Molecular cloning and biochemical characterization of medaka (Oryzias latipes) lysosomal neu4 sialidase

Glycoconjugates are known to be involved in many physiological events in vertebrates. Sialidase is one of the glycosidases, which removes sialic acid from glycoconjugates. In mammals, the properties and physiological functions of sialidases have been investigated, while there is little understanding of fish sialidase. Here, to investigate the significance of fish neu4 sialidase, neu4 gene was cloned from medaka brain mRNA and identified. Sialidase-specific motifs (GPG, YRVP and Asp-Box) were well conserved in the medaka neu4 polypeptide. Optimal pH of medaka neu4 sialidase was 4.6, but its activity was sustained even at neutral and weak alkaline pH. The neu4 considerably cleaved sialic acid from 4-methylumbelliferyl-N-acetyl-α-d-neuraminic acid and sialyllactose, but not from ganglioside and fetuin, which are good substrates for human NEU4. neu4 activity was mostly detected in mitochondria/lysosome fraction after biochemical fractionation, and indirect immunofluorescence assays revealed neu4 localization in lysosome in neu4 overexpressed cells. Next, developmental change in medaka neu4 and other sialidase mRNA levels were estimated by real-time PCR. Each sialidases showed different expression patterns in embryonic development: neu4 was up-regulated at late developmental stage in embryo, and neu3a mRNA level was quite high in 0.5 dpf. On the other hand, neu3b expression was drastically increased after hatching, suggesting that each sialidase may play a different role in embryonic development.     

Purification and Characterization of Cathepsin B from the Muscle of Horse Mackerel Trachurus japonicus

An endogenous protease in fish muscle, cathepsin B, was partially purified and characterized from horse mackerel meat. On SDS-PAGE of the purified enzyme under reducing conditions, main protein bands were detected at 28 and 6 kDa and their respective N-terminal sequences showed high homology to heavy and light chains of cathepsin B from other species. This suggested that horse mackerel cathepsin B formed two-chain forms, similar to mammalian cathepsin Bs. Optimum pH and temperature of the enzyme were 5.0 and 50 °C, respectively. A partial cDNA encoding the amino acid sequence of 215 residues for horse mackerel cathepsin B was obtained by RT-PCR and cloned. The deduced amino acid sequence contains a part of light and heavy chains of cathepsin B. The active sites and an N-glycosylation site were conserved across species. We also confirmed that the modori phenomenon was avoided by CA-074, a specific inhibitor for cathepsin B. Therefore, our results suggest that natural cysteine protease inhibitor(s), such as oryzacystatin derived from rice, can apply to thermal-gel processing of horse mackerel to avoid the modori phenomenon. Meanwhile, this endogenous protease may be used for food processing, such as weaning meal and food for the elderly.     

Seed formation in triploid loquat (Eriobotrya japonica) through cross-hybridization with pollen of diploid cultivars

As the fruits of loquat (Eriobotrya japonica, 2n = 2x = 34) carry large seeds, the breeding of seedless loquat has long been a goal. The recent creation of triploid cultivars (2n = 3x = 51) and the application of gibberellins allow commercial production of seedless loquat, but the possibility of seed formation in triploid loquats has not been carefully investigated. Through crossing experiments and cytological observations of meiosis and pollen tube growth, we found that the triploid line 3N-N28 was essentially self-sterile, but developed seeds on pollination with pollen from diploid cultivars at rates of up to 5.5%. Almost half of the seedlings survived to 5 months, and carried diploid (2n = 34), tetraploid (2n = 68), or aneuploid chromosome numbers. Our results suggest that triploid loquat cultivars might retain the risk of seed formation. Protection from pollination by diploid cultivars or the development of new triploid cultivars will be necessary to ensure the production of seedless loquat fruits.     

