Hepatoprotective effects of a concentrate and components of sake against galactosamine (GalN)-induced liver injury in mice

We investigated the hepatoprotective effects of a concentrate of sake (CS) and its components against D-galactosamine (GalN)-induced liver injury by measuring the plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in mice. CS significantly suppressed the GalN-induced elevation of ALT and AST activities. Each of four concentrated fractions extracted from sake (respectively consisting mainly of basic amino acids, neutral and acidic amino acids, organic acids and sugars) suppressed the GalN-induced elevation of ALT and AST activities. We focused on the sugar fraction containing glucose and ethyl alpha-D-glucoside (alpha-EG), which is a sake-specific sugar, as the major components and demonstrated that only alpha-EG showed significant suppression of the GalN-induced elevation of ALT and AST activities. We compared the effects of the alpha-EG analogues, methyl alpha-D-glucoside and ethyl beta-D-glucoside, on GalN-induced liver injury and confirmed that only alpha-EG significantly suppressed both the ALT and AST activities. Moreover, CS and alpha-EG suppressed the GalN-induced production of interleukin 6 (IL-6) and liver DNA fragmentation. Together these results show that CS and its component, alpha-EG, suppressed GalN-induced liver injury by inhibiting IL-6 production.     

Effect of ethyl alcohol on plasma testosterone level in mice

The effect of ethanol ingestion on testicular steroidogenesis in mice was evaluated by measuring plasma testosterone level. Four groups of CBA/J male mice were treated with one of the following doses of ethanol: 1.240, 0.620, 0.310 or 0.155 g ethanol/Kg body weight. A control group was given water. The data showed no effect of the treatment on testicular weight. The concentration of testosterone in the plasma was significantly reduced in animals treated with alcohol. There was also a significant relationship between the dose of alcohol and the plasma testosterone level, with the decrease in testosterone being from 2 to 18 fold in various groups.     

Ectopic expression of desmin in the epidermis of transgenic mice permits development of a normal epidermis

Cell architecture is largely based on the interaction of cytoskeletal proteins, which include intermediate filaments (IF), microfilaments, microtubules, as well as their type-specific membrane-attachment structures and associated proteins. In order to further our understanding of IF proteins and to address the fundamental issue whether different IF perform unique functions in different tissues, we expressed a desmin transgene in the basal epidermis of mice. Ectopic expression of desmin led to the formation of an additional, keratin-independent IF cytoskeleton and did not interfere with the keratin-desmosome interaction. We show that ectopic expression of a type III IF protein in basal keratinocytes did not interfere with the normal epidermal architecture and the program of terminal differentiation. This demonstrated that keratinocytes suffered no obvious detrimental effects from extra desmin filaments in their cytoplasm. In addition, we asked whether stable expression of desmin could rescue K5 null mice, which served as a model for severe EBS. Transgenic mice ectopically expressing desmin in the basal layer were mated with K5 heterozygous deficient animals to generate desmin rescue mice and analysed. In summary, our study support the notion that the different IF like desmin or keratins composing a IF network in vivo are central to cytoskeletal architecture and design in cells.     

Identification of a nuclear protein ArpN as a component of human SWI/SNF complex and its selective association with a subset of active genes

The hSWI/SNF complex remodels the chromatin structure to modulate gene expression. The hSWI/SNF complex is a multiprotein complex with at least 10 different proteins in mammals. In this study, we identified the 45 kDa subunit of the hSWI/SNF complex as ArpN, an actin-related protein. ArpN has a 36% identity and 50% similarity with the human beta-actin, but cannot be classified into any known class of actin-related proteins. ArpN is exclusively localized within the nucleus and appears as the unbound, chromatin-associated, or nuclear matrix associated forms in the nucleus. In the chromatin immunoprecipitation (ChIP) assay, we found the associations of ArpN with the Ets-2 and c-mycP2 promoter regions in HeLa cells. The promoter regions of the hsp70, cyclophilin, beta-globin, TdT, and cd4 genes, however, were not associated with ArpN. The Ets-2 and c-mycP2 genes are expressed actively in HeLa cells, but beta-globin, TdT, and cd4 genes are inactive. The hsp70 and cyclophilin genes have a feature of stress-inducibility. These selective associations of ArpN with a subset of active genes support the proposition that the requirement of hSWI/SNF complex in gene activation is gene specific.     

