Development of the techniques for nuclear transfer in pigs

Nuclear transfer in pigs was developed in the late 1980's. The techniques were based on previous studies in frogs, mice and cattle. Within stage nuclear transfer, pronuclear exchange, was followed by the transfer of nuclei from cleavage stage embryos. While these have resulted in term development, many problems remain. Recently progress on the problem of inadequate oocyte activation has been made and now there can be a refocus on the other aspects of the nuclear transfer procedure. The emphasis in developing the cloning/transgenic technology is easily justified, not so much by the ability to produce genetically identical animals for production agriculture, but for the potential to use a cell line that can be genetically engineered prior to the nuclear transfer. Pigs with specific genetic modifications will have a great impact on production agriculture as well as human medicine.     

Integral strategy for evaluation of fecal indicator performance in bird-influenced saline inland waters

Wild birds are an important nonpoint source of fecal contamination of surface waters, but their contribution to fecal pollution is mostly difficult to estimate. Thus, to evaluate the relation between feces production and input of fecal indicator bacteria (FIB) into aquatic environments by wild waterfowl, we introduced a new holistic approach for evaluating the performance of FIB in six shallow saline habitats. For this, we monitored bird abundance, fecal pellet production, and the abundance of FIB concomitantly with a set of environmental variables over a 9-month period. For estimating fecal pellet production, a new protocol of fecal pellet counting was introduced, which was called fecal taxation (FTX). We could show that, over the whole range of investigated habitats, bird abundance, FTX values, and FIB abundance were highly significantly correlated and could demonstrate the good applicability of the FTX as a meaningful surrogate parameter for recent bird abundances and fecal contamination by birds in shallow aquatic ecosystems. Presumptive enterococci (ENT) were an excellent surrogate parameter of recent fecal contamination in these saline environments for samples collected at biweekly to monthly sampling intervals while presumptive Escherichia coli and fecal coliforms (FC) were often undetectable. Significant negative correlations with salinity indicated that E. coli and FC survival was hampered by osmotic stress. Statistical analyses further revealed that fecal pollution-associated parameters represented one system component independent from other environmental variables and that, besides feces production, rainfall, total suspended solids (direct), and trophy (indirect) had significant positive effects on ENT concentrations. Our holistic approach of linking bird abundance, feces production, and FIB detection with environmental variables may serve as a powerful model for application to other aquatic ecosystems.     

Quantification of Fusarium graminearum in infected wheat by species specific real-time PCR applying a TaqMan Probe

A new real-time PCR based method was developed for the species-specific detection, identification and quantification of Fusarium graminearum in planta. It utilizes a TaqMan hybridisation probe targeting the beta-tubulin gene and a plasmid standard. The assay is highly specific giving no product with DNA of closely related species. It is very sensitive, detecting down to five gene copies per reaction, and is able to produce reliable quantitative data over a range of six orders of magnitude.     

Dynamics of endogenous cytokinin pools in tobacco seedlings: a modelling approach

Recent advances in cytokinin analysis have made it possible to measure the content of 22 cytokinin metabolites in the tissue of developing tobacco seedlings. Individual types of cytokinins in plants are interconverted to their respective forms by several enzymatic activities (5'-AMP-isopentenyltransferase, adenosine nucleosidase, 5'-nucleotidase, adenosine phosphorylase, adenosine kinase, trans-hydroxylase, zeatin reductase, beta-glucosidase, O-glucosyl transferase, N-glucosyl transferase, cytokinin oxidase). This paper reports modelling and measuring of the dynamics of endogenous cytokinins in tobacco plants grown on media supplemented with isopentenyl adenine (IP), zeatin (Z) and dihydrozeatin riboside (DHZR). Differences in phenotypes generated by the three cytokinins are shown and discussed, and the assumption that substrate concentration drives enzyme kinetics underpinned the construction of a simple mathematical model of cytokinin metabolism in developing seedlings. The model was tested on data obtained from liquid chromatography/tandem mass spectrometry cytokinin measurements on tobacco seedlings grown on Murashige and Skoog agar nutrient medium, and on plants grown in the presence of IP, Z and DHZR. A close match was found between measured and simulated data, especially after a series of iterative parameter searches, in which the parameters were set to obtain the best fit with one of the data sets.     

Epidermal growth factor-receptor tyrosine kinase activity regulates expansion of porcine oocyte-cumulus cell complexes in vitro

We have recently shown that epidermal growth factor (EGF) strongly stimulates expansion of porcine oocyte-cumulus complexes (OCCs) isolated from large follicles (>6 mm) and does not promote expansion of OCCs from small (3-4-mm) follicles. In order to elucidate the role of EGF in OCCs expansion, in the present study, we first examined the presence of EGF receptors (EGFRs) in cumulus cells isolated from follicles of different sizes. Surprisingly, immunoblotting showed that cumulus cells obtained from all follicular size categories contained similar amounts of EGFR protein. On the other hand, we found a dramatic difference in the pattern of protein tyrosine phosphorylation in a comparison of cumulus cells isolated from small and large follicles treated by EGF. Furthermore, tyrosine-phosphorylated EGFR was specifically immunoprecipitated with antiphosphotyrosine antibodies from EGF-treated cumulus cells isolated from the large follicles. This result strongly indicates that only OCCs from the large follicles contain mature EGFRs that are capable of becoming activated by EGF. Remarkably, preincubation of cumulus cells from small follicles (3-4 mm) with FSH strongly increased EGF-stimulated tyrosine phosphorylation to levels comparable with OCCs from large follicles. The FSH-dependent activation of EGFRs was beneficial for expansion of OCCs isolated from the small follicles since OCCs treated sequentially by FSH (3 h) and EGF (1 h) underwent expansion significantly better then OCCs cultured in FSH or EGF alone. We conclude that a FSH-dependent pathway has an important role in the maturation of the EGFR in cumulus cells and that activation of EGFR-dependent signaling is sufficient to induce expansion.     

