Effects of cycloheximide treatment on in-vitro development of porcine parthenotes and somatic cell nuclear transfer embryos

This study aimed to verify the beneficial effect of cycloheximide (CHX) treatment on the development of porcine somatic cell nuclear transfer (NT) embryos, which were constructed with enucleated oocytes and cumulus cells by using a single direct current (DC) pulse. In the first experiment, a single DC pulse applied to the induction of fusion and activation of NT embryos gave a high fusion rate. However, cleavage and subsequent development of fused couplets (NT embryos) to the blastocyst stage were poor. Experiment 2 was conducted to determine whether CHX treatment could enhance metaphase II (M II) oocyte activation and improve the subsequent parthenogenetic development. After giving the DC pulse and incubation with or without CHX, M II oocytes incubated with CHX showed higher cleavage and development to blastocysts compared with those incubated without CHX (P < 0. 05). Experiment 3 was carried out to verify the beneficial effect of CHX on the development of NT embryos. The NT embryos treated with the DC pulse and CHX treatment showed higher cleavage and subsequent development compared with those treated with the DC pulse alone (P < 0.05) . The present study demonstrates that CHX treatment enhances the electrical stimulus-induced activation of oocytes and NT embryos, and improves the subsequent development of parthenotes and NT embryos. The results indicate that protein synthesis inhibition treatment required for the induction of oocyte activation promotes the development of NT embryos.     

Precursor‐targeted immune‐mediated anemia in a dog with a stage IV mast cell tumor and bone marrow infiltration

A 12‐year‐old spayed female Shiba Inu dog was referred to our hospital for a suspected mast cell tumor (MCT) of the bone marrow (BM). Laboratory abnormalities included severe nonregenerative anemia (packed cell volume or PCV: 12.5%; reference interval (RI): 37.3‐61.7%; reticulocytes: 35.1 × 10³/µL; RI: 10‐110 × 10³/µL), and a few mast cells were visualized in the blood smear examination. The BM was hypercellular with hematopoietic cells, a decreased myeloid:erythroid (M:E) ratio (0.77; RI, 0.9‐1.8), and no dysplastic hematopoietic cells. Mast cells accounted for 11.5% of the total nucleated BM cells. Neoplastic mast cells and histiocytes phagocytizing erythroid progenitor cells were occasionally noted. The dog was diagnosed with precursor‐targeted immune‐mediated anemia (PIMA) concurrent and a stage IV MCT infiltrating the BM. Multimodal treatment included toceranib, imatinib, vinblastine, lomustine (CCNU), prednisolone, cyclosporine, mycophenolate mofetil, and a blood transfusion. The dog died due to MCT progression lasting 139 days after the initial BM examination. To the best of our knowledge, this is the first report of a dog presenting with PIMA and a stage IV MCT infiltrating the BM.     

Presumptive hemophagocytic syndrome associated with immune-mediated anemia in two Miniature Dachshunds

This report describes the cases of two Miniature Dachshunds who were suspected to have immune-mediated hemolytic anemia (IMHA) and were treated with immunosuppressive therapy. However, progression of anemia, increases in C-reactive protein (CRP) and total-bilirubin (T-Bil) levels, splenomegaly, transition to nonregenerative anemia, and thrombocytopenia occurred after the treatment. Splenectomy and bone-marrow aspirations were performed subsequently. Both dogs were diagnosed with hemophagocytic syndrome (HPS) associated with IMHA. Unfortunately, they died 9 and 6 days later. These findings indicate that some cases of refractory IMHA have the pathogenicity of HPS. HPS should be included as a differential diagnosis of refractory IMHA concurrent with thrombocytopenia. Continuously elevated CRP and T-Bil levels may be helpful indicators in the detection of HPS associated with IMHA.     

Caulogenic callus induction and adventitious bud formation from embryos of long-term stored seeds ofPicea jezoensis

Caulogenic calli with a high differentiation potency were induced from mature embryos ofPicea jezoensis seeds stored over a long time, for 29 years, resulting in the active formation of adventitious buds. Embryos began to induce calli within 3 weeks of cultivating on LP medium containing 3 μM BAP and 1 μM 2,4-D. Then, the calli proliferated and transformed into caulogenic calli with bud primordia in 8 weeks. The caulogenic calli increased actively with the addition of 500 mg/l ofl-glutamine in the medium. Furthermore, caulogenic calli, induced on LP medium containingl-glutamine, resulted in the formation of adventitious buds, which elongated after transferring the calli into LP medium with 0.1 μM BAP, but withoutl-glutamine. It appears that the number of adventitious buds and the process of shoot elongation are influenced by the kind of nitrogen contained in the medium for callus induction. A part of this study was presented at the 107th Annual Meeting of the Japanese Forestry Society (1996).     

Biomass allocation and nitrogen limitation in a Cryptomeria japonica plantation chronosequence

We investigated soil net nitrogen mineralization rate, above- and belowground biomass allocation, and nitrogen use in a Cryptomeria japonica plantation chronosequence. Total biomass accumulation showed an asymptotic accretion pattern, and the peak total biomass accumulation rate occurred approximately 30 years after afforestation. Soil net nitrogen mineralization rate was lowest 30 years after afforestation. Between years 30 and 88, net nitrogen mineralization increased again. These results indicate that an imbalance in soil nitrogen supply and plant nitrogen demand occurred approximately 30 years after afforestation. Furthermore, leaf nitrogen concentration, which was used as an index of plant nitrogen status, was lower in mature forest than in young forest, suggesting that mature stands did not take up nitrogen as successfully. If soil resources such as nitrogen limit plant growth, plants may increase biomass allocation to fine root structure; however, fine root biomass was not higher in 30- and 88-year-old stands than in younger stands, suggesting that changes in biomass allocation may not be effective against nitrogen deficiency in a C. japonica plantation chronosequence.     

