Influence of maceration temperature in red wine vinification on extraction of phenolics from berry skins and seeds of grape (Vitis vinifera)

The extraction of phenolics from berry skins and seeds of the grape, Vitis vinifera cv. Cabernet Sauvignon, during red wine maceration and the influence of different temperature conditions (cold soak and/or heating at the end of maceration) were examined. Phenolics contained mainly in berry skins, viz., anthocyanin, flavonol, and epigallocatechin units within proanthocyanidins, were extracted during the early stage of maceration, whereas those in seeds, viz., gallic acid, flavan-3-ol monomers, and epicatechin-gallate units within proanthocyanidins, were gradually extracted. In addition to their localization, the molecular size and composition of the proanthocyanidins possibly influenced the kinetics of their extraction. Cold soak reduced the extraction of phenolics from the seeds. Heating at the end of maceration decreased the concentration of proanthocyanidins. Thus, modification of the temperature condition during maceration affected the progress of the concentration of phenolics, resulting in an alteration of their make-up in the finished wine.     

The circadian clock modulates water dynamics and aquaporin expression in Arabidopsis roots

We have developed a plant growth system to analyze water dynamics in the roots of a small model plant, Arabidopsis thaliana, by nuclear magnetic resonance (NMR) microscopic imaging. Using the two-dimensional slice technique, we obtained a series of images with high signal-to-noise ratio indicating the water distribution in the root. To demonstrate light regulation of water transport in the root and involvement of aquaporin gene expression, we visualized the distribution of water in Arabidopsis roots under various light conditions and compared the data with the expression profiles of two aquaporin genes. (1)H-NMR imaging revealed that water content in Arabidopsis roots is lower in the light than in the dark. This diurnal variation in water content was clearly observed in the basal zone of the root. In addition, an autonomous rhythm of water dynamics was observed under continuous light (LL) and darkness (DD). However, the circadian oscillation in water dynamics was obscured in the early-flowering 3 (elf3) mutant under LL. The expression of both the aquaporin genes, AtPIP1;2 and AtPIP2;1, oscillated with the circadian rhythm under LL conditions in wild-type seedlings, but not in the elf3 mutant. These results demonstrate the advantages of our technique for monitoring water dynamics in roots of living Arabidopsis seedlings, and suggest that the circadian clock modulates water dynamics and aquaporin expression.     

Endogenous synthesis of corticosteroids in the hippocampus

Background

Brain synthesis of steroids including sex-steroids is attracting much attention. The endogenous synthesis of corticosteroids in the hippocampus, however, has been doubted because of the inability to detect deoxycorticosterone (DOC) synthase, cytochrome P450(c21).

Methodology/Principal Findings

The expression of P450(c21) was demonstrated using mRNA analysis and immmunogold electron microscopic analysis in the adult male rat hippocampus. DOC production from progesterone (PROG) was demonstrated by metabolism analysis of ³H-steroids. All the enzymes required for corticosteroid synthesis including P450(c21), P450(2D4), P450(11β1) and 3β-hydroxysteroid dehydrogenase (3β-HSD) were localized in the hippocampal principal neurons as shown via in situ hybridization and immunoelectron microscopic analysis. Accurate corticosteroid concentrations in rat hippocampus were determined by liquid chromatography-tandem mass spectrometry. In adrenalectomized rats, net hippocampus-synthesized corticosterone (CORT) and DOC were determined to 6.9 and 5.8 nM, respectively. Enhanced spinogenesis was observed in the hippocampus following application of low nanomolar (10 nM) doses of CORT for 1 h.

Conclusions/Significance

These results imply the complete pathway of corticosteroid synthesis of ‘pregnenolone →PROG→DOC→CORT’ in the hippocampal neurons. Both P450(c21) and P450(2D4) can catalyze conversion of PROG to DOC. The low nanomolar level of CORT synthesized in hippocampal neurons may play a role in modulation of synaptic plasticity, in contrast to the stress effects by micromolar CORT from adrenal glands.     