Principal component analysis of the relationship between the d-amino acid concentrations and the taste of the sake

We performed sensory evaluations on 141 bottles of sake and analyzed the relationship between the d-amino acid concentrations, and the taste of the sake using principal component analysis, which yielded seven principal components (PC1–7) that explained 100 % of the total variance in the data. PC1, which explains 33.6 % of the total variance, correlates most positively with strong taste and most negatively with balanced tastes. PC2, which explains 54.4 % of the total variance, correlates most positively with a sweet taste and most negatively with bitter and sour tastes. Sakes brewed with “Kimoto yeast starter” and “Yamahaimoto” had high scores for PC1 and PC2, and had strong taste in comparison with sakes brewed with “Sokujo-moto”. When present at concentrations below 50 μM, d-Ala did not affect the PC1 score, but all the sakes showed a high PC1 score, when the d-Ala was above 100 μM. Similar observations were found for the d-Asp and d-Glu concentrations with regard to PC1, and the threshold concentrations of d-Asp and d-Glu that affected the taste were 33.8 and 33.3 μM, respectively. Certain bacteria present in sake, especially lactic acid bacteria, produce d-Ala, d-Asp and d-Glu during storage, and these d-amino acids increased the PC1 score and produced a strong taste (Nojun). When d- and l-Ala were added to the sakes, the value for the umami taste in the sensory evaluation increased, with the effect of d-Ala being much stronger than that of l-Ala. The addition of 50–5,000 μM dl-Ala did not effect on the aroma of the sakes at all.     

Pharmacological investigations of the anti-diabetic effect of Cortex Moutan and its active component paeonol

Cortex Moutan (CM, root bark of Paeonia suffruticosa Andr.) is one of the common herbs found in anti-diabetic traditional Chinese medicine formulae. To study the potential anti-diabetic mechanisms of CM, four in vitro models (intestinal brush border membrane vesicles (BBMV), rat hepatoma cell line H4IIE, human skin fibroblasts cell line Hs68 and mouse adipocytes 3T3-L1) were used. CM showed significant in vitro anti-diabetic effects by inhibiting glucose uptake of BBMV and enhancing glucose uptake into Hs68 and 3T3-L1 cells. Using bioassay-guided fractionation, paeonol was confirmed to be one of the active constituents for inhibiting BBMV glucose uptake. With neonatal-streptozotocin diabetic rats, paeonol (200 and 400mg/kgbody wt.) was found to improve oral glucose tolerance in vivo. To the best of our knowledge, this is the first report on the anti-diabetic effect of paeonol.     

Red blood cell fatty acid ethyl esters: a significant component of fatty acid ethyl esters in the blood

Although alcohol abuse is known to cause an array of ethanol-induced red blood cell (RBC) abnormalities, the underlying molecular mechanisms remain poorly understood. Fatty acid ethyl esters (FAEEs) are toxic, nonoxidative ethanol metabolites that have been found in blood, plasma, and tissues. Because FAEEs have been shown to be incorporated into phospholipid bilayers, we conducted a controlled ethanol intake study to test the hypothesis that FAEEs accumulate and persist within RBCs following ethanol ingestion. We demonstrated that RBC FAEEs account for approximately 5% to 20% of total whole-blood FAEEs, and that the fatty acid composition of FAEEs in RBCs and plasma are different and vary differently over time. These data indicate that a significant percentage of FAEEs in the blood is associated with RBCs and that the metabolism of RBC FAEEs and that of plasma FAEEs (bound to albumin or lipoproteins) are largely independent.     

Self-assembly of a group I intron active site from its component tertiary structural domains.

