Mikio Kinoshita

Differences of Rhizopus oryzae strains in organic acid synthesis and fatty acid composition

Two groups of 15 Rhizopus oryzae strains were grown in a liquid medium to analyse metabolic products such as lactic, fumaric, malic and other organic acids and ethanol. Group A produced a large amount of lactic acid, while group B produced a small amount. The addition of calcium carbonate to the medium of group A stimulated the production of lactic acid, but not fumaric and malic acids. As compared with group A, organic acids were only slightly synthesized in group B. Fumaric and malic acids appeared in the medium of group B, which was supplemented with calcium carbonate, but lactic acid was not detected. Myristic, palmitic, palmitoleic, oleic, stearic, linoleic and γ-linolenic acids were determined for total lipids extracted from the fungal cells and subjected to principal component analysis. Group A contained high amounts of unsaturated fatty acids and differed from group B in the proportion of palmitic and γ-linolenic acids. The content of γ-linolenic acid increased by the addition of lactic acid to the medium and not by those of ethanol and NaCl. The physiological differences in R. oryzae strains may depend on the productivity of organic acids, reflecting the composition of fatty acids.

Rapid estimation of peroxide content of soybean oil by measuring thermoluminescence

Thermoluminescence measurements can serve as a simple and rapid procedure for the estimation of peroxide content of soybean oil. The thermoluminescence intensity, measured at 100°C, increases in proportion (r=0.978) to the peroxide value (from 0.5 to 18.0 meq/kg) of soybean oil, without any interference by the tocopherol contents. The emission spectrum had a maximum wavelength at around 440–480 nm, suggesting that excited triplet carbonyls formed during thermal decomposition of hydroperoxides are involved. The thermoluminescence measurement is readily available for the simple and rapid estimation of the peroxide content of soybean oil, with no need for chemical reagents and delicate skills.

Age-related change of phosphatidylcholine hydroperoxide and phosphatidylethanolamine hydroperoxide levels in normal human red blood cells

The age-related occurrence of phosphatidylcholine hydroperoxide (PCOOH) and phosphatidylethanolamine hydroperoxide (PEOOH) in normal human red blood cells (RBC) was confirmed by using chemiluminescence detection-high performances liquid chromatography (CL-HPLC). The concentration (mean +/- S.D.) for the healthy young adult (22-27 of age, n = 20) was 93 +/- 17 pmol PCOOH and 121 +/- 20 pmol PEOOH/ml packed RBC, while for the aged adult (56-92 of age, n = 20) the peroxide content was significantly higher, 162 +/- 52 pmol PCOOH and 186 +/- 40 pmol PEOOH/ml packed RBC. These results indicate that oxidative stress occurs constantly on RBC, even in normal humans, and that the susceptivity significantly increases with age.

Effect of dietary wine pomace extract and oleanolic acid on plasma lipids in rats fed high-fat diet and its DNA microarray analysis.

The aim of this study was to evaluate the concentration of oleanolic acids (OA) in pomace, a winemaking byproduct, and its influence on the levels of plasma lipids in rats fed a high-fat diet and on hepatic gene expression using DNA microarray analysis in vivo. HPLC analyses of pomace ethanol extract (PEE) revealed a high amount of OA ranging from 4 to 11 g/100 g. Male Sprague-Dawley rats were fed a normal-fat diet (NF group), a high-fat diet with 21% lard (HF group), a high-fat diet with 0.05% OA (OA group, 50 mg/kg/day), or a high-fat diet with 0.45% PEE (PEE group, 450 mg/kg/day). Plasma triacylglycerol and phospholipid concentrations were significantly lower in the OA and PEE groups than in the HF group. The microarray analysis of hepatic mRNA revealed reduced expression levels of lipogenic genes including acetyl-CoA carboxylase and glycerol-3-phosphate acyltransferase, probably resulting from the suppression of transcription factor Srebf1 expression. Gene expression of gluconeogensis and inflammatory cytokines was also down-regulated in the OA and PEE groups, suggesting that administration of OA or PEE could ameliorate obesity-induced insulin resistance, as well as prevent hyperlipidemia.

Efflux of Sphingoid Bases by P-Glycoprotein in Human Intestinal Caco-2 Cells

The aim of this study was to determine whether sphingoid bases that originated from various dietary sources, such as mammals, plants, and fungi, are substrates for P-glycoprotein in differentiated Caco-2 cells, which are used as a model of intestinal epithelial cells. In Caco-2 cells, the uptake of sphingosine, the most common sphingoid base found in mammals, was significantly higher at physiological temperatures than those of cis/trans-8-sphingenine, trans-4, cis/trans-8-sphingadienine, 9-methyl-trans-4, trans-8-sphingadienine, or sphinganine. Verapamil, a potent P-glycoprotein inhibitor, increased the cellular accumulation of sphingoid bases, except for sphingosine, in a dose-dependent manner. Incubation with 1 μM digoxin for 48 h caused up-regulation of murtidrug-resistance (MDR)1 mRNA and decreased the accumulation of sphingoid bases in Caco-2 cells, except for sphingosine. Thus P-glycoprotein probably contributes to the selective absorption of sphingosine from dietary sphingolipids in the digestive tract.

