The ex vivo effects of eyestalk peptides on ovarian vitellogenin gene expression in the kuruma prawn Marsupenaeus japonicus

Seven major peptides belonging to the crustacean hyperglycemic hormone family were purified from the sinus gland located in the eyestalk of the kuruma prawn Marsupenaeus japonicus, and their effects on vitellogenin gene expression were examined using the ex vivo ovary incubation system. Six molecular species of crustacean hyperglycemic hormone, Pej-SGP-I, -II, -III, -V, VI, and VII, displayed significant inhibitory effects on vg expression with almost the same efficacies, whereas Pej-SGP-IV (known as molt-inhibiting hormone) did not. Two chromatophorotropic peptides, red pigment-concentrating hormone and pigment-dispersing hormone, which were also present in the sinus glands, did not have a clear effect on the gene expression levels in this incubation system. These results suggest that the six crustacean hyperglycemic hormones are potentially capable of acting as vitellogenesis-inhibiting hormones in M. japonicus.     

Expression of Medicago truncatula ecto-apyrase MtAPY1;1 in leaves of Nicotiana benthamiana restricts necrotic lesions induced by a virulent fungus

Ecto-apyrase(s) participates in cell-wall-associated defense through ATP hydrolysis. Here we analyzed Medicago truncatula genes through cDNA screening and in silico analyses against known databases. This study revealed seven genes, five of which (MtAPY1;1 to MtAPY1;5) are members of a legume-specific family, whereas two genes (MtAPY2;1 and MtAPY2;2) are close to those in other plants. Agrobacterium-based transient expression in Nicotiana benthamiana, combined with a c-myc epitope tag technology, confirmed that the MtAPY1;1 is a secreted protein. Transient expression of MtAPY1;1 in leaves of N. benthamiana restricted disease development by a virulent fungus, suggesting a role in disease resistance.     

Production of a new low-caffeine hybrid coffee and the biochemical mechanism of low caffeine accumulation

The GCAs are new tetraploid interspecific hybrids developed in Madagascar from Coffea eugenioides, C. canephora and C. arabica. Selected GCA having genotype UF1023 contained 0.37% DW caffeine and no detectable theobromine in green beans. Low caffeine accumulation in GCA plants is due mainly to the low biosynthetic activity of purine alkaloids, possibly the extremely weak N-methyltransferase reactions in caffeine biosynthesis. No significant catabolic activity of caffeine was found in GCA-UF1023, in common with almost all coffee plants including C. arabica.     

Relative sensitivity of duckweed Lemna minor and six algae to seven herbicides

We investigated the relative sensitivity of duckweed Lemna minor and six species of algae to seven herbicides, using an efficient high-throughput microplate-based toxicity assay. First, we assessed the sensitivity of L. minor to the seven herbicides, and then we compared its sensitivity to that of previously published data for six algal species based on EC₅₀ values. For five herbicides, the most sensitive species differed: L. minor was most sensitive to cyclosulfamuron: Raphidocelis subcapitata was most sensitive to pretilachlor and esprocarb: Desmodesmus subspicatus was most sensitive to pyraclonil; and Navicula pelliculosa was most sensitive to pyrazoxyfen. Simetryn was evenly toxic to all species, whereas 2,4-D was evenly less toxic, with only small differences in species sensitivity. These results suggested that a single algal species cannot represent the sensitivity of the primary producer assemblage to a given herbicide. Therefore, to assess the ecological effects of herbicides, aquatic plant and multispecies algal toxicity data sets are essential.     

Practical method combining loop-mediated isothermal amplification and bait trap to detect <Emphasis Type="Italic">Pythium helicoides</Emphasis> from hydroponic culture solutions

Two detection methods combining loop-mediated isothermal amplification (LAMP) and a bait trap were developed to detect Pythium helicoides in greenhouses containing roses, miniature roses, and poinsettias in hydroponic culture systems. In “Bait-LAMP”, a crude extract derived from perilla seeds as the bait was used in the LAMP reaction, whereas in the “Bait culture-LAMP”, a crude extract of mycelia grown out from perilla seeds onto Pythium-selective medium served as the bait. The two methods are simple and rapid for practical monitoring of P. helicoides in hydroponic culture systems.     

