Effects of tissue culture on a highly repetitive DNA sequence (E180 satellite) in Medicago sativa

Genomic DNA extracted from leaves of sixteen alfalfa genotypes, six-week-old callus cultures derived from them and from a suspension culture was used in Southern blots and slot blots probed with alfalfa E180 satellite DNA. The restriction patterns revealed by the E180 probe did not differ among alfalfa genotypes and no differences between the leaf and callus restriction patterns were seen. However, the number of copies of the E180 satellite was lower in the callus samples than in the corresponding leaf samples and significant differences in copy number among callus samples were detected. Genomic stress induced by tissue culture may have caused the reduction in copy number. This phenomenon may be important in the generation of somaclonal variation in alfalfa.     

Human Flt3L Generates Dendritic Cells from Canine Peripheral Blood Precursors: Implications for a Dog Glioma Clinical Trial

Background

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and carries a dismal prognosis. We have developed a conditional cytotoxic/immunotherapeutic approach using adenoviral vectors (Ads) encoding the immunostimulatory cytokine, human soluble fms-like tyrosine kinase 3 ligand (hsFlt3L) and the conditional cytotoxic molecule, i.e., Herpes Simplex Type 1- thymide kinase (TK). This therapy triggers an anti-tumor immune response that leads to tumor regression and anti-tumor immunological memory in intracranial rodent cancer models. We aim to test the efficacy of this immunotherapy in dogs bearing spontaneous GBM. In view of the controversy regarding the effect of human cytokines on dog immune cells, and considering that the efficacy of this treatment depends on hsFlt3L-stimulated dendritic cells (DCs), in the present work we tested the ability of Ad-encoded hsFlt3L to generate DCs from dog peripheral blood and compared its effects with canine IL-4 and GM-CSF.

Methodology/Principal Findings

Our results demonstrate that hsFlT3L expressed form an Ad vector, generated DCs from peripheral blood cultures with very similar morphological and phenotypic characteristics to canine IL-4 and GM-CSF-cultured DCs. These include phagocytic activity and expression of CD11c, MHCII, CD80 and CD14. Maturation of DCs cultured under both conditions resulted in increased secretion of IL-6, TNF-α and IFN-γ. Importantly, hsFlt3L-derived antigen presenting cells showed allostimulatory potential highlighting their ability to present antigen to T cells and elicit their proliferation.

Conclusions/Significance

These results demonstrate that hsFlt3L induces the proliferation of canine DCs and support its use in upcoming clinical trials for canine GBM. Our data further support the translation of hsFlt3L to be used for dendritic cells'' vaccination and gene therapeutic approaches from rodent models to canine patients and its future implementation in human clinical trials.     

Identification of myeloid derived suppressor cells in dogs with naturally occurring cancer

Dogs with naturally occurring cancer represent an important large animal model for drug development and testing novel immunotherapies. However, poorly defined immunophenotypes of canine leukocytes have limited the study of tumor immunology in dogs. The accumulation of myeloid derived suppressor cells (MDSCs) is known to be a key mechanism of immune suppression in tumor-bearing mice and in human patients. We sought to identify MDSCs in the blood of dogs with cancer. Peripheral blood mononuclear cells (PBMCs) from dogs with advanced or early stage cancer and from age-matched healthy controls were analyzed by flow cytometry and microscopy. Suppressive function was tested in T cell proliferation and cytokine elaboration assays. Semi-quantitative RT-PCR was used to identify potential mechanisms responsible for immunosuppression. PBMCs from dogs with advanced or metastatic cancer exhibited a significantly higher percentage of CD11b(+)CD14(-)MHCII(-) cells compared to dogs diagnosed with early stage non-metastatic tumors and healthy dogs. These CD11b(+) CD14(-)MHCII(-) cells constitute a subpopulation of activated granulocytes that co-purify with PBMCs, display polymorphonuclear granulocyte morphology, and demonstrate a potent ability to suppress proliferation and IFN-γ production in T cells from normal and tumor-bearing donors. Furthermore, these cells expressed hallmark suppressive factors of human MDSC including ARG1, iNOS2, TGF-β and IL-10. In summary our data demonstrate that MDSCs accumulate in the blood of dogs with advanced cancer and can be measured using this three-marker immunophenotype, thereby enabling prospective studies that can monitor MDSC burden.     

Fish oil decreases matrix metalloproteinases in knee synovia of dogs with inflammatory joint disease

This study was designed to determine whether dietary fish oil affects the expression and activity of matrix metalloproteinases (MMP), tissue inhibitors of MMP-2 (TIMP-2) and urokinase plasminogen activator (uPA) in synovial fluid from dogs with spontaneously occurring stifle (knee) instability in a single hind limb resulting from acute cranial cruciate ligament (CCL) injury. Two groups of 12 dogs were fed diets from 1 week prior to surgery on the affected knee to 56 days post-surgery. The fish oil and control diets provided 90 and 4.5 mg, respectively, of combined eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)/kg body weight per day. Plasma and synovial fluid, from both surgical and nonsurgical knee joints, were obtained at start of the diet (-7), surgery day (0) and 7, 14, 28 and 56 days post-surgery. Plasma total EPA and DHA were significantly increased, and plasma total arachidonic acid (AA) was significantly decreased by the fish oil diet. In synovial fluid from the nonsurgical knee, fish oil treatment significantly decreased proMMP-2 expression at Days 7 and 14, and proMMP-9 expression at Day 56, and uPA activity at 28 days and significantly increased TIMP-2 expression at Days 7 and 28. There were no differences in MMP expression or activity, TIMP-2 expression and uPA activity in the surgical joint synovial fluid at any time throughout the study. These results suggest that dietary fish oil may exert beneficial effects on synovial fluid MMP and TIMP-2 equilibrium in the uninjured stifle of dogs with unilateral CCL injury.     

