Biochemical and genomic analysis of neoculin compared to monocot mannose-binding lectins

Neoculin occurring in an edible tropical fruit is a heterodimeric protein which has both sweetness and a taste-modifying activity that converts sourness to sweetness. Both the primary and the overall tertiary structures of neoculin resemble those of monocot mannose-binding lectins. This study investigated differences in biochemical properties between neoculin and the lectins. Structural comparison between the mannose-binding sites of lectins and the corresponding regions of neoculin showed that there is at least one amino acid substitution at each site in neoculin, suggesting a reason for the lack of its mannose-binding ability. This was consistent with hemagglutination assay data demonstrating that neoculin had no detectable agglutinin activity. DNA microarray analysis indicated that neoculin had no significant influence on gene expression in Caco-2 cell, whereas kidney bean lectin (Phaseolus vulgaris agglutinin) greatly influenced various gene expressions. These data strongly suggest that neoculin has no lectin-like properties, encouraging its practical use in the food industry.     

Photosynthesis and photoassimilate transport during root hypoxia in Melaleuca cajuputi, a flood-tolerant species, and in Eucalyptus camaldulensis, a moderately flood-tolerant species

We compared the photosynthetic and photoassimilate transport responses of Melaleuca cajuputi Powell seedlings to root hypoxia with those of Eucalyptus camaldulensis Dehnh. Control and hypoxia treated roots were maintained in a nutrient solution through which air or nitrogen was bubbled. Under root hypoxic conditions, seedlings of M. cajuputi, a flood-tolerant species, maintained height growth, whereas seedlings of E. camaldulensis, a moderately flood-tolerant species, showed markedly decreased height growth compared with control seedlings. Root hypoxia caused decreases in whole-plant biomass, photosynthetic rate and stomatal conductance in E. camaldulensis, but not in M. cajuputi. Photoassimilate transport to roots decreased significantly in E. camaldulensis seedlings 4 days after treatment and starch accumulated in mature leaves. Photoassimilate supply to hypoxic roots of E. camaldulensis seedlings was, thus, limited by reduced photoassimilate transport rather than by reduced photosynthesis. In contrast, M. cajuputi seedlings showed sustained photoassimilate transport to hypoxic roots and persistent photosynthesis, which together provided a substantial photoassimilate supply to the roots. Sucrose accumulated in hypoxic E. camaldulensis roots, but not in hypoxic M. cajuputi roots. A stable, low sucrose concentration in hypoxic roots would let M. cajuputi seedlings prolong photoassimilate transport to the roots. Photoassimilate partitioning among the water-soluble carbohydrates, starch and structural carbohydrates within the roots was unaffected by root hypoxia in E. camaldulensis, but in M. cajuputi, partitioning was shifted somewhat from structural carbohydrates to water-soluble carbohydrates. This suggests that M. cajuputi seedlings are able to increase photoassimilate utilization in metabolism and sustain energy production under root hypoxic conditions.     

Serotonergic signals enhanced hamster sperm hyperactivation

In the present study, we investigated the regulatory mechanisms underlying sperm hyperactivation enhanced by 5-hydroxytryptamine (5-HT) in hamsters. First, we examined the types of 5-HT receptors that regulate hyperactivation. Hyperactivation was significantly enhanced by 5-HT_2A and 5-HT₄ receptor agonists. Moreover, the results of the motility assay revealed that 5-HT_2A, 5-HT₃, and 5-HT₄ receptor agonists significantly decreased the velocity and/or amplitude of sperm. Under 5-HT₂ receptor stimulation, hyperactivation was associated with phospholipase C (PLC), inositol 1,4,5-trisphosphate (IP₃) receptor, soluble adenylate cyclase (sAC), and protein kinase A (PKA). In contrast, under 5-HT₄ receptor stimulation, hyperactivation was associated with transmembrane adenylate cyclase (tmAC), sAC, PKA, and CatSper channels. Accordingly, under the condition that sperm are hyperactivated, 5-HT likely stimulates PLC/IP₃ receptor signals via the 5-HT_2A receptor and tmAC/PKA/CatSper channel signals via the 5-HT₄ receptor. After sAC and PKA are activated by these stimulations, sperm hyperactivation is enhanced.     