Development of bovine embryos cultured in CR1aa and IVD101 media using different oxygen tensions and culture systems

The aim of the present study was to optimise the culture conditions for the in vitro production of bovine embryos. The development of in vitro fertilised bovine oocytes in CR1aa supplemented with 5% calf serum and IVD101 culture media were compared using traditional microdrops and Well of the Well (WOW) culture systems either under 5% or 20% oxygen tension. After 7 days of culture, a significantly higher blastocyst formation rate was obtained for embryos cultured in CR1aa medium compared to those cultured in IVD101, irrespective of O2 tensions and culture systems. The blastocyst formation in IVD101 was suppressed under 20% O2 compared to 5% O2 . Despite their similar total cell numbers, higher rates of inner cell mass (ICM) cells were observed in blastocysts developed in IVD101 medium than in those developed in CR1aa, irrespective of O2 tensions. There was no significant difference in blastocyst formation, total, ICM and trophectoderm (TE) cell numbers between embryos obtained by microdrop and WOW culture systems irrespective of the culture media and O2 tensions used. In conclusion, CR1aa resulted in higher blastocyst formation rates irrespective of O2 tension, whereas IVD101 supported blastocyst formation only under low O2 levels but enhanced the proliferation of ICM cells.     

In Vitro Maturation and Development of Porcine Oocytes Cultured in a Straw or Dish Using a Portable Incubator with a CO2 Chamber

We investigated the effects of a portable incubator with a CO₂ chamber on the viability and development of porcine oocytes/embryos for their transportation and examined the operational suitability of a straw or dish as a container for culturing the oocytes or embryos in the portable incubator. In the first experiment, the cumulus‐oocyte complexes (COCs) were placed either in a dish or straw; and they were then cultured for 44 h in a standard CO₂ incubator, in the CO₂ chamber in an incubator, or in the CO₂ chamber in a portable incubator. The matured oocytes were fertilized with frozen‐thawed spermatozoa and then cultured in a dish in the standard CO₂ incubator for 8 days. There were no differences in the proportions of oocytes reaching metaphase II stage among the groups. However, the proportions of cleavage and development to blastocysts derived from oocytes matured in a straw were lower than those from oocytes matured in a dish, irrespective of the type of incubator used. In the second experiment, the COCs were matured in a dish in the standard CO₂ incubator, and the matured oocytes were fertilized and then placed either in a dish or straw. These were then cultured for 8 days in the standard CO₂ incubator or portable incubator. Some zygotes cultured in the portable incubator developed to the blastocyst stage. The proportions of cleavage and development to blastocysts were significantly lower for putative zygotes cultured in straw than for those cultured in dish, irrespective of the type of incubator used. Our results indicate that a portable incubator with a CO₂ chamber can maintain the viability and development of oocytes/embryos, but the straw is not a suitable system for in vitro culture of the oocytes/embryos during transportation.     

Systemic injection of FGF-2 stimulates endocortical bone modelling in SAMP6, a murine model of low turnover osteopenia.

The effects of systemically administered fibroblast growth factor-2 (FGF-2) at doses of 0.1 and 0.3 mg/kg/day for 7 days were investigated 5-week-old male SAMP6 mice, a model of low turnover osteopenia. The bone histomorphometry in the distal epiphyseal growth plate of the femur showed that 0.3 mg/kg/day of FGF-2 decreased the longitudinal growth rate and cartilage cell production rate and increased the growth plate width. Growth plate chondrocytes showed the features of defective endochondral ossification at the same dosage level. In the distal one third of the femur, the marrow trabecular area, endocortical mineral apposition rate and/or bone formation rate were increased in both the SAMP6 mice given 0.1 and 0.3 mg of FGF-2/kg/day. In this region, the endocortical osteoblasts were hypertrophied with some layers of overlying proliferated fibroblastic mesenchymal cells. The presence of small foci of bone formation within the layers of these mesenchymal cells indicates their osteogenic potential. On the other hand, the periosteal bone formation rate in the mid-shaft of the femur was depressed in the 0.3 mg/kg/day group. These results suggest that systemically administered FGF-2 may have the possibility to increase the peak bone mass in SAMP6 by stimulating the osteoprogenitor cells to proliferate and differentiate into osteoblasts and enhancing endocortical bone modelling. The higher dose of FGF-2, however, inhibited both endochondral and periosteal bone formation.     

Structural characterization of plancitoxin I, a deoxyribonuclease II-like lethal factor from the crown-of-thorns starfish Acanthaster planci , by expression in Chinese hamster ovary cells

Plancitoxin I, the major lethal factor from the spines of crown-of-thorns starfish Acanthaster planci, is a 37 kDa protein composed of two different subunits, and it has potent hepatotoxicity. It is homologous with mammalian deoxyribonuclease II (DNase II) and exhibits DNase activity responsible for the hepatotoxicity. To obtain information on the structure–activity relationship of plancitoxin I, various mutants were expressed in Chinese hamster ovary cells and examined for DNase activity. The results with deletion mutants revealed the requirement of the signal peptide for the expression of intact plancitoxin I and the inability of each subunit to hydrolyze DNA. Mutation at the N-glycosylation site (Asn-274) did not reduce DNase activity, supporting the absence of carbohydrate moieties in the molecule. The mutant H303A exhibited no DNase activity, suggesting the importance of His-303 for the enzymatic activity. No DNase activity was detected in C29A, C169A, C318A and C337A, indicating that four Cys residues are critical to the enzymatic activity. However, DNase activity was completely maintained in C263A and somewhat reduced in C277A and C357A. Based on the results with the Cys-specific mutants, plancitoxin I was assumed to contain three disulfide bridges (29–169, 277–357 and 318–337) and one free Cys-263.     

