Production of transgenic rabbits using centrifuged pronuclear zygotes

Superovulation of female rabbits was induced by subcutaneous injection(s) of porcine FSH. Zygotes were recovered 17 to 19 hr after hCG injection and were classified into two categories under a microscope equipped with Nomarski interference-contrast optics at x 200 magnification: (A) zygotes with clearly visible pronuclei, or (B) zygotes with visualized pronuclei after 10 min centrifugation at 12,000 x g. No significant difference between strains was found in the proportion of category-A zygotes (JW 72.6% vs NZW 79.3%). Pronuclei of category-A zygotes were located in the center of the cytoplasm, and the pronuclei of category-B zygotes were slightly moved by centrifugation toward the mass of cytoplasmic lipid droplets. Exogenous DNA solution (5 microg/ml of fusion gene composed of bovine alphaS1-casein promoter and human growth hormone structural gene) was microinjected into the pronucleus of the JW zygotes. The pronucleus of category-A zygotes with a mean volume of 7.4 pl swelled up to 16.6 pl (132% increase), while that of category-B zygotes with a mean volume of 6.1 pl swelled up to 15.9 pl (148% increase). Nevertheless, similar proportions of category-A and category-B zygotes developed into offspring after transfer to recipient females (11.1 and 11.2%, respectively). The efficiency to produce hGH-carrying transgenic rabbits was 0.9% (2/235) from category-A zygotes and 0.5% (1/215) from category-B zygotes (P>0.05). To date, transgenic rabbits have been produced without centrifugation of pronuclear zygotes. However approximately 25% of fertilized rabbit zygotes can be used for DNA microinjection after they have been centrifuged to visualize their pronuclei.     

Sympatric distribution of the two morphological types of the common tree shrew in Hat-Yai districts (South Thailand)

The two color types (grayish northern and reddish southern types) of the common tree shrew (Tupaia glis and Tupaia belangeri) were co-distributed in Hat-Yai region (South Thailand). Although the Isthmus of Kra in South Thailand has been considered as distribution barrier of the two types, the sympatric distribution of both types was confirmed in southern side of the Isthmus. In the principal component analysis, the skull measurement character from Hat-Yai region could also be separated into the northern and southern groups according to the skin color identification of corresponding individuals. We could generally distinguish the common tree shrew into two types by skull morphology as well as external skin color.     

Histochemistry of Complex Carbohydrate in the Major Salivary Glands of Hoary Bamboo Rats (Rhizomys purinosus)

The major salivary glands (parotid glands, monostomatic sublingual glands and submandibular glands) were obtained from hoary bamboo rats (Rhizomys purinosus) and fixed in Bouin's solution. Paraffin sections were subjected to a battery of staining methods including lectin staining for demonstration of complex carbohydrates. Among the three major salivary glands, unique histochemical features were observed in the submandibular gland. Different from most myomorpha species, submandibular glands of the hoary bamboo rats have two types of secretory cells in the secretory endpieces. One type of cells showed positive reactions with Alcian blue (AB)(pH2.5), periodic acid-Schiff (PAS) and some lectins (peanuts agglutinin, Griffonia simplicifolia I. Machura pomifera agglutinin). The granular ducts, which exist in animals belonging to suborder myomorpha, were not observed in the submandibular glands of this animal.     

Isolation of Staphylococcus species from the tonsils of healthy cattle and phage patterns of isolates

Staphylococci were found in the tonsils of 121 (75.2%) of 161 cattle. There were 15 different species, 10 belonging to novobiocin-sensitive species. The most predominant species was S. simulans (79.3% of the 121 carriers), followed by S. aureus (20.7%), S. chromogenes (10.7%) and S. epidermidis (8.3%). The other 11 species were present in 0.8 to 5.8%. Twenty-six unidentifiable isolates were isolated from 26 (21.5%) carriers. Sixty-two (51.2%) of the 121 carriers yielded two to five Staphylococcus species together while only one species could be found in each of the other 59 (48.8%). Combinations of S. simulans and other species were most frequently encountered in 50 (41.3%) of the 121 carriers. Twenty-four (96.0%) out of 25 S. aureus isolates, 3 (42.9%) of 7 S. hyicus isolates and 45 (25.4%) of 177 coagulase-negative staphylococci (13 species and unidentifiable isolates) isolates were phage typable. Most of S. aureus isolates were lysed by bovine phages 119 (n = 16) or 116 (n = 5). Thirty-three (25.4%) of 45 coagulase-negative staphylococci typable isolates with Pulverer's phage set showed the phage pattern ph5/ph9/ph10/ph12/ph13/U4/U14/U16/++ +U20/U46. The tonsils of cattle thus appear to be a suitable environment for Staphylococcus species, particularly novobiocin-sensitive species.     

