Potent cellulase activity in the hepatopancreas of mangrove crabs

Mangrove crabs play a crucial role in the carbon cycle in forests by consuming large amounts of mangrove litter, which is mainly composed of cellulose. However, the detailed mechanism of cellulose digestion remains to be elucidated. We tested endogenous hepatopancreatic cellulase activity in eight species of crabs, including three mangrove sesarmid crabs (Episesarma versicolor, Perisesarma indiarum, and Episesarma palawanense) native to Thailand. Endo-??-1,4-glucanase activity was significantly higher in the enzyme extract from mangrove crabs than in that from Japanese marsh crabs. A ??-glucosidase assay revealed particularly high endo-??-1,4-glucanase activity for E. versicolor, whereas little activity was observed for the Japanese marsh crabs. In a zymogram analysis for endo-??-1,4-glucanase activity, endo-??-1,4-glucanase had a similar molecular mass (30.7?C33.1?kDa) among the mangrove crabs, whereas various sizes (44.3?C84.8?kDa) were found in Japanese crabs depending on the species. These results suggest that mangrove crabs efficiently digest cellulose endogenously.     

Determination of genome size of Haliotis discus hannai and H.?diversicolor aquatilis (Haliotidae) and phylogenetic examination of this family

Genome size (C value) is an important index for phylogenetic studies. Haliotidae (abalones) includes many species widely distributed throughout the world??s oceans, which makes this family interesting for phylogenetic studies. To examine Haliotidae phylogeny, we determined the C value and adenine and thymine base pair content (AT?%) of Haliotis discus hannai and H.?diversicolor aquatilis by using flow cytometry. The C values of H.?discus hannai and H.?diversicolor aquatilis were 1.84 and 1.45?pg, with AT?% of 62.3 and 66.3?%, respectively. These data represent the first report of abalones classified as Pacific Northwest (H.?discus hannai) and Indo-Pacific (H.?diversicolor aquatilis) groups, and provides new validation for previous theories related to Haliotidae phylogeny.     

Identification of genes required for the fitness of Rhodococcus equi during the infection of mice via signature-tagged transposon mutagenesis

Rhodococcus equi is a Gram-positive facultative intracellular bacterium that causes pyogranulomatous pneumonia in foals and immunocompromised people. In the present study, signature-tagged transposon mutagenesis was applied for the negative selection of R. equi mutants that cannot survive in vivo. Twenty-five distinguishable plasmid-transposon (plasposon) vectors by polymerase chain reaction (PCR), each containing a unique oligonucleotide tag, were constructed and used to select the transposon mutants that have in vivo fitness defects using a mouse systemic infection model. Of the 4,560 transposon mutants, 102 mutants were isolated via a real-time PCR-based screening as the mutants were unable to survive in the mouse model. Finally, 50 single transposon insertion sites were determined via the self-cloning strategy. The insertion of the transposon was seen on the virulence plasmid in 15 of the 50 mutants, whereas the remaining 35 mutants had the insertion of transposon on the chromosome. The chromosomal mutants contained transposon insertions in genes involved in cellular metabolism, DNA repair and recombination, gene regulation, non-ribosomal peptide synthesis, and unknown functions. Additionally, seven of the chromosomal mutants showed a reduced ability to multiply in the macrophages in vitro. In this study, we have identified several biosynthetic pathways as fitness factors associated with the growth within macrophages and survival in mice.     

Cold tolerance at the early growth stage in wild and cultivated rice

The present study was conducted to understand the pattern of variation and the genetic bases for cold tolerance at the early growth stage in Asian rice. The genetic variation was investigated at the germination, plumule and seedling stages among 57 strains including cultivated rice (Oryza sativa ssp. indica and ssp. japonica) and its wild progenitor (Oryza rufipogon). The significant differentiation of cold tolerance was observed among the taxonomically divided groups. At the germination stage, both indica and japonica subspecies tended to be more tolerant than O. rufipogon, whereas at the plumule and seedling stages, ssp. japonica tended to be more tolerant than ssp. indica and O. rufipogon. Furthermore, in cold tolerance at the plumule stage, the clinal variation across the latitude of origins was observed within O. rufipogon and ssp. japonica, suggesting that the current pattern of variation seems to have been shaped by both their phylogenetic histories and on-going adaptation to the local environments. QTL analysis between O. sativa ssp. japonica (tolerant) and O. rufipogon (susceptible) revealed five putative QTLs for cold tolerance at the plumule and seedling stages but not at the germination stage. Substitution mapping was also carried out to precisely locate the two major QTLs for cold tolerance at the plumule stage, which could be used for improvement of tolerance to cold stress in ssp. indica.     

Study of mechanical properties of wooden bolt-nut connector I: effect of size and shape of thread on withdrawal strength

The withdrawal strength of a bolt-nut connector made from wood-based material was evaluated. The thread strength of the wooden bolt-nut connector was tested to select various parameters of the connector and the type of wood material; the wood materials tested were hard maple, white oak, ebony, glue-laminated bamboo, and densified Japanese cedar. A plane model of wooden threads with various thread angles was also evaluated. The results showed that the maximum failure load of the thread increased with increasing bolt density and connection area, which was calculated from the diameter of the bolt and the thickness of the nut. The withdrawal resistance after reaching the maximum load underwent a graded decrease because the bolt threads were broken one by one. In addition, the thread strength depended on the thread angle. In the model with a thread angle of 90°, compressive deformation in the transverse direction occurred prior to shear deformation along the root of the threads; the model with this thread angle thus had higher strength than those with other angles.     