Effects of Various Growth Conditions in a Chemostat on Expression of Virulence Factors in Porphyromonas gingivalis

Porphyromonas gingivalis, one of the gram-negative organisms associated with periodontal disease, possesses potential virulence factors, including fimbriae, proteases, and major outer membrane proteins (OMPs). In this study, P. gingivalis ATCC 33277 was cultured in a chemostat under hemin excess and presumably peptide-limiting conditions to better understand the mechanisms of expression of the virulence factors upon environmental changes. At higher growth rates, the amounts of FimA and the 75-kDa protein, forming long and short fimbriae, respectively, increased significantly, whereas gingipains decreased in amount and activity. In a nutrient-limited medium, lesser amounts of the above two fimbrial proteins were observed, whereas clear differences were not found in the amounts of gingipains. In addition, two-dimensional electrophoresis revealed that proteins in cells were generally fewer in number during nutrient-limited growth. Under aeration, a considerable reduction in gingipain activity was found, whereas several proteins associated with intact cells significantly increased. However, the expression of major OMPs, such as RagA, RagB, and the OmpA-like proteins, was almost constant under all conditions tested. These results suggest that P. gingivalis may actively control expression of several virulence factors to survive in the widely fluctuating oral environment.     

Comparison of cell lines for stable production of fucose-negative antibodies with enhanced ADCC

Several methods have been described to enhance antibody-dependent cellular cytotoxicity (ADCC) using different host cells that produce antibody with reduced levels of fucose on their carbohydrates. We compared the suitability of these methods for the serum-free fed-batch production of antibody for clinical trials and commercial uses. Recombinant anti-human CD20 chimeric IgG1-producing clones were established from host-cells that have been shown to produce more than 90% fucose-negative antibody. The cell lines were a FUT8 (alpha-1,6-fucosyltransferase) knockout Chinese hamster ovary (CHO) cell line, Ms704, and two Lens culinaris agglutinin (LCA)-resistant cell lines, one derived from a variant CHO line, Lec13 and the other from a rat hybridoma cell line, YB2/0. The amount of fucose-negative antibody produced by Lec13 and YB2/0 significantly decreased with the culture. The increase in fucosylation was due to remaining synthesis of GDP-fucose via de novo pathway for the CHO line and the elevation of FUT8 expression by the YB2/0 cells. In contrast, Ms704 cells stably produced fucose-negative antibody with a consistent carbohydrate structure until the end of the culture. The productivity of the Ms704 cells reached 1.76 g/L with a specific production rate (SPR) of 29 pg/cell/day for 17 days in serum-free fed-batch culture using a 1 L spinner bioreactor. Our results demonstrate that FUT8 knockout has the essential characteristics of host cells for robust manufacture of fucose-negative therapeutic antibodies with enhanced ADCC.     

Effects of various growth conditions in a chemostat on expression of virulence factors in Porphyromonas gingivalis

Porphyromonas gingivalis, one of the gram-negative organisms associated with periodontal disease, possesses potential virulence factors, including fimbriae, proteases, and major outer membrane proteins (OMPs). In this study, P. gingivalis ATCC 33277 was cultured in a chemostat under hemin excess and presumably peptide-limiting conditions to better understand the mechanisms of expression of the virulence factors upon environmental changes. At higher growth rates, the amounts of FimA and the 75-kDa protein, forming long and short fimbriae, respectively, increased significantly, whereas gingipains decreased in amount and activity. In a nutrient-limited medium, lesser amounts of the above two fimbrial proteins were observed, whereas clear differences were not found in the amounts of gingipains. In addition, two-dimensional electrophoresis revealed that proteins in cells were generally fewer in number during nutrient-limited growth. Under aeration, a considerable reduction in gingipain activity was found, whereas several proteins associated with intact cells significantly increased. However, the expression of major OMPs, such as RagA, RagB, and the OmpA-like proteins, was almost constant under all conditions tested. These results suggest that P. gingivalis may actively control expression of several virulence factors to survive in the widely fluctuating oral environment.     

