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.     

Cloning of DNA fragments specific to the pathotype and race of <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

Japanese isolates of Verticillium dahliae, a causal agent of wilt disease in many plants, are classifiable into pathotypes based on their pathogenicity. Because these pathotypes are morphologically indistinguishable, establishing a rapid identification method is very important for the control of this pathogen in Japan. For cloning DNA fragments that are useful for identification and specific detection of V. dahliae pathotypes, we performed random amplified polymorphic DNA (RAPD) analyses using various isolates. One polymerase chain reaction (PCR) product, E10-U48, was specific to isolates pathogenic to sweet pepper. The other product, B68-TV, was specific to race 1 of isolates pathogenic to tomato. The specificity of these sequences was confirmed by genomic Southern hybridization. Further analyses revealed that the region peripheral to B68-TV obtained from the genomic DNA library includes the sequence specific to all isolates pathogenic to tomato (races 1 and 2). Moreover, sequence tagged site (STS) primers designed from B68-TV and its peripheral region showed race-specific and pathotype-specific amplification in a PCR assay. The probes and primers obtained in this study are likely to be useful tools for the identification and specific detection of pathotypes and races of V. dahliae. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession number AB095266.     

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.     

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.     

Effects of current-year and previous-year PPFDs on shoot gross morphology and leaf properties in Fagus japonica

We investigated how shoot gross morphology and leaf properties are determined in Fagus japonica Maxim., a deciduous species with flush-type shoot phenology, in which all leaves are produced in a single flush at the start of each season. We examined relationships between current-year shoot properties and local light environment in a 14-m tall beech tree growing in a deciduous forest. Leaf number (LN), total leaf area (TLA), and total leaf length (SL) of the current-year shoot increased with increasing photosynthetic photon flux density (PPFD). Leaf thickness, dry mass per leaf area and nitrogen content on a leaf area basis increased, whereas the chlorophyll/N ratio decreased with increasing PPFD. To separate the effects of current-year PPFD from those of previous year(s), we artificially shaded a part of the uppermost leaf tier. Reciprocal transfers of beech seedlings between controlled PPFD regimes were also made. Characteristics of shoot gross morphology such as LN, TLA and SL were largely determined by the PPFD of the previous year. The exception was the length of the longest "long shoots" with many leaves, in which elongation appeared to be influenced by both previous-year and current-year PPFD. In contrast, leaf properties were determined by current-year PPFD. The ecological implications of our findings are discussed.     

Characterization of Aerococcus viridans isolated from milk samples from cows with mastitis and manure samples

Thirty-eight Aerococcus viridans isolates were obtained from milk from 478 cows with clinical mastitis in a farm during the periods between November 2011 and February 2012, and between December 2012 and March 2013. Additional isolates were obtained from processed manure (a mixture of composted manure, straw and hydrated lime) and bedding materials. The processed manure was later used to cover the floor of the stalls in barns as bedding materials. The temperatures recorded in the composted and processed manure were not as high as those generally observed during satisfactory composting. To reveal the association of A. viridans in manure-related products with intramammary infection in cows, isolates were characterized by their DNA fragment patterns as determined by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. Isolates obtained from milk, processed manure and bedding materials had identical DNA fragment patterns. Antimicrobial susceptibilities were determined for 29 isolates from milk, processed manure and bedding materials. Of these, 26 (89.7%) were resistant to clindamycin, whereas virtually all the isolates were susceptible to 12 other antimicrobials including cefalosporins that have been used to treat bovine mastitis in Japan. In vitro, three A. viridans isolates from milk and an isolate from processed manure survived for 3 hr in Good’s buffer (pH 9) at high temperature (50°C). The results suggest that the processed manure and bedding materials in this farm were possible sources of A. viridans that caused infection in the cows with mastitis.