Masuo Nakano

Cholesterol-Lowering Effects of Maitake (Grifola frondosa) Fiber, Shiitake (Lentinus edodes) Fiber, and Enokitake (Flammulina velutipes) Fiber in Rats:

The effects of mushroom fibers on serum cholesterol and hepatic low-density lipoprotein (LDL) receptor mRNA in rats were investigated. Rats were fed a cholesterol-free diet with 50 g/kg cellulose powder (CP), 50 g/kg maitake (Grifola frondosa) fiber (MAF), 50 g/kg shiitake (Lentinus edodes) fiber (SF), or 50 g/kg enakitake (Flammulina velutipes) fiber (EF) for 4 weeks. There were no significant differences in the body weight, food intake, liver weight, cecum weight, and cecum pH among the groups. Cecal acetic acid, butyric acid, and total short-chain fatty acid (SCFA) concentrations in the SF and EF groups were significantly higher than those in the other groups. The serum total cholesterol concentration in the CP group was significantly higher than that in the MAF and EF groups. The very LDL (VLDL) + intermediate-density lipoprotein (IDL) + LDL-cholesterol concentration in the CP group was significantly higher than that in the MAF, SF, and EF groups, whereas the high-density lipoprotein (HDL)-cholesterol concentration in the EF group was significantly lower than that in the other groups at the end of the 4-week feeding period. The hepatic LDL receptor mRNA level in the EF group was significantly higher than that in the CP group. The fecal cholesterol excretion in the MAF, SF, and EF groups was significantly higher than that in the CP group. The results of this study demonstrate that MAF and EF lowered the serum total cholesterol level by enhancement of fecal cholesterol excretion, and in particular, by enhancement of hepatic LDL receptor mRNA in EF group.

Effects of a mixture of organisms, Lactobacillus acidophilus or Streptococcus faecalis on cholesterol metabolism in rats fed on a fat- and cholesterol-enriched diet

The effect of a mixture of organisms (a probiotic mixture) comprising Bacillus, Lactobacillus, Streptococcus, Clostridium, Saccharomyces and Candida (10(7-8) colony-forming units/g rice bran of each component) on lipid metabolism was compared with that of L. acidophilus and that of S. faecalis. There were four treatment groups: rice bran (control), the mixture of organisms, L. acidophilus or S. faecalis (30 g/kg) were given to rats in a fat- and cholesterol-enriched diet for 4 weeks. The serum total cholesterol concentration of the group fed on the mixture of organisms was reduced by 15-33% compared with the other groups at the end of the 4-week feeding period (P < 0.05). This group also had a lower hepatic cholesterol concentration (36-44%) than the two single-bacteria groups (P < 0.05). 3-Hydroxy-3-methylglutaryl-Co A reductase (NADPH; EC 1.1.1.34) activities of the mixed-organism and L. acidophilus groups were significantly lower (61-63%) than those of the other groups (P < 0.05); the activity of the S. faecalis group was also significantly lower (42%) than that of the control group (P < 0.05). The faecal cholesterol and bile acid concentrations of the mixed-organism group increased compared with those of the L. acidophilus and S. faecalis groups (P < 0.05). The capacity of the mixed-organism cells to bind bile salt in vitro was significantly higher (approximately 50%) than that of the single-bacteria cells (P < 0.05). On the other hand, cholesterol micelle formation for the mixed-organism cells was significantly (approximately 9%) lower than that of the single-bacteria cells (P < 0.05). These results indicate that the mixture of organisms decreased the synthesis of cholesterol in the liver and increased the loss of steroids from the intestine, in rats. Thus, the mixture of organisms had a hypocholesterolaemic role.

The effect of a probiotic on faecal and liver lipid classes in rats

The effect of a probiotic composed of Bacillus, Lactobacillus, Streptococcus, Saccharomyces and Candida species (each at 10(7-8) colony-forming units (cfu)/g rice bran), given at a level of 150 g/kg diet for 6 weeks, on lipid metabolism was examined in the faeces, serum and liver of male rats. Liver weight decreased 35% in the rats fed on a high-fat, high-cholesterol diet containing the probiotic. Total cholesterol concentration in the serum was significantly lower in the probiotic group than in the control group throughout the experimental period in rats fed on the high-fat, high-cholesterol diet, and HDL-cholesterol concentration was significantly higher (P < 0.05) in the probiotic group than in the control group which was fed for the 6 week experimental period on a basal diet. The serum VLDL+IDL+LDL cholesterol concentration in the probiotic groups were reduced compared with those of the corresponding control groups. The probiotic groups fed on the high-fat, high-cholesterol diet and the basal diet had lower hepatic cholesterol concentrations than did the corresponding control groups (P < 0.05). Hydroxymethylglutaryl coenzyme A reductase (NADPH) (EC 1.1.1.34) activity in the liver was lower in rats fed on the high-fat, high cholesterol diet with the probiotic. The neutral and acidic steroid concentrations in faeces were higher in the probiotic group than in the control group fed on the high-fat, high-cholesterol diet. Escherichia coli decreased and Bifidobacterium and Eubacterium increased in the faecal microflora of rats fed on the dietary probiotic. Lactobacillus in the probiotic groups was higher than that in the control groups. The present study shows that the probiotic promotes Bifidobacterium and Eubacterium in the faecal microflora, and reduces cholesterol levels in the serum and liver of rats.