Molecular cloning and sequence analysis of full-length cDNA for rabbit liver NADPH-cytochrome P-450 reductase mRNA

The nucleotide sequence of the mRNA for NADPH-cytochrome P-450 reductase from rabbit liver was determined from a full-length cDNA clone (pFP105). The clone contains 2,269 nucleotides complementary to rabbit liver reductase mRNA. The single open reading frame of 2,037 nucleotides codes for a 679-amino acid polypeptide with a calculated molecular weight of 76,583 daltons. The cloned cDNA contains the complete 3'-noncoding region of 193 nucleotides, including 68 nucleotides of poly(A), and 39 nucleotides of the 5'-noncoding region. The nucleotide sequence in the coding region of cDNA of rabbit reductase (pFP105) showed 85% homology to that of rat reductase (Porter, T.D. & Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U.S. 82, 973-977, and Murakami, H. et al. (1986) DNA 5, 1-10). Rabbit reductase has one more amino acid residue than the rat enzyme, and the amino acid compositions of the two enzymes are similar. The amino acid sequence of the rabbit enzyme showed 91% identity with that of the rat enzyme. The segment related to binding of FMN and FAD was well conserved among rabbit, rat, and pig reductases. The sequence related to AMP moiety-binding was also conserved among these species, and was found in the amino acid sequence of NADH-cytochrome b5 reductase, another flavoenzyme in the microsomal electron transport system.     

Both low and high concentrations of staurosporine induce G1 arrest through down-regulation of cyclin E and cdk2 expression

Staurosporine has been reported to cause arrest of cells in G1 phase at low concentration and in G2 phase at high concentration. This raises the question of why the effects of staurosporine on the cell cycle depend on the applied concentration. In order to verify these multiple functions of staurosporine in Meth-A cells, we used cyclin E as a landmark of G1/S transition, cyclin B as a landmark of G2/M transition and MPM2 as a hallmark of M phase. We found that staurosporine arrested cells in G1 phase at a low concentration (20 nM) and in G2/M phase at a high concentration (200 nM). However, 200 nM staurosporine increased the expression of cyclin B and cdc2 proteins, suggesting that the cells progressed through the G2/M transition, and increased the expression of MPM2 protein, indicating that the cells entered M phase. Moreover, 200 nM staurosporine increased the expression of p53 and p21 proteins and inhibited the expression of cyclin E and cdk2 proteins, suggesting that the cells were arrested in the G1 phase of the next cycle. Morphological observation showed similar results as well. These data suggest that the G2/M accumulation induced by 200 nM staurosporine does not reflect G2 arrest, but rather results from M phase arrest, followed by progression from M phase to the G1 phase of the next cycle without cytokinesis, and finally arrest of the cells in G1 phase.     

Silymarin attenuated the amyloid β plaque burden and improved behavioral abnormalities in an Alzheimer's disease mouse model

Alzheimer's disease (AD) is characterized by progressive cognitive impairment and the formation of senile plaques. Silymarin, an extract of milk thistle, has long been used as a medicinal herb for liver diseases. Here we report marked suppression of amyloid β-protein (Aβ) fibril formation and neurotoxicity in PC12 cells after silymarin treatment in vitro. In vivo studies had indicated a significant reduction in brain Aβ deposition and improvement in behavioral abnormalities in amyloid precursor protein (APP) transgenic mice that had been preventively treated with a powdered diet containing 0.1% silymarin for 6 months. The silymarin-treated APP mice also showed less anxiety than the vehicle-treated APP mice. These behavioral changes were associated with a decline in Aβ oligomer production induced by silymarin intake. These results suggest that silymarin is a promising agent for the prevention of AD.