Genomic variations among Bartonella henselae isolates derived from naturally infected cats

The purpose of this study was to understand the mechanisms of persistent infection with Bartonella henselae in cats. Blood samples were collected from three naturally infected cats for 24 months. These cats were confirmed to be persistently infected with B. henselae by serological and bacteriological examination. Relapsing bacteremia was found in all three cats with intervals of 3-19 months. Following the peaks of bacteremia, increases of specific antibody titer were observed in these cats. To examine the genetic differences among the isolates derived from the first and following bacteremia, the genome DNA patterns of the restriction enzyme fragment length polymorphism (RFLP) of the isolates were examined by pulsed field gel electrophoresis. The isolates derived from the first bacteremia showed an identical RFLP pattern in each of the three cats. The isolates derived from the following peaks, however, showed 1-3 of different RFLP patterns in these cats. Furthermore, the isolates showing different RFLP patterns from those of the first bacteremia were also detected at the following bacteremic peaks in all three cats examined. The 16S ribosomal RNA (rRNA) gene type of all isolates was found to be 16S rRNA type I. The emergence of genetically distinct organisms at various peaks of bacteremia may contribute to the establishment of persistent infection in the naturally infected cats.     

Partial characterization of structure and function of a xylanase gene from the rumen hemicellulolytic bacterium Eubacterium ruminantium

A gene encoding for xylanase activity in the rumen hemicellulolytic bacterium Eubacterium ruminantium was cloned into pBR322 in Escherichia coli (E. coli ). The primary clone had a 5.7 kb insert produced by Eco RI partial digestion. Subcloning followed by sequencing allowed for the discovery that this enzyme has a glycosyl‐hydrolase family 10 catalytic domain with a family 9 carbohydrate binding module at C‐terminus and a region partially homologous to a family 22 carbohydrate binding module at N‐terminus. Cloned xylanase is specifically active against xylan and oligoxyloside to produce xylobiose and xylotriose, showing optimal pH and temperature at 7.0 and 50°C, respectively. Molecular size of the xylanase (91 kDa) was confirmed by zymogram analysis of the E. coli clone, which agreed with the predicted size from the DNA sequence. Functions of the two modules at C‐ and N‐termini were evaluated by using xylanase variants with and without the respective module and the C‐terminal module was found to be functional in binding to acid‐swollen cellulose and insoluble oat‐spelt xylan, whereas the N‐terminal module was inactive for binding them.     

Characterization of tetraploid plants regenerated via protoplast culture of Iris fulva and their crossability with Japanese irises

Characteristics such as flower form, size and color of outer and inner perianths, anthocyanins in outer perianths, size, color and fertility of pollen and self-fertility of diploid and tetraploid lines regenerated via protoplast culture of Iris fulva were examined and compared with those of the diploid wild line. Among these characteristics, flower form, inner and outer perianth sizes of the tetraploid lines were noticeable, because these lines had upward flower forms and bigger flowers than diploid lines. Furthermore, reciprocal crosses between diploid or tetraploid lines of I. fulva and I. ensata and those of I. fulva and I. laevigata were performed. Three seedlings were obtained from the cross of tetraploid I. fulva × diploid I. laevigata through embryo rescue. One of them was identified as the interspecific hybrid between tetraploid I. fulva and I. laevigata by flow cytometoric (FCM), cytological and molecular (RAPD) analyses. This is the first report on production of hybrids from these lines. I. fulva has unique brown flowers, and this trait could be very useful for flower color breeding of I. laevigata which lacks this color. Therefore, the hybrid of I. fulva (4×) × I. laevigata may be the best available gene source for brown color breeding of this species.     

Evaluation of the live vaccine efficacy of virulence plasmid-cured,and phoP- or aroA-deficient Salmonella enterica serovar Typhimurium in mice