Mortality processes of hatchery‐reared Pacific bluefin tuna Thunnus orientalis (Temminck et Schlegel) larvae in relation to their piscivory

In mass culture of Pacific bluefin tuna Thunnus orientalis, yolk‐sac larvae of other species are fed as a major prey item to tuna larvae from 7 to 8 mm in total length. Marked growth variations in tuna larvae are frequently observed after feeding of yolk‐sac larvae, and this variation in the growth of tuna larvae is subsequently a factor leading to the prevalence of cannibalistic attacks. To elucidate details of the mortality process of hatchery‐reared tuna larvae after the initiation of yolk‐sac larvae feeding, we compared the nutritional and growth histories of the surviving (live) tuna larvae to those of the dead fish, found dead on the bottom of the tank, as direct evidence of their mortality processes. Cause of mortality of tuna larvae 3 and 5 days after the initiation of feeding of yolk‐sac larvae was assessed from nitrogen stable isotope and otolith microstructure analyses. Stable isotope analysis revealed that the live fish rapidly utilized prey fish larvae, but the dead fish had depended more on rotifers relative to the live fish 3 and 5 days after the initiation of feeding of yolk‐sac larvae. The growth histories based on otolith increments were compared between the live and dead tuna larvae and indicated that the live fish showed significantly faster growth histories than dead fish. Our results suggest that fast‐growing larvae at the onset of piscivory could survive in the mass culture tank of Pacific bluefin tuna and were characterized by growth‐selective mortality.     

Changes in the morphology and functions of vacuoles during the death of ray parenchyma cells in <Emphasis Type="Italic">Cryptomeria japonica</Emphasis>

Changes in the morphology and functions of vacuoles provide useful information about the mechanism of cell death. In the present study, we monitored the morphology and contents of vacuoles during the death of ray parenchyma cells in the conifer Cryptomeria japonica. In differentiating xylem, ray parenchyma cells had large central vacuoles. In sapwood, vacuoles in ray parenchyma cells contained proteins, an indication that one of the main functions of these vacuoles might be protein storage. A dramatic decrease in the protein content of some vacuoles was detected in the intermediate wood before the initiation of vacuole rupture. Although vacuole rupture was detected from the intermediate wood to the outermost heartwood, some vacuoles were obviously enlarged in the inner intermediate wood. Condensed nuclei were first observed after the rupture of these large vacuoles in ray parenchyma cells. It seems plausible that the autolysis of the contents of ray parenchyma cells might be caused by the rupture of the enlarged vacuoles in the inner intermediate wood.     

Weeds as the potential inoculum source of <Emphasis Type="Italic">Colletotrichum fructicola</Emphasis> responsible for strawberry anthracnose in Nara,Japan

Colletotrichum fructicola is a major causal agent among anthracnose pathogens of strawberry in Nara, Japan. We hypothesized that a wide range of weeds growing in and around strawberry fields are inoculum sources of the disease and investigated their potential as hosts of C. fructicola. We also examined the influence of herbicide treatment on C. fructicola sporulation on weeds. The fungus was detected on 31 of 541 (5.7%) leaves sampled from 13 weed species from 2005 to 2008. The fungus was most frequently isolated from leaves of Amaranthus blitum with an isolation frequency of 17.9%; inoculation of A. blitum with the pathogen caused brown leaf spots. Other weeds such as Digitaria ciliaris, Galinsoga ciliata, Solidago altissima, Erigeron annuus, and Sonchus oleraceus were found to harbor the fungus at lower rates (4.3–8.1%) without symptoms. C. fructicola formed acervuli on leaves of A. blitum, D. ciliaris, and S. oleraceus after plants were killed by a herbicide (glyphosate). These results demonstrated that infected weeds associated with strawberry cultivation are potential inoculum sources of C. fructicola, especially after herbicide treatment.     

Lack of Modifying Effects of Intratracheal Instillation of Quartz or Dextran Sulfate Sodium (DSS) in Drinking Water on Lung Tumor Development Initiated with 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in Female A/J Mice

The purpose of the present study was to investigate the effects of inflammation, induced by intratracheal instillation (i.t.) of quartz as an environmental factor in the lung or drinking of dextran sulfate sodium (DSS) as an environmental factor in the colon on lung tumors in female A/J mice initiated with NNK. For comparison, colonic preneoplastic lesions, aberrant crypt foci (ACF), were also assessed. A/J mice at 6 weeks of age were divided into 5 groups, and Groups 1, 2 and 3 were pretreated with NNK (2 mg / 0.1 ml saline / mouse, intraperitoneal injection) at week 0. For a week, 2% DSS in drinking water was administered to the mice in Groups 2 and 4 beginning in week 1. In week 2, the mice of Groups 3 and 5 were exposed to intratracheal instillation of quartz (0.1 mg/rat) suspended in 25 μl saline. The experiment was terminated after 16 weeks. The results for the lung tumors and colonic ACFs showed a lack of modifying effects of the inflammation in either site. Hematologically and histopathologically, the inflammation induced by 0.1 mg quartz in the lung and 2% DSS in the colon was lacking or only mild at the end of 16 weeks. These results suggest that there may be differences in sensitivity to inflammation that determine tumor promoting potential.     