Enzymatic aminolysis of lactones in aqueous miniemulsion: Catalysis through a novel pathway

Lipase-catalyzed aminolysis of lactones under aqueous conditions usually leads to the hydrolysis of the ester bond with only a minor content of the corresponding amide. However, the aminolysis of pentadecanolide (PD) and hexadecanolide (HD), respectively, with oleylamine (OA) in aqueous miniemulsion under optimized conditions (temperature, concentration of enzyme, reaction time) yields >90% amide. Kinetic investigations performed with OA and PD reveal that the lipase catalyzes a novel reaction pathway, i.e., the hydrolysis of a lactone followed by the amidation requiring 30 min and 8 days reaction time, respectively. The demands of a high amount of lipase as well as the long reaction time are caused by the low reactivity of the carboxylic group and the formation of salt with the amine. Similar reactions were performed with PD and other amines such as dodecyl, decyl, octyl, benzyl and hexyl amine resulting the analogous amide compounds.     

AT2-AT3-profiling: a new look at synonymous codon usage

The teleology of synonymous codon usage (SCU) still awaits a unifying concept. Here the 2nd codon letter of human mRNA-codons was graphically, aided by a computer program, put in relation to the 3rd codon letter, the carrier of SCU: AT2, the density of A+T in 2nd codon position, behaves to AT3, the analogous density of the 3rd codon position, mostly in an inverse fashion that can be expressed as typical figures: mRNAs with an overall AT-density below 50% have a tendency to produce bulky figures called "red dragons" (when redness is attributed to graph-areas, where AT3< AT2), while mRNAs with an AT-density above 50% produce a pattern called "harlequin" consisting of alternating red and blue (blueness, in analogy, when AT3>AT2) diamonds. With more diversion of AT3 from AT2, the harlequin patterns can assume the pattern of a "blue dragon". By analysing the mRNA of known proteins, these patterns can be correlated with certain functional regions: proteins with multiple transmembrane passages show bulky "red dragons", structural proteins with a high glycine- and proline content such as collagen result in "blue dragons". Non-coding mRNAs tend to show a balance between AT2 and AT3 and hence "harlequin patterns". Signal peptides usually code red due to a low AT3 with an AT2-density at the expectance level. With this technique DNA-sequences of as yet unknown functional meaning were scanned. When stretches of harlequin patterns appear interrupted by red or blue dragons, closer scrutiny of these stretches can reveal ORFs which deserve to be looked at more closely for their protein-informational content. At least in humans, SCU appears to follow protein-dependent AT2-density in a reciprocal fashion and does not seem to serve the purpose of influencing mRNA secondary structure which is discussed in depth.     

Genetic diversity impacts of forest fires,forest harvesting,and alternative reforestation practices in black spruce (Picea mariana)

Benchmarks were established for genetic diversity inherent in natural mature populations, and genetic diversity impacts of forest fires, clearcut harvesting and alternative natural and artificial silvicultural regeneration practices were determined in black spruce (Picea mariana). Allozymes of 32 loci were used to determine and compare genetic diversity and genetic relationships of adjacent or nearby four stand types: post-fire natural mature (FNM), post-fire natural young (FNR), post-harvest natural young (HNR) and post-harvest plantation (PLT), of black spruce at each of the four study sites located in two ecoregions in Manitoba: Ecoregion 90-Lac Seul Upland (Eastern) and Ecoregion 158 - Mid-Boreal Lowland (Northern). Both allelic- and genotypic-based genetic diversity parameters, as well as latent genetic potential, were determined. Black spruce populations showed typical moderate to high levels of allozyme genetic diversity. The mean genetic diversity parameters over the 16 black spruce populations sampled were as follows: percent loci polymorphic - 67%, mean number of alleles per locus - 2.52, effective number of alleles per locus - 1.70, observed heterozygosity - 0.222, expected heterozygosity - 0.308, mean number of observed genotypes per locus - 3.65, mean number of expected genotypes per locus - 5.03, genotype additivity (observed) - 116.8, genotype additivity (expected) - 161, genotype multiplicity (observed) - 6.16 x 10(15), genotype multiplicity (expected) - 2.06 x 10(19) and latent genetic potential - 26.12. The four stand types (FNM, FNR, HNR and PLT) had comparable and statistically similar genetic diversity levels at each of the four study sites as well as overall. No significant differences in black spruce genetic diversity levels were observed between the two ecoregions in Manitoba, as well as between the post-fire and post-harvest regenerated stands. No particular order of genetic relatedness among the four stand types was observed. Black spruce populations showed some sort of site-related differentiation in their genetic constitution. Allelic heterogeneity and genetic distances among populations within stand types and among four stand types suggest that the genetic diversity was maintained at the landscape level in black spruce. The results of our study demonstrate that forest fires and currently used clearcut harvesting, and alternative natural and artificial silvicultural regeneration practices, do not adversely affect genetic diversity in black spruce, and that the genetic diversity effects of clearcut harvesting are not significantly different from those due to forest fires in black spruce.