Detailed Analysis of Japanese Population Substructure with a Focus on the Southwest Islands of Japan

Uncovering population structure is important for properly conducting association studies and for examining the demographic history of a population. Here, we examined the Japanese population substructure using data from the Japan Multi-Institutional Collaborative Cohort (J-MICC), which covers all but the northern region of Japan. Using 222 autosomal loci from 4502 subjects, we investigated population substructure by estimating FST among populations, testing population differentiation, and performing principal component analysis (PCA) and correspondence analysis (CA). All analyses revealed a low but significant differentiation between the Amami Islanders and the mainland Japanese population. Furthermore, we examined the genetic differentiation between the mainland population, Amami Islanders and Okinawa Islanders using six loci included in both the Pan-Asian SNP (PASNP) consortium data and the J-MICC data. This analysis revealed that the Amami and Okinawa Islanders were differentiated from the mainland population. In conclusion, we revealed a low but significant level of genetic differentiation between the mainland population and populations in or to the south of the Amami Islands, although genetic variation between both populations might be clinal. Therefore, the possibility of population stratification must be considered when enrolling the islander population of this area, such as in the J-MICC study

Hydrolysis of CMC, filter paper, and seaweeds by the synergistic action of cellulases and ß-glucosidases.

<p>(<b>A</b>) CMC (1 mL, 1% in 50 mM acetate, pH 5.5) was incubated with various combinations of purified enzymes (2 µg) as indicated at 37°C for 1 h. Reaction products were analyzed by TLC. (<b>B</b>) Filter paper (60 mg) was digested with various combinations of purified enzymes (10 µg) as indicated at 37°C for 48 h, and reaction products were analyzed by TLC. (<b>C</b>) Filter paper (60 mg) was digested with 21 K and 45 K cellulase (2 µg) in the presence of 110 K or 210 K ß-glucosidase (2 µg) at 37°C for 16 h. Reaction products were analyzed by TLC. (<b>D</b>) Seaweed, sea lettuce (<i>Ulva pertusa</i>), <i>Eisenia bicyclis,</i> and <i>Lessonia nigrescens</i> (20 mg in 50 mM acetate, pH 5.5) were incubated with purified enzymes (10 µg) at 37°C for 24 h. Glucose and reducing sugar content were then determined. (<b>E, F</b>) TLC analysis of reaction products of sea lettuce treated with purified enzymes or <i>Trichoderma</i> cellulase. Control sea lettuce (<b>E</b>) and sea lettuce treated with steam explosion (<b>F</b>) (20 mg in 50 mM acetate, pH 5.5) were incubated with purified cellulase (20 µg) in the presence of ß-glucosidase (20 µg) at 37°C for 15 h. As controls, 50 µg of <i>Trichoderma</i> cellulases, meicelase, and onozuka R-10 were used. The data shown are from one of three independent experiments with similar results.</p

Mode of action of purified cellulases and ß-glucosidases from sea hare.

<p>(<b>A</b>) Effect of treatment with purified cellulases or 210<b> </b>K ß-glucosidase on CMC viscosity. CMC (2 mL, 40 mg/mL in 50 mM acetate, pH 5.5) was incubated at 37°C for 4, 8, 10, 12, and 24 h in the absence or presence of the enzyme (0.2 µg) and the viscosity of the CMC solution was then measured as described in Materials and Methods. (<b>B</b>) Degradation of cello-oligosaccharides by purified cellulase. Cello-oligosaccharides (50 mL, 20 mg/mL in 20 mM acetate buffer, pH 5.5) were incubated with the enzyme (0.1 µg) at 37°C for 24 h and then subjected to TLC as described in Materials and Methods. G1, glucose; G2, cellobiose; G3, cellotriose; G4, cellotetraose; G5, cellopentaose; G6, cellohexaose. (<b>C</b>) Degradation of filter paper and CMC by purified cellulase and ß-glucosidases. Filter paper (60 mg) was incubated with 10 µg of the purified enzyme at 37°C for 15 h in 1 mL of 50 mM acetate buffer (pH 5.5). Furthermore, 1% CMC in 1 mL of 50 mM acetate buffer (pH 5.5) was incubated with the purified enzymes (2 µg) at 37°C for 1 h. The reaction mixture (2 mL) was subjected to TLC. (<b>D</b>) Digestion of cello-oligosaccharides with 210<b> </b>K or 110<b> </b>K ß-glucosidase. Cello-oligosaccharides (50 µL, 2 mg/mL in 10 mM acetate buffer, pH 5.5) were incubated with the enzyme (0.2 µg) at 37°C for 4 h and then subjected to TLC. (<b>E</b>) Time-course of hydrolysis of cellohexaose by the enzyme. Purified ß-glucosidase (BGL, 0.2 µg) was incubated with cellohexaose (50 µL, 20 mg/mL in 20 mM acetate, pH 5.5) for the time indicated. (<b>F</b>) Hydrolysis of lactose with 210<b> </b>K or 110<b> </b>K ß-glucosidase. Lactose (50 µL, 20 mg/mL in 20 mM acetate, pH 5.5) was incubated with the enzyme (0.2 µg) at 37°C for 4 h.</p

SDS-PAGE and amino acid sequence of purified enzymes.

