The action of phorone and other compounds in controlling superficial scald of apples

A new inhibitor of superficial scald, phorone (2,6-dimethylhepta-2,5-dien-4-one) is reported. Phorone reduced scald on ‘Granny Smith’ apples when applied by injection or as a vapour. Phorone was found to limit the accumulation of α-farnesene during storage and the amounts of conjugated triene oxidation products derived from α-farnesene. Monoterpenes also reduced α-farnesene, oxidation products and scald, but these compounds were less effective than phorone. In contrast, diphenylamine had little effect on the amount of α-farnesene in the fruit but it prevented the autoxidation of α-farnesene and controlled scald.     

In planta transient expression as a system for genetic and biochemical analyses of chlorophyll biosynthesis

Background  

Mg chelatase is a multi-subunit enzyme that catalyses the first committed step of chlorophyll biosynthesis. Studies in higher plants and algae indicate that the Mg chelatase reaction product, Mg-protoporphyrin IX plays an essential role in nuclear-plastid interactions. A number of Mg chelatase mutants have been isolated from higher plants, including semi-dominant alleles of ChlI, the gene encoding the I subunit of the enzyme. To investigate the function of higher plant CHLI, bacterial orthologues have been engineered to carry analogous amino acid substitutions to the higher plant mutations and the phenotypes examined through in vitro characterization of heterologously produced proteins. Here, we demonstrate the utility of a transient expression system in Nicotiana benthamiana for rapidly assaying mutant variants of the maize CHLI protein in vivo.     

Optimizing Electrical Activation of Porcine Oocytes by Adjusting Pre‐ and Post‐Activation Mannitol Exposure Times

Modifying electrical activation conditions have been used to improve in vitro embryo production and development in pigs. However, there is insufficient information about correlations of porcine embryo development with oocyte pre‐ and post‐activation conditions. The purpose of this study was to compare the developmental rates of porcine oocytes subjected to different mannitol exposure times, either pre‐ or post‐electrical activation, and to elucidate the reason for the optimal mannitol exposure time. Mannitol exposure times around activation were adjusted as 0, 1, 2 or 3 min. Blastocyst development were checked on day 7. Exposure of oocytes to mannitol for 1 or 2 min before electrical activation produced significantly higher blastocyst rates than exposure for 0 or 3 min. There was no significant difference in blastocyst rates when activated oocytes were exposed to mannitol for 0, 1, 2 or 3 min after electrical activation. While exposure of oocytes to mannitol for 1 min pre‐ and 3 min post‐activation showed significantly higher blastocyst development than 0 min pre‐ and 0 min post‐activation. It also showed higher maintenance of normal oocyte morphology than exposure for 0 min pre‐ and 0 min post‐activation. In conclusion, exposure of oocytes to mannitol for 1 min pre‐ and 3 min post‐activation seems to be optimal for producing higher in vitro blastocyst development of porcine parthenogenetic embryos. The higher blastocyst development is correlated with higher maintenance of normal morphology in oocytes exposed to mannitol for 1 min pre‐ and 3 min post‐activation.     

Pond-Level Risk Factors Associated with Columnaris Disease on Mississippi Commercial Catfish Farms

Abstract

A large commercial catfish enterprise encompassing over 500 food fish ponds from five farms covering multiple counties in the Mississippi Delta was included in this analysis of columnaris risk factors. A gram-negative bacterium, Flavobacterium columnare, is the cause of columnaris disease and is considered the second-most prevalent bacterial disease in farm-raised catfish. The objective of this study was to determine if pond-level risk factors reported by farm personnel were associated with columnaris disease mortalities. To identify risk factors affecting susceptibility of farm-raised channel catfish Ictalurus punctatus to columnaris disease, a Catfish Management database was developed. Logistic regression was used to model the relationships between probability of columnaris in ponds and risk factors examined. Generalized linear mixed models incorporating hierarchically structured random effects of ponds and one or more fixed-effects risk factors were fitted. In the screening process, each risk factor was evaluated in the basic model as a single fixed-effects factor, and if associated with the outcome (P ≤ 0.20), was retained for development of multivariable models. Two multivariable logistic regression models were constructed from data collected at the pond level by producers. The first was constructed from data in which water quality was not considered. Pond depth and reduced feed consumption for a 14-d period prior to disease outbreaks measured on a per hectare basis were significantly (P ≤ 0.05) associated with columnaris disease. The second, in which water quality variables were also considered, pond depth, reduced feed consumption, shorter intervals from stocking to disease outbreaks, and total ammonia nitrogen were significantly (P ≤ 0.05) associated with columnaris occurrence. This study showed some commonly recorded production variables were associated with columnaris disease outbreaks and, if monitored, could help identify “at risk” ponds before disease outbreaks occur.