Selection for increased adult body weight in mouse lines with and without the rat growth hormone transgene

SUMMARY: Four lines of mice with and without the rat growth hormone (rGH) transgene were developed to measure responses to selection for increased 42-day body weight and evaluate fitness of mice with and without the rGH transgene. Each line contained selected and unselected (control) sublines. At the last three generations of selection (Generations 12-14), selected sublines differed from unselected controls by 3.8 to 4.7 g (14.8 to 19.8%) in 42-day weight, -0.5 to -8.3% in fertility, and 0.5 to 1.6 in litter size at birth. The origin of the lines (W: previously selected for 42-day weight and C: unselected) affected 42-day weight, i. e. 42-day weight of mice originating from W was significantly (P < 0.01) heavier than that of mice originating from C. Responses to selection, as measured by the deviation of the selected subline from the control, continued to be positive over 14 generations. Realized heritability of 42-day weight ranged from 0.30 to 0.42. The rGH transgene that increased 63-day weight by 54% was not found at Generation 12. The unexpected loss of rGH transgene was due to poor fitness of mice with the rGH transgene. Mice with the transgene had lower fertility rate than those without the transgene (50.0 to 73.7% vs. 95.0%), smaller litter size (6.8 to 7.8 vs. 8.6) and poorer survival of the progeny (69.2 to 74.5% vs. 88.3%). Based on these data, selective advantage/disadvantage of the rGH transgene in the fitness traits was estimated quantitatively. The results from the study on growth and reproductive traits suggest that desirable effects of gene transfer on a specific trait (42- and 63-day weight in the present study) might be offset by undesirable effects on other traits (e. g., reproduction and survival) in some cases of transgenic animals. ZUSAMMENFASSUNG: Selektion auf hohes adultes Gewicht in M?uselinien mit und ohne Rattenwachstumshormon-Transgenen Vier M?uselinien mit und ohne das Rattenwachstumshormon (rGH) Transgen wurden zur Messung des Selektionserfolges auf gesteigertes 42-Tage-K?rpergewicht entwickelt, um auch Fitne? zu prüfen. Jede Linie bestand aus einer selektierten und aus einer unselektierten (Kontroll-)Unterlinie. In den drei letzten Selektionsgenerationen (Generationen 12-14) unterschieden sich die selektierten Unterlinien um 3,8 bis 4,7 g (14,8 bis 19,8%) im 42-Tage-Gewicht, -0,5 bis -8,3% in Fruchtbarkeit und 0,5 bis 1,6 in Wurfgr??e bei Geburt. Der Ursprung der Linien (W: früher auf 42-Tage-Gewicht selektiert, C: unselektiert) beeinflu?te das 42-Tage-Gewicht. M?use aus der ersten waren signifikant (p < 0,01) schwerer als jede aus C. Selektionserfolg gemessen als Abweichung von der Kontrolle war über 14 Generationen positiv, realisierte Heritabilit?t variierte von 0,3 bis 0,42. Das rGH-Transgen steigerte das 63-Tage-Gewicht um 54%, aber war in der Generation 12 nicht mehr vorhanden. Der unerwartete Verlust des RGH-Transgens war auf schlechte Fitne? der besitzenden M?use zurückzuführen. Transgene M?use hatten geringere Fruchtbarkeit als jene ohne das Transgen (50 bis 73,7% im Vergleich zu 95%), kleinere Wurfgr??e (6,8 bis 7,8 gegenüber 8,6) und schlechtere überlebensrate der Nachkommen (69,2 bis 74,5% im Vergleich zu 88,3%). Die Ergebnisse erlauben die quantitative Sch?tzung der selektiven Vorteile/Nachteile des rGH-Transgens im Bezug auf Fitne?. Erwünschte Wirkungen von Gentransfer auf spezifische Merkmale (41- und 63-Tage-Gewicht in dieser Untersuchung) k?nnen durch unerwünschte Wirkungen von Transgenen auf andere Merkmale (Reproduktion und überleben) neutralisiert werden.