Yield-enhancing and tuber-downsizing effects of transplantation cultivation method of case-held tuber seedlings in the sweet potato cultivar Beniharuka

We developed transplantation cultivation method of case-held tuber seedlings (CTS), which was derived from direct planting method of seed tubers, and applied this method to the sweet potato cultivar Beniharuka. A plastic case made of polypropylene was designed for cultivation of CTS. Seed tubers of cultivar Beniharuka in the range of 30–80 g were cut in half. The half-cut tubers were placed inside the plastic cases, and the cases were filled with a commercial soil mix. The case-held tubers were incubated under natural sunlight in a glass house. After 3–4 wk, the CTS were transplanted into a field. Mother tuber (seed tuber) enlargement was suppressed by the plastic confinement of the cases, and daughter tubers were formed above the case as vine-root-originated tubers. In the field experiments in 2012 and 2013, daughter tuber yields were increased 19% and 21% by case-held tuber seedling transplanting (CTST) over conventional vine-planting (VP), the number of daughter tubers per plant in CTST were 36 and 68% higher than in VP, and the mother tuber yields were limited to 2.1 and 4.3% of the total fresh yield of mother and daughter tubers, respectively in 2012 and 2013. Application of CTST method to cultivar Beniharuka enhanced tuber yield, increased the number of daughter tubers per plant, downsized daughter tubers compared to VP, and mother tuber enlargement was suppressed by case-holding. The CTST method is expected to produce more and smaller good in shape tubers of cultivar Beniharuka compared to VP.     

Effects of anthocyanins on psychological stress-induced oxidative stress and neurotransmitter status

There is strong evidence that oxidative stress participates in the etiology of neurodegenerative diseases such as Parkinson's, and Alzheimer's diseases. Moreover, emotional stress effects in the central nervous system play a vital role in homeostasis. The protective effect of anthocyanins on the cerebral oxidative stress was studied using the whiskers cut model. In mice, such treatment causes psychological or emotional distress leading to oxidative stress in tissues. To investigate the in vivo antioxidant activity of anthocyanins, an extract of Vaccinium myrtillis L., an anthocyanin mixture, was orally administered (100 mg/kg of body weight.) to mice for 7 days, and then psychological stress was assessed by cutting off their whiskers. Whisker removal increased both protein carbonyl formation and lipid peroxidation in the brain, heart, kidney, and liver. Further, the levels of oxidative markers showed regional differences in the brain. Concomitantly, dopamine neurotransmitter levels were altered in both the midbrain and the brain cortex. Orally administered anthocyanins were also active in the brain, suppressing stress-induced cerebral oxidative stress and dopamine abnormalities in distressed mice. These effects of anthocyanin treatment suggest their possible usefulness for the treatment of cerebral disorders related to oxidative stress.     

Dietary phosphatidylinositol prevents the development of nonalcoholic fatty liver disease in Zucker (fa/fa) rats

Recent studies have shown that dietary phospholipids, especially phosphatidylcholine and phosphatidylserine, have various beneficial biological effects. However, there are not enough data concerning the physiological function of dietary phosphatidylinositol (PI). The metabolic syndrome, a cluster of metabolic abnormalities such as dyslipidemia, diabetes mellitus, and hypertension, is a widespread and increasingly prevalent disease in industrialized countries. Nonalcoholic fatty liver disease (NAFLD) is often associated with features of the metabolic syndrome. NAFLD describes the spectrum of liver damage ranging from hepatic steatosis to steatohepatitis, liver fibrosis, and cirrhosis, and it is emerging as the most common liver disease worldwide. The present study examined whether dietary PI protects Zucker ( fa/ fa) rats from the metabolic syndrome. For 4 weeks, rats were fed semisynthetic diets containing either 7% soybean oil or 5% soybean oil plus 2% PI. Dietary PI markedly prevented the development of hepatomegaly and hepatic steatosis and lowered hepatic injury markers in serum. Additionally, hyperinsulinemia was relieved by the feeding of dietary PI in Zucker rats. These effects were attributable to an increase in serum adiponectin, enhancement of fatty acid beta-oxidation, and suppression of mRNA expression of inflammatory genes in the liver. This is the first report that dietary PI increases serum adiponectin level and prevents the development of NAFLD in a rat model of the metabolic syndrome.     

d -Alanine-d -alanine ligase gene ( ddl ) of Erwinia chrysanthemi strain EC16 II: analysis of pectate lyase regulation using ddl ? mutant

 Erwinia chrysanthemi (Ech) triggers soft rot disease mainly by secreting pectate lyase (Pel), which is regulated in a complex manner by many regulatory genes. In a previous study, we used a gene dosage method to show that the ddl gene, which encodes d-alanine-D-alanine ligase, reduced Pel production and tissue maceration by Ech strain EC16n. In this study, the ddl ⁻ marker-exchanged mutant was shown to overcome the long growth lag caused by various salts in the growth medium and to increase Pel production over that by EC16n, especially in a medium containing magnesium salts. Thus, ddl seems to regulate Pel production in a negative manner. Because the profiles of a gel shift assay using the pelE promoter region as the target DNA with crude extracts of EC16n and ddl ⁻ mutant were distinguishable, Ddl is thought to affect the binding of other regulatory proteins. Expression of the ddl gene was induced in the medium containing a low-molecular-weight fraction of potato extract, but it was reduced in that containing both polygalacturonic acid (PGA) and the fraction. The repression of ddl expression by PGA should contribute in part to the in planta hyperinduction of Pel. Received: May 15, 2002 / Accepted: June 20, 2002