转基因枳橙中GA20ox1与rol基因互作关系的研究

为分析转rol ABC基因枳橙GA20ox1基因与rol基因表达的互作关系,进一步阐释其矮化性状形成的分子机制。以转rol基因枳橙实生苗为试验材料,研究其对赤霉素的敏感反应,用荧光定量RT-PCR分析GA20ox1基因和rol基因的表达,并检测幼芽中POD酶活性和植物内源激素含量的变化。结果表明转rol基因枳橙既不属于GA缺陷型,也不属于GA不敏感型,喷施GA3能促进其茎伸长生长,但恢复不到野生型水平,幼芽中IAA（P<0.01）、GA1和GA4（P<0.05）显著降低,POD酶活性显著提高（P<0.01）。转rol基因枳橙幼芽中GA20ox1基因mRNA水平相比对照显著下调（P<0.01）。B、D系与野生型嫩茎中无明显差异,B、D系老叶中明显降低,E系中嫩茎和老叶中均明显增加。B、D和E系嫩叶中GA20ox1基因转录表达均较野生型高。在幼芽、嫩茎中,rol C基因与GA20ox1表达负相关。rol基因通过在幼芽中的高表达下调了GA20ox1基因转录表达,进而抑制了活性GAs在幼芽中的合成,顶端分生组织较低量的活性GAs限制植物茎伸长,在转rol ABC基因枳橙矮化性状建成中发挥重要作用。     

Nitrogen and carbon mineralisation of leather meal in soil as affected by particle size of fertiliser and microbiological activity of soil

Carbon and nitrogen mineralisation of leather meal fertilisers were studied in two soils characterised by different respiration activity. Both C and N mineralisation were highest in the most active soil, and when leather meal was added as a powder rather than as 2- to 4- and 4- to 6-mm particles. Fast and slow soluble N pools were determined after extraction with cold water and with hot buffer, respectively. The N remaining after the second extraction with hot buffer was named slow-release N. The percentage of slow-release N rose as the size of the applied leather meal particles increased, whereas fast soluble N was highest in the coarsest (4-6 mm) fertiliser.     

Gene expression study of two widely used pig intestinal epithelial cell lines: IPEC-J2 and IPI-2I

The intestinal epithelial cells (IEC) play an important role in the immune system of swine, protecting against infectious and non-infectious environmental insults. The IEC participate in the innate immune response of the intestine through different mechanisms such as barrier function, mucus secretion, antibacterial peptide synthesis and participation in the cytokine/chemokine networks.Most of the current knowledge of intestinal cell functions has come from studies conducted on cell cultures generated from human cancers or from classical animal models. However, because the molecular and cellular elements of the immune system have been selected over evolutionary time in response to the species-specific environment, models of immune function based on mouse and human need to be applied cautiously in pig. Few models of swine small intestine epithelium exist and these are poorly characterised. In the present study we characterised the basal expression of epithelial and immune-related genes of two pig small intestine cell lines, IPEC-J2 and IPI-2I, under different culture conditions. These data represent essential background information for future studies on pig-intestinal pathogen interactions.     

Defining output-based standards to achieve and maintain tuberculosis freedom in farmed deer, with reference to member states of the European Union

