In vivo gene transfer into skeletal muscle of neonatal chicks by electroporation

Chicks (Gallus gallus domesticus) show considerable growth of skeletal muscle during the neonatal period. The in vivo gene transfer method is useful for studying gene function and can be employed to elucidate the molecular mechanisms of skeletal muscle growth in chicks. We evaluated the following conditions for gene transfer to the skeletal muscle of neonatal chicks by electroporation: (i) voltage; (ii) age of the chick; (iii) plasmid DNA injected amount; and (iv) duration of gene expression. The results obtained from this study indicate that the most efficient gene transfer condition was as follows: 75 µg of plasmid DNA encoding β‐galactosidase was injected into the gastrocnemius muscle of chicks at 4 days of age electroporated at 50 V/cm. In addition, peak transferred gene expression was observed from 3 days to 5 days after electroporation. Our results provide optimal electroporation conditions for elucidating the gene function related to skeletal muscle growth and development in neonatal chicks.     

Guar foaming albumin: a low molecular mass protein with high foaming activity and foam stability isolated from guar meal

The water extract of guar meal ( Cyamopsis tetragonolobus) was examined for its foamability. Compared with egg white, the extract showed an extraordinary foam stability: no drainage after 3 h of standing in contrast to 65% drainage for egg white at the same protein concentration. The acid-precipitated protein from the extract was responsible for the high foamability and designated guar foaming albumin (GFA). The foaming activity of GFA was 20 times higher than that of egg white. GFA consisted of two subunits with molecular masses of 6 and 11 kDa linked to each other through disulfide bonds. The cleavage of disulfide bonds in GFA affected the foamability only slightly. GFA remarkably decreased the surface tension of water at low protein concentrations. Immunoblotting analysis demonstrated that GFA did not react to the antisera from allergic patients against plant food. These results suggest that GFA serves as an effective food additive in developing protein-stabilized foam.     

Tastes and structures of bitter peptide, asparagine-alanine-leucine-proline-glutamate, and its synthetic analogues

Asn-Ala-Leu-Pro-Glu (NALPE) is a strong bitter peptide with a minimum response threshold (MRT) of 0.074 mM. To elucidate the relationship of spatial structure and bitterness on peptides, NALPE and its analogues, NALPW, NALPS, NALPL, NALPP, NALPD, and NALPR, were synthesized and sensorially evaluated. Structural analysis using computer simulation for each peptide revealed that the presence of a polar group and hydrophobic bitter amino acids, the composition of hydrophobic regions, the spatial orientation of the polar group and hydrophobic regions, and the proximity between polar groups and hydrophobic regions faced within the same plane space may be the major determinants for the taste type and intensity of peptide bitterness.     

Conditioning of karyoplasts for producing somatic nuclear transferred gonadal germ cells in domestic chickens

The objective of this study was to establish a protocol for generating karyoplasts that can be used to produce somatic nuclear transferred gonadal germ cells (snt-GGCs) in domestic chickens. Karyoplasts were produced by centrifuging cultured fibroblasts from 10-day-old chick embryos at 10,000 x g in the presence of 1.0 microg/ml cytochalasin B. The number of karyoplasts was significantly (P<0.05) higher and the diameters of the karyoplasts were significantly (P<0.05) smaller when fibroblasts were centrifuged for 60 min than for 10 or 30 min. It was possible to generate snt-GGCs by electrofusion of GGCs with karyoplasts produced from cryopreserved or serum-starved fibroblasts. These results indicate that karyoplasts generated from 10-day-old chick embryos can be used to produce snt-GGCs even after cryopreservation and serum starvation of the fibroblasts.     

Allantoin production and its utilization in relation to nodule formation in soybeans

A remarkable accumulation and utilization of allantoin are observed in soybean plant bearing modules (Glycine max variety A62-1). To study ita physiological role, changes in activities and distribution of the enzymes concerning the purine catabolism (i.e., xanthine oxidase, uricase, allantoinase and allantoicase) were measured during development.

Uricase activity was found in the radicles in the early stage of seedling development, and no difference of the enzyme activity was detected between the radicles of the nodulating variety (A62-) and non-nodulating variety (A62-2). On maturation the activity disappeared rapidly in radicles and appeared in leaves, pods and nodules. The activity in nodules was much higher than that in the leaves and the pods throughout the culture period.

Xanthine oxidase activity was found in the radicles and nodules.

Allantoinase activity was present in all regions of both varieties, and the specific activity in the nodules Was 2-10 times that in other regions.

Allantoicase activity was detected in the leaves, but not in the nodules.

The distribution of these enzymes suggested that allantoin is formed in the nodules and utilized mainly in the leaves after translocation.

Changes in the allantoin concentration and in the activities of uricase and allantoicase were studied in non-nodulating soybean (A62-2) grown in a culture solution containing various nitrogenous compounds. The allantoin concentration in the leaves increased as the plants were cultured in the solution containing 100 ppm of nitrate-N, urea-N or allantoin-N, whereas no uricase activity in the root was detected. The specific activity of uricase decreased in the process of plant development in all cultures. Allantoicase activity increased in the roots and leaves when the plants were cultured in this solution containing 100 ppm of urea-N or allantoin-N, indicating that allantoin is utilized easily in the soybean plants.