Distribution of PCDDs/PCDFs and Co-PCBs in human maternal blood, cord blood, placenta, milk, and adipose tissue: dioxins showing high toxic equivalency factor accumulate in the placenta.

To assess levels of dioxin background contamination and transfer of dioxins from mothers to unborn children and infants, concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and coplanar-polychlorinated biphenyls (Co-PCBs) were measured in human samples from expectant and nursing mothers living in Nara, Japan. The average toxic equivalency quantities (TEQs) of PCDDs/PCDFs and Co-PCBs from circulating maternal blood, cord blood, placenta, milk taken 3–10 d after delivery, milk taken one month after delivery, and adipose tissue were 26 and 9.3, 15 and 2.3, 31 and 1.2, 16 and 5.4, 18 and 8.8, and 16 and 7.7 pg-TEQ/g-fat, respectively. Among the various PCDD/PCDF congeners, 1,2,3,7,8-PeCDD and 2,3,4,7,8-PeCDF contributed most heavily to the TEQs of all maternal samples. Among the various Co-PCB congeners, 3,3′,4,4′,5-PeCB (#126), 2,3,3′,4,4′,5-HxCB (#156), and 2,3′,4,4′,5-PeCB (#118) contributed most heavily to the TEQs of all maternal samples. But, the concentrations and relative percentages of congeners differed among the various samples, suggesting that congeners showing high toxic equivalency factor accumulate in the placenta.

Genetic relationship and distribution of the Japanese wild boar (Sus scrofa leucomystax) and Ryukyu wild boar (Sus scrofa riukiuanus) analysed by mitochondrial DNA

Mitochondrial genetic variations were used to investigate the relationships between two Japanese wild boars, Japanese wild boar (Sus scrofa leucomystax) and Ryukyu wild boar (S.s. riukiuanus). Nucleotide sequences of the control (27 haplotypes) and cytochrome b (cyt‐b) regions (19 haplotypes) were determined from 59 Japanese wild boars, 13 Ryukyu wild boars and 22 other boars and pigs. From phylogenetic analyses, the mtDNA of Ryukyu wild boar has a distinct lineage from that of Japanese wild boar, which was classified into the Asian pig lineage. This result suggests that the Ryukyu wild boar has a separate origin from the Japanese wild boar.

Geographic population structure and sequence divergence in the mitochondrial DNA control region of the Japanese wild boar (Sus scrofa leucomystax), with reference to those of domestic pigs

Mitochondrial DNA (mtDNA) control regions from 40 Japanese wild boars were examined by direct sequencing after amplification by PCR. From the DNA sequences obtained, we found eight haplotypes, whose differences arose via transitions. The geographical distribution of these different haplotypes indicated that wild boar populations inhabited limited areas and that there was some restricted gene flow between local populations. Eight mtDNA haplotypes from Eastern and Western domestic pigs and the Ryukyu wild boar were also analyzed as references to those from Japanese wild boars. The cluster analyses of the control-region sequences showed that those from Japanese wild boards belong to the Asian type as do those from Eastern domestic pigs and the Ryukyu wild boar, which differed from the European type (Western domestic pigs).

Effects of a mixture of organisms, Lactobacillus acidophilus or Streptococcus faecalis on δ6-desaturase activity in the livers of rats fed a fat- and cholesterol-enriched diet

The effect of a mixture of organisms (a probiotic mixture) comprising Bacillus, Lactobacillus, Streptococcus, Clostridium, Saccharomyces, and Candida (10(7-8) colony-forming units/g rice bran of each component) on delta6-desaturase activity in liver microsomes was compared with those of Lactobacillus acidophilus and Streptococcus faecalis. There were four treatment groups. Each group of these rats received rice bran (control), the mixture of organisms, L. acidophilus, or S. faecalis (30 g/kg) along with a fat- and cholesterol-enriched diet for 4 wk. The serum total cholesterol concentration of the group fed the mixture of organisms was reduced by 15-33% compared with the other groups at the end of the 4-wk feeding period (P<0.05). The proportion of palmitic acid in the serum phosphatidylcholine (PC) for the control group was significantly higher than those of the other groups. The proportion of arachidonic acid in the serum PC for the mixed-organism group was also significantly higher than those of the other groups. The proportion of arachidonic acid in the liver PC for the mixed-organism group was significantly higher than those of the control and S. faecalis groups. The ratio of arachidonic acid/linoleic acid was significantly higher in the liver PC of rats fed the mixed organisms compared with the control group (P<0.05). The delta6-desaturase activity in the liver microsomal fraction of the mixed-organism group was significantly higher than those of the other groups. The delta6-desaturase activity correlated positively with the ratio of arachidonic acid/linoleic acid of liver PC, the correlation coefficient (r) being 0.819 (P<0.001). The results indicate that the effect of the mixture of organisms was to increase delta6-desaturase activity and serum arachidonic acid and decrease cholesterol compared to the other organisms and control, but the mechanism whereby the enzyme activity was related to serum cholesterol does not appear to have been explored.