Genomic evaluation using SNP‐ and haplotype‐based genomic relationship matrices in a closed line of Duroc pigs

A simulation analysis and real phenotype analysis were performed to evaluate the impact of three different relationship matrices on heritability estimation and prediction accuracy in a closed‐line breeding of Duroc pigs. The numerator relationship matrix (NRM), single nucleotide polymorphism (SNP)‐based genomic relationship matrix (GRM) (G_S), and haplotype‐based GRM (G_H) were applied in this study. We used PorcineSNP60 genotype array data (38 114 SNPs) of 831 Duroc pigs with four selection traits. In both heritability estimation and prediction accuracy, the accuracy depended on the number of animals with records. For heritability estimation, a large difference in the results among three relationship matrices was not shown, but the trend of the estimated heritabilities between GRMs, that is G_S < G_H, was shown in this population. For the accuracy of prediction values in test animals, the accuracies of prediction values obtained by two GRMs were higher than that by the NRM in this population. The accuracies obtained by GRMs using animals with no records were lower than that by the NRM using animals with their performance records, but were close to that by the NRM using animals with full‐sib testing records.     

Failure to gulp surface air induces swim bladder adenomas in Japanese medaka (Oryzias latipes)

In order to elucidate the effects of swim bladder inflation failure on swim bladder carcinogenesis, we investigated the sequential histopathological changes of swim bladders at 13, 24, 35, and 53 days post-hatch (dph) in medakas with an uninflated swim bladder, which was experimentally induced by denying access to the air–water interface between 0 and 6 dph. The reactive oxygen species (ROS) levels were measured at 24 dph. An uninflated swim bladder was induced in 47.3% of the fish denied access to the air–water interface (the denied group). The total incidence of swim bladder adenoma was 54.1% in the denied group; however, these tumors were observed in all fish with an uninflated swim bladder. In fact, these tumors were observed from 13 dph and onwards. The TBARS levels of the juveniles showed a 2.6-fold increase in fish with an uninflated swim bladder in the denied group compared to that in the control group. It is speculated that swim bladder inflation failure has some effects on the gas gland to produce ROS, leading to DNA damage in the gas glandular epithelium, which develops into swim bladder adenomas. Consequently, it is concluded that denying access to the air-water interface between 0 and 6 dph in medaka is an easy method of inducing swim bladder tumors in a short-term period, and is a useful method for producing tumor-bearing fish.     

Diagnostic value of recombinant nanoluciferase fused Toxoplasma gondii antigens in Luciferase-linked Antibody Capture Assay (LACA) for Toxoplasma infection in pigs

Toxoplasmosis is a widespread protozoan zoonosis. Since ingesting undercooked meat harboring Toxoplasma gondii cyst is considered one of the major transmission routes to humans, the screening of T. gondii in meat-producing animals can reduce the risk of food-borne toxoplasmosis in humans. Among serological diagnostic methods, Luciferase-linked Antibody Capture Assay (LACA) has been found to be a promising platform with high sensitivity and specificity. In this study, we aimed to evaluate recombinant nanoluciferase fused-T. gondii antigens (rNluc-GRA6, rNluc-GRA7, rNluc-GRA8 and rNluc-BAG1) for their potential use in LACA for pigs. As a result, the sensitivity of GRA6-, GRA7-, GRA8- and BAG1-LACA were 70.0%, 80.0%, 80.0% and 30.0% with specificity 87.0%, 81.5%, 74.1% and 50.0%, respectively. The cocktail LACA using a mixture of rNluc-GRA6, rNluc-GRA7 and rNluc-GRA8 indicated higher sensitivity (90.0%) and a similar specificity (96.3%) in comparison with the commercial ELISA kit. Compared to the Dye-Test as a reference test, cocktail LACA showed strong agreement (kappa value=0.811) when we assessed pig sera collected at the slaughterhouse. In addition, we also successfully established the rapid LACA format for the detection of Toxoplasma infection in pigs (called Rapid-LACA) in which the test could be performed within 30 min. In Rapid-LACA, the protein A pre-coated/blocked plates could be preserved at −30°C, 4°C or room temperature conditions for at least two months without compromising on the quality of assay.