of patients allergic to mites and house dust in the previous studies. We examined the effect of LH2171 on cytokine production by antigen-stimulated murine naïve splenocytes in vitro and demonstrated that it inhibited IL-4 and IL-13 production while enhancing IFN-γ and IL-10 production. Then, we examined the anti-allergic effect of LH2171 in vivo using a murine model of pollen allergy and found that LH2171 reduced the sneezing frequency when orally administered to mice. We successfully confirmed the immune modulatory activity of LH2171 and its anti-allergic activity against inhaled antigens. These evidences would contribute to identifying the anti-allergic mechanism of LH2171.

Abbreviations: ALDH: aldehyde dehydrogenase; EGCG: epigallocatechin gallate; LAB: lactic acid bacteria; LH2171: Lactobacillus helveticus SBT2171; NALT: nasal-associated lymphoid tissue; OVA: ovalbumin