Ancient Mitochondrial DNA Reveals the Origin of Sus scrofa from Rebun Island, Japan

Abstract. The Kabukai A site (5 to 8C A.D.) of the Okhotsk cultural area is on Rebun Island, a small island near the coast, north–northwest of Hokkaido, Japan. Specimens of Sus scrofa, called the Sakhalin pig, were discovered in five cultural layers at the Kabukai A site. Ancient DNA was extracted from the remains of 42 Sakhalin pig bones. Thirty-nine nucleotide sequences of the 574-bp mitochondrial DNA control region, estimated to have originated from at least 21 individuals, were amplified and analyzed phylogenetically. Nine distinct haplotypes (A1, A2, A3, B1, B2, C1, C2, D1, and D2) from this site were classified into four haplotype groups (A, B, C, and D) by parsimonious network analysis. Phylogenetic analysis of 9 ancient and 55 modern haplotypes indicated that the population of Sakhalin pigs at the Kabukai A site belonged to two distinct clusters; haplotype groups A and B formed a cluster comprised only of themselves, and haplotype groups C and D belonged to the cluster of one of the two genetic groups of Japanese wild boars uniquely distributed in the western part of Japan, including one northeast Mongolian wild boar. Analysis of the haplotype distribution among three archaeological sites and their historical transitions among the five layers reflecting the cultural periods at the Kabukai A site suggests that the Sakhalin pig populations were introduced from Sakhalin island and the Amur River basin in the northeastern Eurasian continent together with some cultural influences.

Phylogeography and population structure of the Japanese wild boar Sus scrofa leucomystax: mitochondrial DNA variation.

Abstract Phylogeographic characteristics and population structure of Japanese wild boar (Sus scrofa leucomystax) were investigated using mitochondrial DNA (mtDNA) sequence data. Sixteen Japanese wild boar haplotypes detected from partial sequences of the mtDNA control region (574-bp) from 180 Japanese wild boar specimens from 10 local populations on Honshu, Shikoku, and Kyushu islands and 41 haplotypes from other S. scrofa were analyzed using the neighbor-joining method. The Japanese wild boars were more closely related to Northeast Asian wild boars from Mongolia than to the other Asian continental S. scrofa. The Japanese and Northeast Asian wild boars were not significantly distinguished by corrected average pairwise difference analysis. The ancestors of Japanese wild boars are suggested to have been part of the continental S. scrofa population that spread from Southeast to Northeast Asia during the Middle to Late Pleistocene. The Japanese wild boar mtDNA haplotype cladogram shows 95% parsimoniously plausible branch connections supporting three sympatric clades. Nested clade analysis indicates that these three clades are the result of distinct historical events or gene flow. The present population of Japanese wild boars may have been formed by a few independent migrations of distinct clades from the continent with subsequent mixing on the Japanese Islands.

Prehistoric introduction of domestic pigs onto the Okinawa Islands: Ancient mitochondrial DNA evidence

Abstract. Ancient DNAs of Sus scrofa specimens excavated from archaeological sites on the Okinawa islands were examined to clarify the genetic relationships among prehistoric Sus scrofa, modern wild boars and domestic pigs inhabiting the Ryukyu archipelago, the Japanese islands, and the Asian continent. We extracted remain DNA from 161 bone specimens excavated from 12 archaeological sites on the Okinawa islands and successfully amplified mitochondrial DNA control region fragments from 33 of 161 specimens. Pairwise difference between prehistoric and modern S. scrofa nucleotide sequences showed that haplotypes of the East Asian domestic pig lineage were found from archaeological specimens together with Ryukyu wild boars native to the Ryukyu archipelago. Phylogenetic analysis of 14 ancient sequences (11 haplotypes; 574 bp) indicated that S. scrofa specimens from two Yayoi-Heian sites (Kitahara and Ara shellmiddens) and two Recent Times sites (Wakuta Kiln and Kiyuna sites) are grouped with modern East Asian domestic pigs. Sus scrofa specimens from Shimizu shellmidden (Yayoi-Heian Period) were very closely related to modern Sus scrofa riukiuanus but had a unique nucleotide insertion, indicating that the population is genetically distinct from the lineage of modern Ryukyu wild boars. This genetic evidence suggests that domestic pigs from the Asian continent were introduced to the Okinawa islands in the early Yayoi-Heian period (1700–2000 BP), or earlier.