Forest fires and vegetation during the Holocene in central Yakutia, eastern Siberia

In order to investigate the relationship between vegetation change and fire history in the Siberian boreal forest, fossil pollen and charcoal from two lakes in central Yakutia, eastern Siberia, were analyzed. The vegetation change inferred from the pollen analysis was similar to that found in previous research in the region. Open larch forest covered this region during the late Glacial and early Holocene periods. Later, during the mid-Holocene, Scots pine expanded its range. The low levels of charcoal in the lake deposits represent surface forest fires, suggesting that the present-day surface fire regime has been taking place since at least 6,500 calibrated years before the present (cal yr BP) and that stand-replacing fire has not occurred during the Holocene. Larch and Scots pine forests, which are characterized by surface fire regimes, have been predominant since the early Holocene.     

Contributions of salmon-derived nitrogen to riparian vegetation in the northwest Pacific region

We examined the relationship between the annual escapement of salmon and the δ ¹⁵N of willow (Salix spp.) leaves to evaluate the contribution of marine-derived nutrients (MDN) to riparian vegetation around the Pacific Northwest and Northeast regions. Foliar δ ¹⁵N values ranged from −3.42‰ to 4.65‰. The value increased with increasing density of carcasses up to 500 fish/km and 1500 fish/km. δ ¹⁵N values were variable at carcass densities below 500 fish/km. Possible factors affecting the fluctuation of δ ¹⁵N at reference sites are: (1) denitrification; (2) the presence of N₂-fixing trees, such as alder; and (3) agricultural runoff. δ ¹⁵N values at the sites with carcass densities over 500 fish/km were consistently high, while a value of δ ¹⁵N below zero was observed at only one site (Rusha River; δ ¹⁵N = −1.87‰). At this site, most adult pink salmon returned to limited locations near the estuary because steeper channel gradients acted as a migration barrier, resulting in the negative δ ¹⁵N value. Nevertheless, we concluded that our results showed evidence of the feedback of MDN to terrestrial vegetation, although the use of the δ ¹⁵N value as a terrestrial end member at spawning sites is limited. If the relationship between the enrichment index, which is expressed as the values using a mixing model, and salmon abundance was estimated, the availability of MDN in riparian ecosystems could possibly be evaluated and will lead to the establishment of escapement goals. An erratum to this article is available at .     

Similarity between Copper Resistance Genes from Pseudomonas syringae pv. actinidiae and P. syringae pv. tomato

Twenty-eight strains of Pseudomonas syringae pv. actinidiae isolated in 1984, 1987 and 1988 from kiwifruit orchards in Japan were tested for their resistance to copper sulfate. All strains isolated in 1984 were copper sensitive with a minimum inhibitory concentration (MIC) of cupric sulfate of 0.75 mM. However, some strains isolated in 1987 and 1988 were resistant, with the MIC ranging from 2.25 to 3.0 mM. All copper-resistant strains contained at least one of two plasmids, pPaCul (about 70.5 kb) or pPaCu2 (about 280 kb), or both. In a copper-resistant strain Pa429, the location of the copper-resistance gene(s) was examined by insertional inactivation with Tn5. The MIC of copper sulfate in the copper-sensitive mutant obtained by Tn5 tagging decreased from 2.75 to 0.75 mM. The 14.5 kb BamHI fragment, designated pPaCuB14, containing the same locus mutagenized with Tn5 was cloned from pPaCu1. However, pPaCuB14 did not confer copper resistance in the transformant of copper-sensitive strain Pa21R, suggesting that this clone did not contain a full set of copper-resistance gene(s). Then a cosmid library of pPaCu1 was constructed and six cosmid clones hybridized with pPaCuB14 were selected. One of the six cosmids, designated pPaCuC1, conferred a near wild-type level of copper resistance in the transformant of the copper-sensitive strain. pPaCuC1 had a homologous region that hybridized with all of the PCR-amplifled fragments of copA, copB, copR, and copS genes of P. syringae pv. tomato. DNA sequence analysis of the homologous region revealed the existence of four open reading frames (ORF A, B, R and S) oriented in the same direction. The predicted amino acid sequences of ORF A, B, R and S had 80, 70, 97 and 95% identity with CopA, B, R and S of P. syringae pv. tomato, respectively. Received 5 July 2001/ Accepted in revised form 27 September 2001     

Effects of low temperatures on pupal survival of the stone leek leafminer Liriomyza chinensis (Diptera: Agromyzidae)

Abstract

The cold-hardiness of Liriomyza chinensis pupae was measured in the laboratory by observing pupal mortality at low temperatures. Pupal mortality increased with decreasing temperature and with extended cold exposure time. No recently pupated pupae (6 h) were able to survive after 16 days of chilling at 0, 2.5 and 5°C, but 42.9% survival was observed at 10°C. Pupae at different developmental stages showed significant difference in mortality, with very low levels of mortality observed for older pupae (4 and 7 days) after exposure to 0°C for 16 days. The lethal time for 50% survival (LT₅₀) increased with increasing age of pupae. LT₅₀ for 4-day-old pupae exposed to 0°C was 52.1 days. Analysis of mean temperatures at several localities indicated that L. chinensis is able to overwinter outdoors in southern regions, but is unable to overwinter in open fields in northern regions of Japan, suggesting that overwintering in these regions would only occur in greenhouses.