Isolation of Cerebroside from Pea Seeds

Cerebroside was isolated from pea (Pisum sativum L.) seeds by solvent extraction, mild alkaline hydrolysis and silicic acid column chromatography. The purified material was identified as cerebroside by thin-layer chromatography and infrared spectrometry. Hydrolysates of the cerebroside were divided into fatty acid, sphingosine base and sugar fractions, and analysed, mainly by gas-liquid chromatography. The major fatty acid components were hydroxytricosanoic, hydroxydocosanoic and hydroxytetracosanoic acids. Dihydrosphingosine was the predominant sphingosine base. Only glucose was detected in the sugar fraction. Based on these results, one of the major species of pea cerebroside is suggested to be N-hydroxytricosanoyl-glucopyranosyl-dihydrosphingosine.     

Novel methods for nucleotide length control in double-stranded polyinosinic-polycytidylic acid production using uneven length components

ABSTRACT

Polyinosinic-polycytidylic acid (PIC), a double-stranded RNA that induces innate immunity in mammals, is a candidate immunopotentiator for pharmaceuticals. The potency and adverse effects of PIC are strongly correlated with the nucleotide length, and the inability to precisely control the length in PIC production limits its practical use. Length extension during the annealing process is the major factor underlying the lack of control, but tuning the annealing conditions is insufficient to resolve this issue. In this study, we developed a novel method to produce accurate nucleotide length PIC at an industrial scale. The length extension was significantly suppressed by the assembly of multiple short polyinosinic acid molecules with one long polycytidylic acid molecule. A newly developed PIC, uPIC100-400, demonstrated a reproducible length and better storage stability than that of corresponding evenly structured PIC. Human dsRNA receptors exhibited equivalent responsiveness to uPIC100-400 and the evenly structured PIC with the same length.     

Rapid Fabricating Technique for Multi-Layered Human Hepatic Cell Sheets by Forceful Contraction of the Fibroblast Monolayer

Cell sheet engineering is attracting attention from investigators in various fields, from basic research scientists to clinicians focused on regenerative medicine. However, hepatocytes have a limited proliferation potential in vitro, and it generally takes a several days to form a sheet morphology and multi-layered sheets. We herein report our rapid and efficient technique for generating multi-layered human hepatic cell (HepaRG® cell) sheets using pre-cultured fibroblast monolayers derived from human skin (TIG-118 cells) as a feeder layer on a temperature-responsive culture dish. Multi-layered TIG-118/HepaRG cell sheets with a thick morphology were harvested on day 4 of culturing HepaRG cells by forceful contraction of the TIG-118 cells, and the resulting sheet could be easily handled. In addition, the human albumin and alpha 1-antitrypsin synthesis activities of TIG-118/HepaRG cells were approximately 1.2 and 1.3 times higher than those of HepaRG cells, respectively. Therefore, this technique is considered to be a promising modality for rapidly fabricating multi-layered human hepatocyte sheets from cells with limited proliferation potential, and the engineered cell sheet could be used for cell transplantation with highly specific functions.     

Analysis of internal structure changes in black human hair keratin fibers with aging using Raman spectroscopy

To investigate the internal structure changes in virgin black human hair keratin fibers due to aging, the structure of cross-sections at various depths of virgin black human hair (sections of new growth hair: 2 mm from the scalp) from a group of eight Japanese females in their twenties and another group of eight Japanese females in their fifties were analyzed using Raman spectroscopy. For the first time, we have succeeded in recording the Raman spectra of virgin black human hair, which had been impossible due to high melanin granule content. The key points of this method are to cross-section hair samples to a thickness of 1.50-microm, to select points at various depths of the cortex with the fewest possible melanin granules, and to optimize laser power, cross slit width as well as total acquisition time. The reproducibility of the Raman bands, namely the alpha-helix (alpha) content, the beta-sheet and/or random coil (beta/R) content, the disulfide (--SS--) content, and random coil content of two adjoining cross-sections of a single hair keratin fiber was clearly good. The --SS-- content of virgin black human hair from the Japanese females in their fifties for the cortex region decreased compared with that of the Japanese females in their twenties. On the other hand, the beta/R and alpha contents of the cortex region did not change.     