<p>(<b>A</b>) SDS-PAGE of purified enzymes (2 µg protein). The marker proteins were as follows: myosin heavy chain (200 kDa), ß-galactosidase (116 kDa), phosphorylase b (97 kDa), BSA (67 kDa), ovalbumin (45 kDa), and glyceraldehyde-3-phosphate dehydrogenase (36 kDa). (<b>B</b>) Alignment of N-terminal and internal sequences of purified enzymes with other endo-ß-1,4-glucanases from freshwater snail (UniProt: A7KMF0, A0SGK2), brackish water clam (B9X0W1), abalone (B6RB06, Q86M37), and scallop (C6L866) and ß-glucosidases from brackish water clam (B5U9B3) and termite (D0VYS0). The molecular mass of A7KMF0, B6RB06, B9X0W1, Q86M37, C6L866, A0SGK2, B5U9B3, and D0VYS0 is 19 kDa, 21 kDa, 22.6 kDa, 66 kDa, 64 kDa, 66 kDa, 110 kDa, and 55 kDa, respectively. The internal sequences of fragments (LEP#37 from 21<b> </b>K cellulase, LEP#30 from 45<b> </b>K cellulase, LEP#5 from 65<b> </b>K cellulase, LEP#59 from 110<b> </b>K ß-glucosidase, LEP#33 and 43 from 210<b> </b>K ß-glucosidase) generated by lysyl endopeptidase digestion of purified enzymes were determined as described in Materials and Methods. The amino acid residue numbers of other endo-ß-1,4-glucanases and ß-glucosidases are indicated on both sides of the corresponding sequences.</p

Comprehensive Enzymatic Analysis of the Cellulolytic System in Digestive Fluid of the Sea Hare <i>Aplysia kurodai</i>. Efficient Glucose Release from Sea Lettuce by Synergistic Action of 45 kDa Endoglucanase and 210 kDa ß-Glucosidase

<div><p>Although many endo-ß-1,4-glucanases have been isolated in invertebrates, their cellulolytic systems are not fully understood. In particular, gastropod feeding on seaweed is considered an excellent model system for production of bioethanol and renewable bioenergy from third-generation feedstocks (microalgae and seaweeds). In this study, enzymes involved in the conversion of cellulose and other polysaccharides to glucose in digestive fluids of the sea hare (<i>Aplysia kurodai</i>) were screened and characterized to determine how the sea hare obtains glucose from sea lettuce (<i>Ulva pertusa</i>). Four endo-ß-1,4-glucanases (21<b> </b>K, 45<b> </b>K, 65<b> </b>K, and 95<b> </b>K cellulase) and 2 ß-glucosidases (110<b> </b>K and 210<b> </b>K) were purified to a homogeneous state, and the synergistic action of these enzymes during cellulose digestion was analyzed. All cellulases exhibited cellulase and lichenase activities and showed distinct cleavage specificities against cellooligosaccharides and filter paper. Filter paper was digested to cellobiose, cellotriose, and cellotetraose by 21<b> </b>K cellulase, whereas 45<b> </b>K and 65<b> </b>K enzymes hydrolyzed the filter paper to cellobiose and glucose. 210<b> </b>K ß-glucosidase showed unique substrate specificity against synthetic and natural substrates, and 4-methylumbelliferyl (4MU)-ß-glucoside, 4MU–ß-galactoside, cello-oligosaccharides, laminarin, and lichenan were suitable substrates. Furthermore, 210<b> </b>K ß-glucosidase possesses lactase activity. Although ß-glucosidase and cellulase are necessary for efficient hydrolysis of carboxymethylcellulose to glucose, laminarin is hydrolyzed to glucose only by 210<b> </b>K ß-glucosidase. Kinetic analysis of the inhibition of 210<b> </b>K ß-glucosidase by D-glucono-1,5-lactone suggested the presence of 2 active sites similar to those of mammalian lactase-phlorizin hydrolase. Saccharification of sea lettuce was considerably stimulated by the synergistic action of 45<b> </b>K cellulase and 210<b> </b>K ß-glucosidase. Our results indicate that 45<b> </b>K cellulase and 210<b> </b>K ß-glucosidase are the core components of the sea hare digestive system for efficient production of glucose from sea lettuce. These findings contribute important new insights into the development of biofuel processing biotechnologies from seaweed.</p></div

Substrate specificities of 110 K and 210 K ß-glucosidase.