Within the European Union (EU), detailed legislation has been developed for cattle, but not deer, to minimise disease risks associated with trade in animals and animal products. This legislation is expressed as input-based standards, providing a detailed outline of the activity required (for example, testing of animals and application of defined control measures), on the expectation that an adequate output (for example, confidence in freedom) will be achieved. Input-based standards are at odds with the increasing shift towards output-based standards, particularly in OIE rules governing international trade. In this paper, we define output-based standards to achieve and maintain freedom from tuberculosis (TB) in farmed deer, with reference to EU member states. After considering the probability of freedom achieved for cattle under existing EU legislation, we defined a ‘free farmed deer holding’ as one with a probability of freedom from infection of at least 99%. We then developed an epidemiological model of TB surveillance systems for deer holdings, incorporating different surveillance strategies, including combinations of diagnostic tests, and a variety of different scenarios relating to the potential for introduction of infection. A range of surveillance strategies were identified to achieve and maintain a free farmed deer holding, and worked examples are presented. The surveillance system sensitivity for varying combinations of screening and confirmatory tests in live animals, animals at slaughter and on-farm deaths is also presented. Using a single test at a single point in time, none of the TB tests routinely used in farmed deer is able to achieve an acceptable probability of TB freedom. If repeat testing were undertaken, an acceptable probability of TB freedom could be achieved, with differing combinations of the surveillance system sensitivity, frequency of testing and risk of introduction. The probability of introduction of infection through the importation of infected deer was influenced by the use of a pre-movement test (assumed 90% test sensitivity and negative test results), the TB prevalence in the source herd and the number of animals imported. A surveillance system sensitivity of at least 81% was achieved with different combinations of annual live animal surveillance and surveillance of animals at slaughter or on-farm deaths. This methodology has broad applicability and could also be extended to other diseases in both deer and other species with relevance to trade in animals and animal products.     

Undifferentiated tumor in the ovary of a corn snake (Elaphe guttata guttata)

A 6-year-old intact female corn snake (Elaphe guttata guttata) was presented with a 3-week history of anorexia. Coelomic radiographs revealed a 9 x 4 cm soft tissue opacity suggestive of a right ovarian enlargement. The mass (9 x 5 x 4 cm) was surgically removed, and multiple smears from tissue sections were stained with Diff-Quik. Multiple tissue samples from the mass were collected and fixed in formalin. Cytologic specimens were moderately cellular and contained light pink amorphous background material. The cells were primarily spindle-shaped with moderate to marked anisocytosis and anisokaryosis. Cells sometimes were round to polygonal, and rarely were arranged in small clusters. Macrophages occasionally were observed. Histologic specimens consisted of a highly cellular mass composed of pleomorphic, spindle-shaped cells and, occasionally, round to polygonal cells arranged in irregular fascicles. The neoplastic cells were immunoreactive for cytokeratin (AE1/AE3), smooth muscle actin, and skeletal muscle actin, but did not stain for vimentin or desmin. On the basis of the morphologic and immunohistochemical results, a diagnosis of ovarian undifferentiated carcinoma was made. In this report, we describe the challenges of using immunohistochemistry to diagnose this uncommon type of tumor in reptiles.     

Dissection of antioxidant activity of durum wheat (Triticum durum Desf.) grains as evaluated by the new LOX/RNO method

Antioxidant activity (AA) of durum wheat (Triticum durum Desf.) grains was studied using the innovative LOX/RNO method, able to simultaneously detect different antioxidant mechanisms, and the TEAC assay, one of the most widely used assays. Insoluble-bound and free-soluble phenols, hydrophilic and lipophilic compounds were extracted from eight different whole flour samples; extracts were analyzed for AA and their content in several antioxidants. The LOX/RNO method measured very high AA values, with the highest ones [850–1500 μmol Trolox eq./g whole flour (dry weight)] for insoluble-bound phenolic extracts, highly correlated to total phenolic (r = 0.761, P < 0.001) and ferulic acid (r = 0.816, P < 0.001) contents. Hydrophilic and lipophilic extracts showed lower AA [70-140 and 40–60 μmol Trolox eq./g (dry weight), respectively], highly correlated to flavonoid (r = 0.583, P < 0.01) and protein (r = 0.602, P < 0.01), as well as β-tocotrienol (r = 0.684, P < 0.05) contents, respectively. Interestingly, the LOX/RNO method suggests that insoluble-bound phenolic compounds may exert very strong synergistic interactions within the extract. Contrarily, the TEAC assay did not correlate to any antioxidant content, resulted unable to highlight differences among samples, measured much lower AA values and did not suggest synergism. The use of the LOX/RNO method is useful to unearth new properties of phytochemicals from durum wheat grains, potentially giving health benefits.     