Efficient release of overproduced gene products from Escherichia coli BL21(DE3) by lytic infection with newly isolated bacteriophages

Overproduced proteins from Escherichia coli BL21(DE3) were efficiently released with virulent bacteriophages. Leviviridae-like bacteriophages were isolated from soil and used to lyse BL21(DE3) cells transformed with beta-glucosidase, chitinase, or chitosanase genes. This method caused lysis of bacterial cells similar to that by conventional sonication and enabled us to effectively recover and purify the enzymes.     

Synthesis of a callosic substance during rhizoid differentiation in Spirogyra

Spirogyra living in running water is anchored to the substratum by rhizoids that form at the ends of the filaments. A new terminal cell differentiates into a rhizoid cell if the filament is injured. The mode of growth changes from diffuse to tip growth when rhizoid differentiation begins. In this study, we found that a callosic substance was synthesized during rhizoid differentiation. Decreasing the cell turgor, lowering extracellular Ca2+ or adding Gd3+, all inhibited the commencement of rhizoid differentiation as well as synthesis of the callose-like substance at the tip of the terminal cell. A callosic substance was also synthesized during formation of the conjugation tube.     

Upregulation of uncoupling proteins by oral administration of capsiate, a nonpungent capsaicin analog.

Capsiate is a nonpungent capsaicin analog, a recently identified principle of the nonpungent red pepper cultivar CH-19 Sweet. In the present study, we report that 2-wk treatment of capsiate increased metabolic rate and promoted fat oxidation at rest, suggesting that capsiate may prevent obesity. To explain these effects, at least in part, we examined uncoupling proteins (UCPs) and thyroid hormones. UCPs and thyroid hormones play important roles in energy expenditure, the maintenance of body weight, and thermoregulation. Two-week treatment of capsiate increased the levels of UCP1 protein and mRNA in brown adipose tissue and UCP2 mRNA in white adipose tissue. This dose of capsiate did not change serum triiodothyronine or thyroxine levels. A single dose of capsiate temporarily raised both UCP1 mRNA in brown adipose tissue and UCP3 mRNA in skeletal muscle. These results suggest that UCP1 and UCP2 may contribute to the promotion of energy metabolism by capsiate, but that thyroid hormones do not.     

Hygrolidin induces p21 expression and abrogates cell cycle progression at G1 and S phases

Hygrolidin family antibiotics showed selective cytotoxicity against both cyclin E- and cyclin A-overexpressing cells. Among them, hygrolidin was the most potent and inhibited growth of solid tumor-derived cell lines such as DLD-1 human colon cancer cells efficiently more than that of hematopoietic tumor cells and normal fibroblasts. FACS analysis revealed that hygrolidin increased cells in G1 and S phases in DLD-1 cells. While hygrolidin decreased amounts of cyclin-dependent kinase (cdk) 4, cyclin D, and cyclin B, it increased cyclin E and p21 levels. Hygrolidin-induced p21 bound to and inhibit cyclin A-cdk2 complex more strongly than cyclin E-cdk2 complex. Furthermore, hygrolidin was found to increase p21 mRNA in DLD-1 cells, but not in normal fibroblasts. Thus, hygrolidin inhibited tumor cell growth through induction of p21. In respect to p21 induction, inhibition of vacuolar-type (H+)-ATPase by hygrolidin was suggested to be involved.     

Augmentation of Interferon Production after Cell-Differentiation of U937 Cells by TPA

Persistent infections with mumps virus were established in several human lymphoid cells of T-cell origin (Molt-4, TALL-1, and CCRF-CEM) and human monocyte cells (U937 and THP-1). 2′,5′-Oligoadenylate synthetase (2–5AS) activity was demonstrated to be only slightly induced by interferon (IFN) or TPA (12-O-tetradecanoyl-phorbol-13-acetate) treatment in these cells. Treatment of the persistently infected cells with IFN or TPA did not stimulate an increase in the amount of synthetase mRNA. Induction of cell differentiation and augmentation of IFN production by TPA were demonstrated in U937 cells persistently infected with mumps virus (U937-MP). Similar results for IFN production were obtained from differentiated U937 cells. It is suggested that cell differentiation of U937 cells might be associated with the development of IFN inducibility.