Novel plasmodesmata association of dehydrin-like proteins in cold- acclimated Red-osier dogwood (Cornus sericea)

Dehydrins are proteins associated with conditions affecting the water status of plant cells, such as drought, salinity, freezing and seed maturation. Although the function of dehydrins remains unknown, it is hypothesized that they stabilize membranes and macromolecules during cellular dehydration. Red-osier dogwood (Cornus sericea L.), an extremely freeze-tolerant woody plant, accumulates dehydrin-like proteins during cold acclimation and the presence of these proteins is correlated with increased freeze tolerance (Karlson 2001, Sarnighausen et al. 2002, Karlson et al. 2003). Our objective was to determine the location of dehydrins in cold-acclimated C. sericea stems in an effort to provide insight into their potential role in the freeze tolerance of this extremely cold hardy species. Abundant labeling was observed in the nucleus and cytoplasm of cold-acclimated C. sericea stem cells. In addition, labeling was observed in association with plasmodesmata of cold-acclimated vascular cambium cells. The unique association of dehydrin-like proteins with plasmodesmata has not been reported previously.     

Distribution of boron in wood treated with aqueous and methanolic boric acid solutions

The distributions of boron in Japanese cedar (Cryptomeria japonica D. Don) sapwood blocks treated with aqueous or methanolic boric acid [B(OH)₃] solutions were explored through Raman spectroscopy and prompt gamma-ray analysis (PGA). B(OH)₃ was the sole boron species observed in Raman spectra of the wood blocks treated with either solution. Plots of weight gain of the treated wood blocks versus boron concentration in treatment solutions were found to be linear. The results indicated that the methanolic solution makes it possible to impregnate wood with much larger amounts of boron than the aqueous solution. PGA confirmed that B(OH)₃ was highly enriched near the end grains of the treated wood blocks. Raman measurements suggested that boron content in the bulk of the wood block is not as large as expected from the weight gain of the treated wood blocks when an ordinary air-drying method is used. It was concluded that the aqueous solution impregnates the cell walls of wood with boron more easily than the methanolic solution. Part of this report was presented at the 55th (Kyoto, March 2005) and 56th (Akita, August 2006) Annual Meetings of the Japan Wood Research Society. This article follows the previous rapid communication “Analysis of boron in wood treated with boric acid solutions using Doppler broadening method of prompt gamma-rays.” J Wood Sci (2006) 52:279–281     

First report of trunk rot caused by <Emphasis Type="Italic">Fomitiporia torreyae</Emphasis> in Kyoto prefecture on cultivars of Japanese cedar with no relatedness to ‘Sanbu-sugi’

Trunk rot, caused by Fomitiporia torreyae, is one of the most economically important sap rot diseases on Cryptomeria japonica, especially on a cutting cultivar ‘Sanbu-sugi’. This disease had been reported only from Chiba and Ibaraki prefectures in Japan; however, a similar trunk rot on Japanese cedars was found recently in Kyoto prefecture. We identified the causal agent of the trunk rot on Japanese cedars in Kyoto prefecture, clarified the genotype of ‘Sanbu-sugi’ (sensu stricto) using microsatellite markers, and then investigated the relatedness of these infected cedars with ‘Sanbu-sugi’. The pathogen was identified as F. torreyae by its DNA sequences and morphological characteristics of the fruiting body. The 25 ramets of ‘Sanbu-sugi’ had only one genotype based on eight microsatellite loci. Infected Japanese cedars in Kyoto were comprised of different genotypes and showed no close relatedness to ‘Sanbu-sugi’. This is the first report of trunk rot caused by F. torreyae on cultivars of Japanese cedar with no relatedness to ‘Sanbu-sugi’ outside of Chiba and Ibaraki prefectures and showed the potential for expansion of this disease to Japanese cedar plantations in other areas.     

Stem water transport of Lithocarpus edulis, an evergreen oak with radial-porous wood

The stem water conducting system of an evergreen broad-leaved oak, Lithocarpus edulis (Mak.) Nakai, was investigated. Evergreen broad-leaved oaks (Lithocarpus, Castanopsis, Cyclobalanopsis, Quercus) belonging to the Quercoideae are a major component of Asian monsoon forests, and are characterized by the possession of radial-porous wood. A characteristic of radial-porous wood is the development of aggregate rays between radially oriented files of vessels. We measured the distribution of vessel lumen diameters in a stem cross section and calculated the theoretical water conductivity of the wood. The radial profile of the heat pulse velocity (HPV) was measured for an intact whole tree under field conditions and compared with the theoretical distribution of water conductivity. Soft X-ray photographs of frozen stem sections indicated that most of the vessel lumina were filled with water, including those of vessels more than 20 years old. Even when vessels were relatively wide (lumen diameters > 100 microm), cavitation was negligible. The rate of water uptake from the cut stem base correlated closely with HPV (r = 0.96), and HPV closely reflected the mean volume flow per stem sectional area (SFVS) around the sensor probes. However, the ray tissue sharply inhibited heat transfer, and the positioning of the probes strongly affected the absolute value of HPV. It was also found that HPV more closely reflected the mean sap flow velocity in the vessels than did SFVS.