Exotic and invasive species compromise the seed bank and seed rain dynamics in forests undergoing restoration at urban regions

The control of exotic and invasive species in areas undergoing recovery is a challenge for ecological restoration and this problem may be even greater in urbanized areas. This study evaluated the seed bank and seed rain of a 5-year-old riparian forest located at the metropolitan region of Belo Horizonte in southeast Brazil, and verified changes over time in the abundance and species richness and whether planted species already were providing propagules to the site. At the forest, fifteen parcels of 100 m² were distributed randomly and soil samples were collected thrice (October 2011, March 2012 and July 2012) to evaluate the seed bank. Seed traps were installed in nine parcels to collect propagules over 1 year. Propagules were segregated and germinate at greenhouse. Species richness and abundance were estimated, and variance and similarity were quantified. In total, 86 species were recorded in the seed banks, 41.9% natives and 33.7% exotics, with the predominance of herbs (87.2%) and only 7% of trees. Species classified as weeds amounted to 52.3%. In the seed rain 642 seedlings germinated but they belonged to only 10 species, half of them were exotics. Regarding life forms, half of the species were arboreal and dominated the samples, highlighting the exotic species Melia azedarach. The species richness of the seed bank did not differ by season, unlike the seed rain which varied significantly by month. Seed bank and seed rain species had a very low floristic similarity with the list of planted species, suggesting that few planted trees had dispersed or were stocking propagules. Many non-planted species were recorded, mostly exotics and/or invasive that were probably derived from the surrounding urban matrix. These might interrupt the successional dynamics of the restoration forest and compromise the restoration process in the medium and long-terms. Our results highlight: (1) the importance of monitoring areas undergoing restoration; (2) that restoration methods and management actions directed to control exotic and invasive species are particularly important for sites in urban regions; and (3) the relevance of conserving forest fragments as sources of propagules for nearby areas.     

Antimycotic effectiveness against dermatophytes: comparison of two in vitro tests

Seven antimycotic drugs (econazole, enilconazole, fluconazole, griseofulvin, itraconazole, ketoconazole, and miconazole) were tested against 36 dermatophyte strains (19 M. canis, 7 T. mentagrophytes, 5 M. gypseum, 2 M. cookei, 1 T. rubrum, 1 T. ajelloi, and 1 T. terrestre) isolated from animals, humans, and the environment. Two in vitro methods were compared: a micro-dilution test based on the CLSI M38-A method, and a disk-diffusion test. Fluconazole was not effective in vitro against the dermatophytes. Econazole and enilconazole were the most effective. Thirteen strains were griseofulvin-resistant. The correlation between the two methods was statistically significant for enilconazole, griseofulvin, itraconazole, and miconazole.     

Susceptibility of grapevine viruses to thermotherapy on in vitro collection of Kober 5BB

A number of virus elimination protocols have been developed to obtain healthy plants but their success rates have been variable due to the strong virus–host relationship, treatment performances and experimental condition. An in vitro collection of plants of Vitis berlandieri × Vitis riparia Kober 5BB single-infected by Grapevine vitivirus A (GVA), Grapevine fanleaf nepovirus (GFLV), Grapevine fleck maculavirus (GFkV), Grapevine leafroll ampelovirus 1 (GLRaV-1) and Grapevine leafroll ampelovirus 3 (GLRaV-3) was established to carry out thermotherapy treatments. The experimental system allowed to control the effects of host genotype by using the same genetic background, and the environmental condition such as relative humidity, soil and temperature conditions. Trials were conducted in a growth chamber set at 37 ± 0.5 °C for 48 days using in vitro cultures for all infections. ELISA and RT-PCR were used to check sanitary conditions. Results showed complete eradication of GFLV and no effect against GFkV. Different sanitation rates were obtained for other phloematic viruses: 70.2% for GVA, 25.1% for GFLaV-1 and 24.7% for GLRaV-3. The results on phloematic viruses showed a gradient of elimination efficiency which could not be completely explained with the location of viral particles that seems to be only one of the several factors affecting virus susceptibility to heat stress. Moreover, the advantages of this experimental system provide the opportunity to obtain a greater understanding of the thermotherapeutic effects on phloematic viruses.