<p>Substrate specificities of 110 K and 210 K ß-glucosidase.</p

Enzyme activities toward CMC and lichenan of 21<b> </b>K, 45<b> </b>K, 65<b> </b>K, and 95<b> </b>K cellulase.

<p>Enzyme activities toward CMC and lichenan of 21<b> </b>K, 45<b> </b>K, 65<b> </b>K, and 95<b> </b>K cellulase.</p

Mode of action of 210 K or 110 K ß-glucosidase on CMC, laminarin, and lichenan.

<p>(<b>A</b>) Digestion of CMC with 210 K or 110 K ß-glucosidase in the absence or presence of 21 K or 45 K cellulase. CMC (1 mL, 1% in 50 mM acetate, pH 5.5) was incubated with 2 µg of 210 K or 110 K ß-glucosidase and 5 mg of 21 K or 45 K cellulose, as indicated, at 37°C for 10, 20, 30, and 60 min. Reducing sugar (1) and glucose (2) in the reaction mixture were determined. Reaction products were analyzed by TLC (3). (<b>B</b>) Laminarin (1 mL, 1% in 50 mM acetate, pH 5.5) was incubated with 2 µg of 110 K or 210 K ß-glucosidase at 37°C for 10, 20, and 60 min. The glucose content in the reaction mixture was then determined. Reaction products were analyzed by TLC. (<b>C</b>) Lichenan (1 mL, 1% in 50 mM acetate, pH 5.5) was incubated with 2 µg of 110 K or 210 K ß-glucosidase at 37°C for 10, 20, and 60 min.</p

Comprehensive enzymatic analysis of the amylolytic system in the digestive fluid of the sea hare, Aplysia kurodai: Unique properties of two α-amylases and two α-glucosidases

Sea lettuce (Ulva pertusa) is a nuisance species of green algae that is found all over the world. East-Asian species of the marine gastropod, the sea hare Aplysia kurodai, shows a clear feeding preference for sea lettuce. Compared with cellulose, sea lettuce contains a higher amount of starch as a storage polysaccharide. However, the entire amylolytic system in the digestive fluid of A. kurodai has not been studied in detail. We purified α-amylases and α-glucosidases from the digestive fluid of A. kurodai and investigated the synergistic action of these enzymes on sea lettuce. A. kurodai contain two α-amylases (59 and 80 kDa) and two α-glucosidases (74 and 86 kDa). The 59-kDa α-amylase, but not the 80-kDa α-amylase, was markedly activated by Ca2+ or Cl−. Both α-amylases degraded starch and maltoheptaose, producing maltotriose, maltose, and glucose. Glucose production from starch was higher with 80-kDa α-amylase than with 59-kDa α-amylase. Kinetic analysis indicated that 74-kDa α-glucosidase prefers short α-1,4-linked oligosaccharide, whereas 86-kDa α-glucosidase prefers large α-1,6 and α-1,4-linked polysaccharides such as glycogen. When sea lettuce was used as a substrate, a 2-fold greater amount of glucose was released by treatment with 59-kDa α-amylase and 74-kDa α-glucosidase than by treatment with 45-kDa cellulase and 210-kDa β-glucosidase of A. kurodai. Unlike mammals, sea hares efficiently digest sea lettuce to glucose by a combination of two α-amylases and two α-glucosidases in the digestive fluids without membrane-bound maltase–glucoamylase and sucrase–isomaltase complexes

Survival Rate of Cells Sent by a Low Mechanical Load Tube Pump: The “Ring Pump”

A ring pump (RP) is a useful tool for microchannels and automated cell culturing. We have been developing RPs (a full-press ring pump, FRP; and a mid-press ring pump, MRP). However, damage to cells which were sent by the RP and the MRP was not investigated, and no other studies have compared the damage to cells between RPs and peristaltic pumps (PPs). Therefore, first, we evaluated the damage to cells that were sent by a small size FRP (s-FRP) and small size MRPs (s-MRPs; gap = 25 or 50 &mu;m, respectively). &ldquo;Small size&rdquo; means that the s-FRP and the s-MRPs are suitable for microchannel-scale applications. The survival rate of cells sent by the s-MRPs was higher than those sent by the s-FRP, and less damage caused by the former. Second, we compared the survival rate of cells that were sent by a large size FRP (l-FRP), a large size MRP (l-MRP) (gap = 50 &mu;m) and a PP. &ldquo;Large size&rdquo; means that the l-FRP and the l-MRP are suitable for automated cell culture system applications. We could not confirm any differences among the cell survival rates. On the other hand, when cells suspended in Dulbecco&rsquo;s phosphate-buffered saline solution were circulated with the l-MRP (gap = 50 &mu;m) and the PP, we confirmed a difference in cell survival rate, and less damage caused by the former