Intracellular localization of uricase in the nodules was studied by the method of linear sucrose density gradient centrifugation. The uricase activity was associated with the bacteroids, density 1.25.

The uricase activity peak was coincident with the peak of fonnic dehydrogenase activity (bacteria type), and not with peaks of catalase activity, cytochrome oxidase activity and absorbance at 280 nm. This evidence together with the observation by electron microscopy suggested that at uricase is associated with bacteroids.     

Seasonal and interannual variation in net ecosystem production of an evergreen needleleaf forest in Japan

Carbon dioxide (CO₂) flux was measured above the forest at the Fujiyoshida site on the northern slope of Mount Fuji in Japan in 2000?C2008 using an eddy covariance technique. The forest mainly consists of Japanese red pine (Pinus densiflora) and Japanese holly (Ilex pedunculosa). The 9-year average of monthly mean net ecosystem production (NEP) ranged from ?0.1?g?C?m^?2?day^?1 in January to 2.5?g?C?m^?2?day^?1 in May. The maximum net uptake was observed in May, although gross primary production (GPP) was highest in July. Variation in the leaf amount did not notably affect seasonal variation in GPP. This site was characterized by carbon uptake even in winter, if the meteorological conditions were conducive for photosynthesis and a resulting long period of carbon uptake. The 9-year averages of annual NEP, GPP, and ecosystem respiration (RE) were 388, 1,802, and 1,413?g?C?m^?2?year^?1, respectively. The annual NEP was lowest in 2003 and highest in 2004 over the 9?years. Year-to-year variability of NEP mainly depended on air temperature and photosynthetically active radiation in summer, and the dependence of the deviation of annual NEP on that of GPP was greater than that of RE. Long-term observational data indicated that the carbon uptake ability at the study site was at a moderate level in comparison with other temperate humid evergreen forests around the world. These data also indicated that the site had a high carbon uptake ability compared with other deciduous forests in Japan because of the duration of carbon uptake.     

Effect of the timing of first cleavage on in vitro developmental potential of nuclear-transferred bovine oocytes receiving cumulus and fibroblast cells

The aim of the present study was to examine whether cumulus and fibroblast cell nuclear-transferred oocytes, which have high and low potential to develop into normal calves, respectively, are different in terms of in their patterns of timing of first cleavage and in their relationships between timing of first cleavage and in vitro developmental potential. The timing of first cleavage was similar in both types of nuclear-transferred and in vitro fertilized oocytes. More than 86% of the oocytes cleaved within 24 h after activation or in vitro fertilization; these oocytes contributed to more than 98% of the total number of blastocysts in all three groups. The potential of oocytes that cleaved at different intervals to develop into blastocysts differed among the groups. The developmental potential of the cumulus cell nuclear-transferred oocytes and in vitro fertilized oocytes decreased with the increase in time required for cleavage. Fibroblast cell nuclear-transferred oocytes that cleaved at 20 h, an intermediate cleaving time, had higher potential to develop into blastocysts. The results of the present study suggest that the type of donor nucleus used for nuclear transfer affects the timing of first cleavage.     

An avirulence gene to rice cultivar K60 is located on the 1.6-Mb chromosome in <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> isolate 84R-62B

A Japanese differential rice cultivar K60 was tested with 114 F₁ cultures of Magnaporthe oryzae from a cross between isolates 84R-62B and Y93-245c-2. Segregation patterns of avirulence and virulence in the progeny suggested that avirulence on cv. K60 was controlled by a single gene derived from 84R-62B and tentatively named AvrK60. In the F₁ population, AvrK60 cosegregated with avirulence gene AvrPik on a small 1.6-Mb chromosome of 84R-62B and with the 1.6-Mb chromosome itself. Therefore, we suggest that, along with AvrPik, AvrK60 is also located on the 1.6-Mb chromosome of 84R-62B.     

Interspecific and intergeneric hybridization and chromosomal engineering of Brassicaceae crops

In Brassicaceae crop breeding programs, wild relatives have been evaluated as genetic resources to develop new cultivars with biotic and abiotic stress resistance. This has become necessary because of the diversification of ecotypes of diseases and pests, changing food preferences, advances in production technology, the use of new approaches such as in vitro breeding programs, and the need for economical production of F₁ seed. To produce potential new cultivars, interspecific and intergeneric hybridizations have been performed between cultivated species and between cultivated species and their wild relatives. Furthermore, interspecific and intergeneric hybrids have been successfully produced using embryo rescue techniques. In this paper, we review the interspecific and intergeneric incompatibilities between Brassicaceae crops and their wild relatives, and the production, characterization, and improvement of synthetic amphidiploid lines, alien gene introgression lines, alloplasmic lines, monosomic alien chromosome addition lines, and monosomic alien chromosome substitution lines. The goal is to provide useful materials to support practical breeding strategies and to study the genetic effects of individual chromosomes on plant traits, the number of genes that control a trait, their linkage relationships, and genetic improvement in Brassicaceae crops.