Anti-Inflammatory Effect of Buckwheat Sprouts in Lipopolysaccharide-Activated Human Colon Cancer Cells and Mice

In conducting an in vitro screening of ethanol extracts from various natural foods using a human colon cancer cell line (CoLoTC cells), an extract of buckwheat sprouts (ExtBS) was found to express significant anti-inflammatory activity. The anti-inflammatory activity of ExtBS was confirmed by oral administration of lipopolysaccharide (LPS) to mice. Inflammatory cytokines (interleukin 6 and tumor necrosis factor alpha) were markedly up-regulated in the spleen and liver from LPS-administrated mice, and combinatory treatment with LPS and ExtBS decreased up-regulation of them in both cytokines. Both serum cytokine levels corresponded to their gene expressions in tissues, but no anti-inflammatry effect in mice was observed when ExtBS was treated intraperitoneally. ExtBS oral administration also showed protective activity as to hepatic injury induced by galactosamine/LPS treatment. Based on these data, we suggest that ExtBS contains anti-inflammatory compounds.

Apoptosis Induction by Wheat-flour Sphingoid Bases in DLD-1 Human Colon Cancer Cells

The apoptotic effects of plant sphingoid bases prepared from wheat-flour cerebroside on human colorectal cancer DLD-1 cells were examined. The viability of DLD-1 cells treated with such plant sphingoid bases was reduced in a dose-dependent manner and was similar to that of cells treated with sphingosine. Morphological changes such as condensed chromatin fragments were found, so those sphingoid bases reduced cell viability through causing apoptosis in these cells.

Isolation and Characterization of the Genes Encoding Δ8-Sphingolipid Desaturase from Saccharomyces kluyveri and Kluyveromyces lactis

Saccharomyces kluyveri IFO 1685 and Kluyveromyces lactis IFO 1090 synthesize cerebroside containing 9-methyl-trans-4, trans-8-sphingadienine as a sphingoid base. From the genome of the two strains, the regions encompassing Δ8-sphingolipid desaturase were amplified and sequenced. The nucleotide sequences of these regions revealed single open reading frames of 1707 bp for S. kluyveri and 1722 bp for K. lactis, encoding polypeptides of 568 and 573 amino acids with molecular weights of 66.5 and 67.1 kDa, respectively. Conversion of 4-hydroxysphinganine to 4-hydroxy-trans-8-sphingenine in the cells of Saccharomyces cerevisiae was observed by the expressed gene from K. lactis and not by that from S. kluyveri. These findings may be explained by the difference in substrate specificity for the sphingoid base moiety between Δ8-sphingolipid desaturases of S. kluyveri and K. lactis.

Presence of higher alcohols as ferulates in potato pulp and its radical-scavenging activity.

Higher alcohols with a carbon length ranging from 16 to 30 found in the lipophilic fraction from potato pulp were shown to be present as ferulate and in a free form, but not as wax. Thin-layer chromatography of the neutral lipids in potato pulp indicated a few spots with scavenging activity toward the 1,1-diphenyl-2-picrylhydrazyl (DPPH) stable radical, the major active component being characterized as alkyl ferulate which showed almost the same level of activity as γ-oryzanol.

Alterations in the Levels of Amyloid-β, Phospholipid Hydroperoxide, and Plasmalogen in the Blood of Patients with Alzheimer’s Disease: Possible Interactions between Amyloid-β and These Lipids

Aside from accumulation of amyloid-β (Aβ) peptide in the brain, Alzheimers disease (AD) has been reported as being associated with peroxidation of major phospholipids (e.g., phosphatidylcholine (PtdCho)) and degradation of antioxidative phospholipids (e.g., ethanolamine plasmalogen (PlsEtn)). In addition to its presence in the brain, Aβ is also found in blood; however, there is still little information about the levels of PtdCho hydroperoxide (PCOOH) and PlsEtn in the blood of patients with AD. In this study, by assuming a possible interaction among Aβ, PCOOH, and PlsEtn in blood circulation, we evaluated the levels of these molecules and correlations in blood samples that had been obtained from our former AD study for PCOOH measurement (Kiko et al., J Alzheimers Dis28, 593-600, 2012). We found that when compared to controls, plasma from patients with AD showed lower concentrations of PlsEtn species, especially PlsEtn bearing the docosahexaenoic acid (DHA) moiety. In addition, lower PlsEtn and higher PCOOH levels were observed in red blood cells (RBCs) of patients with AD. In both AD and control blood samples, RBC PCOOH levels tended to correlate with plasma levels of Aβ40, and each PlsEtn species showed different correlations with plasma Aβ. These results, together with in vitro data suggesting Aβ aggregation due to a decrease in levels of PlsEtn having DHA, led us to deduce that Aβ is involved in alterations in levels of PCOOH and PlsEtn species observed in the blood of patients with AD.