We evaluated the protective efficacy of 94-kb virulence plasmid-cured, and phoP- or aroA-deficient strains of Salmonella enterica serovar Typhimurium (ΔphoP or ΔaroA S. Typhimurium) as oral vaccine candidates in BALB/c mice. Two weeks after the completion of 3 oral immunizations with 1 × 10⁸ colony-forming units (CFU) of virulence plasmid-cured, and ΔphoP or ΔaroA S. Typhimurium at 10-day intervals, S. Typhimurium lipopolysaccharide (LPS)-specific mucosal secretory immunoglobulin A (s-IgA) antibody titers were detected in the cecal homogenate, bile and lung lavage fluid, but not in the intestinal lavage fluid. In addition, the increases in S. Typhimurium LPS-specific immunoglobulin G (IgG) and IgA antibody titers in the serum were also observed 2 weeks after completing 3 oral immunizations with virulence plasmid-cured, and ΔphoP or ΔaroA S. Typhimurium. The series of 3 oral immunizations protected the mice against an oral challenge with 5 × 10⁸ CFU of the virulent strain of S. Typhimurium, suggesting that both the virulence plasmid-cured, and ΔphoP and ΔaroA S. Typhimurium strains are promising candidates for safe and effective live S. Typhimurium vaccines.     

Effect of shelter acclimation on the post-release movement and putative predation mortality of hatchery-reared black-spot tuskfish <Emphasis Type="Italic">Choerodon schoenleinii</Emphasis>, determined by acoustic telemetry

In this study, the effect of shelter acclimation on the post-release movement and putative predation mortality of hatchery-reared black-spot tuskfish Choerodon schoenleinii was examined using acoustic telemetry. We acclimated four 1-year-old fish to shelters in cages before release and compared their movements with six nonacclimated fish. Since it was not possible to compare the behavioral pattern between the former and the latter fish due to the short periods the latter fish were available to be monitored, we also compared their movements with those of large nonacclimated fish that were less likely to be preyed upon. Sixty-seven percent of the nonacclimated fish showed atypical movements before the signals ceased to be detected, a pattern that suggested a predation event had occurred, whereas none of the acclimated and large nonacclimated fish showed the atypical movements. In addition, the probability of detection cessation was about 13 times lower in the acclimated than nonacclimated fish. The signal detection patterns suggest that the acclimated fish utilized night-time shelters from the first night after release, while the large nonacclimated fish started to utilize shelters several days after release. Therefore, it is likely that the shelter acclimation enhanced the shelter utilization by tuskfish, possibly decreasing post-release predation mortality.     

Nitrogen fixation in paddy soils

Several important features of the N. fixation in paddy fields which were reported previously were confirmed and some new additional results regarding the evaluation of the N₂ fixation in the rhizosphere were obtained by reinvestigation in the fields. In addition, rice plants were cultivated in the submerged soil in pots and various parts of the soil were analyzed for the N₂-fixing activity as well as several other properties. The results of the pot experiments were found to be fairly similar to those observed in the field investigations, indicating the validity of the submerged soil in a pot as a rather simulated model for the actual paddy field. By using this model system, the following facts were ascertained: (1) Water-percolation had almost no effect on the N₂-fixing activities of both the rhizosphere and the non-rhizosphere soils. (2) Suppressing effect of washing the root of rice plant on the N₂-fixing activity was slight in the seedling stage and marked in the tillering and flowering stages. (3) The N₂-fixing activity of a single rice root varied from tip to base.     

QTL analysis for soil‐surface roots originating from a New Plant Type rice (Oryza sativa L.)

The introgression line YTH16 harbouring chromosome segments from the New Plant Type cultivar IR65600–87–2‐2–3 with genetic background of an Indica Group rice IR 64 forms soil‐surface roots. To clarify the genetic mechanism, QTL analysis was performed using hybrid populations derived from a cross between IR 64 and YTH16. A total of eight QTLs were detected in the three introgressed segments on chromosomes 2, 5 and 7. Seven chromosome segment lines (CSLs) combining these three QTL regions were selected from the progenies. The two CSLs harbouring a single region (excluding the CSL with a region on chromosome 5) showed high scores and low means of root angle distribution in comparison with IR 64. Four CSLs harbouring two or three regions showed high scores and low means of root angle distribution in comparison with YTH16 and the CSLs harbouring a single QTL region. These results indicated that the soil‐surface rooting of YTH16 was controlled by the three QTLs’ regions and that chr. 5 particularly played a role in supporting the effect with others.