The catalytic core of Group I self-splicing introns has been proposed to consist of two structural domains, P4-P6 and P3-P9. Each contains helical segments and conserved unpaired nucleotides, and the isolated P4-P6 domain has been shown to have substantial native tertiary structure. The proposed tertiary structure domains of the Tetrahymena intron were synthesized separately and shown to self-assemble into a catalytically active complex. Surprisingly, the concentration dependence of these reactions revealed that the domains interact with nanomolar apparent dissociation constants, even though there is no known base pairing between P4-P6 and P3-P9. This suggests that the domains interact through multiple tertiary contacts, the nature of which can now be explored in this system. For example, a circularly permuted version of the P4-P6 domain, which folds similarly to the native P4-P6 molecule, formed a stable but inactive complex. Interestingly, activity was demonstrated with the permuted molecule when nucleotides proposed to form a triple-strand interaction with P4 and P6 were restored as part of the P1-P3 substrate or as part of the P3-P9 RNA. Thus, beyond stabilization of the P4-P6 domain, the triple-strand region may facilitate correct orientation of the RNA domains or participate more directly in catalysis.     

Effect of L-proline on sake brewing and ethanol stress in Saccharomyces cerevisiae

During the fermentation of sake, cells of Saccharomyces cerevisiae are exposed to high concentrations of ethanol, thereby damaging the cell membrane and functional proteins. L-proline protects yeast cells from damage caused by freezing or oxidative stress. In this study, we evaluated the role of intracellular L-proline in cells of S. cerevisiae grown under ethanol stress. An L-proline-accumulating laboratory strain carries a mutant allele of PRO1, pro1(D154N), which encodes the Asp154Asn mutant gamma-glutamyl kinase. This mutation increases the activity of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase, which catalyze the first two steps of L-proline synthesis and which together may form a complex in vivo. When cultured in liquid medium in the presence of 9% and 18% ethanol under static conditions, the cell viability of the L-proline-accumulating laboratory strain is greater than the cell viability of the parent strain. This result suggests that intracellular accumulation of L-proline may confer tolerance to ethanol stress. We constructed a novel sake yeast strain by disrupting the PUT1 gene, which is required for L-proline utilization, and replacing the wild-type PRO1 allele with the pro1(D154N) allele. The resultant strain accumulated L-proline and was more tolerant to ethanol stress than was the control strain. We used the strain that could accumulate L-proline to brew sake containing five times more L-proline than what is found in sake brewed with the control strain, without affecting the fermentation profiles.     

Effect of carbon dioxide concentration on the bioleaching of a pyrite-arsenopyrite ore concentrate

The effect of carbon dioxide concentration on the bacterial leaching of a pyrite-arsenopyrite ore concentrate was studied in continuous-flow reactors. Steady-state operation with two feed slurry densities, 6 wt% and 16 wt% solids, were tested for the effect of carbon dioxide concentration. Bacterial growth rates were estimated via the measurement of carbon dioxide consumption rates. Aqueous-phase carbon dioxide concentrations in excess of 10 mg/L were found to be inhibitory to bacterial growth. (c) 1993 John Wiley & Sons, Inc.     

Analysis of free fatty acids in sake by an enzymatic method and its application for estimating ethyl caproate and selecting yeast with high productivity of the ester

We show that the concentration of total free fatty acids (FFAs) in sake produced by yeast with high productivity of ethyl caproate could be approximated by the concentration of 2 FFAs, caproic and caprylic acids. Measurement of the total FFAs concentration by an enzymatic method proved useful for both estimating the ethyl caproate concentration in sake and also for yeast breeding.     

Identification of a bactericidal component active against Gram-positive and Gram-negative bacteria in commercial ovomucoid and ovoinhibitor as lysozyme

During an investigation of the effect of protease inhibitors on the growth of Grampositive and -negative bacteria, commercial ovoinhibitor and ovomucoid were found to be strongly bactericidal. Both commercial inhibitors were fractionated by reversed phase chromatography and the bactericidal substance was identified as chicken egg white lysozyme. Lysozyme inhibited the growth of Gram-negative bacteria without addition of complement or EDTA. The use of ovoinhibitor and ovomucoid must be cautioned by the fact that the commercial preparations are contaminated with lysozyme.