Porcine oocyte activation: differing roles of calcium and pH

Intracellular pH has recently been shown to increase during parthenogenetic activation of the porcine oocyte. In the following set of experiments, intracellular pH was monitored during activation and pronuclear development was assessed following activation treatments with calcium, in the absence of calcium, and in oocytes loaded with the calcium chelator BAPTA-AM in calcium-free medium. Intracellular pH increase was not different among groups when treating with 7% ethanol or 50 microM calcium ionophore, or during treatment with thimerosal for 12 or 25 min. Activation with thimerosal (200 microM, 12 min) followed by 8 mM dithiothreitol (DTT, 30 min) resulted in a decreased pronuclear development in calcium-free medium with or without BAPTA-AM loaded oocytes as compared to controls. Activation with 50 microM calcium ionophore resulted in pronuclear development that was different between the calcium-free and BAPTA-AM loaded oocytes in calcium-free medium. Similar incidences of pronuclear formation were observed in all ethanol treatment groups. It was concluded that external calcium as well as large changes in intracellular free calcium are not necessary for the increase in intracellular pH, but normal intracellular calcium signaling is critical for normal levels of pronuclear development. Finally, oocytes were measured for intracellular pH changes for 30 min following subzonal sperm injection. Intracellular pH did not increase, although pronuclear formation was observed 6 hr post SUZI. This suggested that major differences were still present between sperm-induced and parthenogenetic activation of the porcine oocyte.     

The Hypocrea jecorina HAP 2/3/5 protein complex binds to the inverted CCAAT-box (ATTGG) within the cbh2 (cellobiohydrolase II-gene) activating element

The 5' regulatory region of the chh2 gene, encoding cellobiohydrolase II, of the filamentous fungus Hypocrea jecorina contains the cbh2 activating element (CAE) which is essential for cbh2 expression. The CAE consists of two separate, adjacent motifs, a CCAAT box on the template strand (ATTGG) and a GTAATA box on the coding strand, which co-operate in the induction of the gene by cellulose or sophorose. EMSA supershift experiments using an antibody against Aspergillus nidulans HAPC suggested that the complex which binds to the H. jecorina CCAAT box contains a HAPC homolog. To obtain direct evidence for this, we have cloned the hap2, hap3 and hap5 genes from H. jecorina. They encode proteins whose core regions display great similarity to Aspergillus HAPB, HAPC and HAPE and to known HAP homologs from other organisms. All three genes are transcribed in a carbon source-independent manner. A. nidulans deltahap strains were functionally complemented in vitro by the overexpressed H. jecorina HAP2, HAP3 and HAP5 proteins, and they thus represent subunits of the CCAAT-binding complex. Furthermore, all three proteins (HAP2, HAP3 and HAP5) were needed to bind to the CAE in the H. jecorina cbh2 gene promoter in vitro. We conclude that the CCAAT box on the template strand in CAE is bound by the H. jecorina equivalent of the HAP protein complex.     

Phosphorylation positively regulates DNA binding of the carbon catabolite repressor Cre1 of Hypocrea jecorina (Trichoderma reesei)

Cre1 of the ascomycete Hypocrea jecorina is a Cys(2)His(2) zinc finger DNA-binding protein functioning as regulator for carbon catabolite repression. It represents the functional equivalent of yeast Mig1, known to be negatively regulated by the Snf1-kinase at the nuclear import level. We demonstrate that Cre1 is also a phosphoprotein, and identify Ser(241) within an acidic protein region as phosphorylation target. In contrast to Mig1 phosphorylation is required for DNA binding of Cre1. A S241E mutation mimics phosphorylation, whereas a S241A mutant protein shows phosphorylation-independent DNA binding activity, suggesting that phosphorylation is required to release Cre1 from an inactive conformation involving unphosphorylated Ser(241). Retransformation of a H. jecorina cre1-non functional mutant with Cre1-S241A leads to permanent carbon catabolite repression in cellobiohydrolase I expression. Contrary to Mig1, the amino acid sequence surrounding Ser(241) (HSNDEDD) suggests that phosphorylation may occur by a casein kinase II-like protein. This is supported by a mutation of E244V leading to loss of phosphorylation, loss of DNA binding, and gain of carbon catabolite derepression. Our results imply that the regulation of carbon catabolite repression at the level of DNA binding strongly differs between Saccharomyces cerevisiae and H. jecorina.     

NK cell activity during human cytomegalovirus infection is dominated by US2-11-mediated HLA class I down-regulation

A highly attractive approach to investigate the influence and hierarchical organization of viral proteins on cellular immune responses is to employ mutant viruses carrying deletions of various virus-encoded, immune-modulating genes. Here, we introduce a novel set of deletion mutants of the human CMV (HCMV) lacking the UL40 region either alone or on the background of a deletion mutant devoid of the entire US2-11 region. Deletion of UL40 had no significant effect on lysis of infected cells by NK cells, indicating that the expected enhancement of HLA-E expression by specific peptides derived from HCMV-encoded gpUL40 leader sequences was insufficient to confer target cell protection. Moreover, the kinetics of MHC class I down-regulation by US2-11 genes observed at early and late phases postinfection with wild-type virus correlated with increased susceptibility to NK lysis. Thus, the influence of HCMV genes on NK reactivity follows a hierarchy dominated by the US2-11 region, which encodes all viral genes capable of down-modulating expression of classical and non-classical MHC class I molecules. The insights gained from studies of such virus mutants may impact on future therapeutic strategies and vaccine development and incorporate NK cells in the line of defense mechanisms against HCMV infection.