Received September 16, 2011; accepted February 9, 2012     

Immunomagnetic isolation of canine circulating endothelial and endothelial progenitor cells

Background: Increased concentrations of circulating endothelial cells (CECs) are thought to be a biomarker of vascular injury in human patients with cardiovascular disease, neoplasia, vasculitis, sickle cell anemia, shock, and sepsis. Immunomagnetic isolation is a technique currently used to enumerate human CECs and can detect low numbers of cells. Objectives: The purpose of this study was to determine whether a standard protocol for immunomagnetic isolation could be used to obtain and enumerate CECs and a subpopulation of endothelial progenitor cells (EPCs) from canine whole blood. Methods: Cultured canine aortic endothelial cells were stained immunohistochemically with von Willebrand factor to verify morphology and number. Using magnetic beads conjugated with anti‐CD146, CECs/EPCs were isolated in culture and in canine whole blood. CD146‐positive cells were stained with fluorescein‐conjugated Ulex europaeus agglutinin 1 (UEA‐1) to confirm endothelial origin and cells were counted manually using a fluorescent microscope. The method was then applied to EDTA‐anticoagulated whole blood samples from 10 healthy client‐owned dogs. Results: The anti‐CD146–coated magnetic beads (>5/cell) bound the cultured canine aortic endothelial cells. Only rare UEA‐1–positive cells were obtained from whole blood, while >85–90% of cultured canine aortic endothelial cells were UEA‐1 positive. The percentage recovery of cultured canine aortic endothelial cells was >86%. CECs in canine whole blood had >8 beads attached to the surface and were 10–40 μm in size. Using immunomagnetic isolation, 43.4 ± 15.6 CECs/mL (range 24–70/mL) were isolated from canine whole blood samples. Conclusions: Immunomagnetic isolation is an acceptable method for enumerating canine CECs/EPCs in whole blood. Further studies are warranted to evaluate the clinical significance of CEC/EPC concentration in different canine diseases.     

The influence of age and Rhodococcus equi infection on CD1 expression by equine antigen presenting cells

There is a distinct age-associated susceptibility of horses to Rhodococcus equi infection. Initial infection is thought to occur in the neonatal and perinatal period, and only foals less than 6 months of age are typically affected. R. equi is closely related and structurally similar to Mycobacterium tuberculosis, and causes similar pathologic lesions. Protective immune responses to M. tuberculosis involve classical major histocompatibility complex (MHC)-restricted T cells that recognize peptide antigen, as well as MHC-independent T cells that recognize mycobacterial lipid antigen presented by CD1 molecules. Given the structural similarity between these two pathogens and our previous observations regarding R. equi-specific, MHC-unrestricted cytotoxic T lymphocytes (CTL), we developed 3 related hypotheses: (1) CD1 molecules are expressed on equine antigen presenting cells (APC), (2) CD1 expression on APC is less in foals compared to adults and (3) infection with live virulent R. equi induces up-regulation of CD1 on both adult and perinatal APC. CD1 expression was examined by flow cytometric analysis using a panel of monoclonal CD1 antibodies with different species and isoform specificities.

Results

Three CD1 antibodies specific for CD1b showed consistent cross reactivity with both foal and adult monocyte-derived macrophages (MDM). CD1b and MHC class II expression were significantly higher on adult MDM compared with foals. R. equi infected MDM showed significantly lower expression of CD1b, suggesting that infection with this bacterium induces down-regulation of CD1b on the cell surface. Histograms from dual antibody staining of peripheral blood mononuclear cells also revealed that 45–71% of the monocyte population stained positive for CD1b, and that the majority of these also co-expressed MHC II molecules, indicating that they were APC. The anti-CD1 antibodies showed no binding or minimal binding to bronchoalveolar lavage (BAL)-derived macrophages.

Conclusion

The CD1b isoform is evolutionarily conserved, and is present on equine MDM, as well as on circulating blood monocytes. The unique susceptibility of foals to R. equi infection may be due in part to lower expression of CD1 and MHC class II, as observed in this study. The data also suggests that infection with R. equi induces down-regulation of CD1b on equine MDM. This may represent a novel mechanism by R. equi to avoid detection and killing of infected cells by the immune system, similar to that observed when human APC are infected with M. tuberculosis.     

Effect of Transgene Introduction and Recloning on Efficiency of Porcine Transgenic Cloned Embryo Production In Vitro

Retrovirus-mediated exogenous gene transfection of somatic cells is an efficient method to produce transgenic embryos by somatic cell nuclear transfer (SCNT). This study evaluated whether efficiency of transgenic embryos production, by SCNT using fibroblast cells transfected by retrovirus vector, is influenced by the introduced transgene and whether recloning could further improve its efficiency. Transgenic cloned embryos were produced by SCNT of porcine foetal fibroblast cells transfected by either LNβ-Z or LNβ-enhanced green fluorescent protein (EGFP) retrovirus vector and evaluated for their developmental ability in vitro . Blastomeres from four-cell stage porcine embryos, produced by SCNT of foetal fibroblast cells transfected with LNβ-EGFP retroviral vector, were subsequently recloned into enucleated metaphase II oocytes and evaluated for changes in chromatin configuration, in vitro embryo development and gene expression. Analysis of results showed that cleavage and blastocyst rates of porcine SCNT embryos, using LacZ (53.6 ± 6.4%; 12.0 ± 5.7%) or EGFP (57.5 ± 6.3%; 10.1 ± 4.1%) transfected fibroblasts, did not differ (p > 0.05) from those of non-transfected controls (60.9 ± 8.2%; 12.3 ± 4.0%). Recloning of blastomeres did not further improve the in vitro development rate. Interestingly, the nuclei of blastomere underwent slower remodelling process than somatic cell nuclei. Both cloned and recloned embryos showed 100% transgene expression and there were no evidence of mosaicism. In conclusion, our data shows that the efficiency of transgenic cloned embryos production by SCNT of somatic cells transfected with replication-defective retrovirus vector is not influenced by the transgene introduction into donor cells and recloning of four-cell stage blastomere could not further improve its efficiency.