Life-long learning in laboratory animal science and ethics for veterinary and para-veterinary professionals in South Africa.

Veterinary and para-veterinary professionals working in the animal research sector are critical to ensure scientific quality and the humane care and use of animals. However, there are few focused education and training opportunities available for these professionals in South Africa. A survey of veterinarians working in animal research, undertaken by the South African Association for Laboratory Animal Science, identified the need for more advanced education and training opportunities beyond the routine Day 1 Skills currently provided for in undergraduate education. These could be broadly categorised into knowledge and skills relating to species-specific husbandry, procedures and clinical approaches, research-related biosecurity and biosafety, and study-specific ethical and animal welfare considerations. A subsequent workshop, attended by 85 veterinary and para-veterinary professionals in the animal research sector, identified 53 life-long learning needs, each with an associated learning outcome, for this professional community. These were grouped into five overarching themes: Personal development (9); Leadership and management skills (12); Education and training skills (5); Welfare, ethics and clinical skills (20); and Regulations and quality-assurance (7). Of the 53 learning outcomes, 14 were knowledge-based, ten were competencies, and 29 both knowledge and competence. These life-long learning opportunities, if available and implemented, will address important needs of veterinary and paraveterinary professionals in the animal research sector in South Africa. This would empower these professionals, assist in improving animal and human wellbeing, support high-quality ethical science, and maintain public confidence in the sector, thus enabling a more satisfactory career environment.

IL-15 improves skeletal muscle oxidative metabolism and glucose uptake in association with increased respiratory chain supercomplex formation and AMPK pathway activation.

BACKGROUND: IL-15 is believed to play a role in the beneficial impact of exercise on muscle energy metabolism. However, previous studies have generally used supraphysiological levels of IL-15 that do not represent contraction-induced IL-15 secretion. METHODS: L6 myotubes were treated acutely (3 h) and chronically (48 h) with concentrations of IL-15 mimicking circulating (1-10 pg/ml) and muscle interstitial (100 pg/ml -20 ng/ml) IL-15 levels with the aim to better understand its autocrine/paracrine role on muscle glucose uptake and mitochondrial function. RESULTS: Acute exposure to IL-15 levels representing muscle interstitial IL-15 increased basal glucose uptake without affecting insulin sensitivity. This was accompanied by increased mitochondrial oxidative functions in association with increased AMPK pathway and formation of complex III-containing supercomplexes. Conversely, chronic IL-15 exposure resulted in a biphasic effect on mitochondrial oxidative functions and ETC supercomplex formation was increased with low IL-15 levels but decreased with higher IL-15 concentrations. The AMPK pathway was activated only by high levels of chronic IL-15 treatment. Similar results were obtained in skeletal muscle from muscle-specific IL-15 overexpressing mice that show very high circulating IL-15 levels. CONCLUSIONS: Acute IL-15 treatment that mimics local IL-15 concentrations enhances muscle glucose uptake and mitochondrial oxidative functions. That mitochondria respond differently to different levels of IL-15 during chronic treatments indicates that IL-15 might activate two different pathways in muscle depending on IL-15 concentrations. GENERAL SIGNIFICANCE: Our results suggest that IL-15 may act in an autocrine/paracrine fashion and be, at least in part, involved in the positive effect of exercise on muscle energy metabolism.

Atmospheric benzenoid emissions from plants rival those from fossil fuels.

Despite the known biochemical production of a range of aromatic compounds by plants and the presence of benzenoids in floral scents, the emissions of only a few benzenoid compounds have been reported from the biosphere to the atmosphere. Here, using evidence from measurements at aircraft, ecosystem, tree, branch and leaf scales, with complementary isotopic labeling experiments, we show that vegetation (leaves, flowers, and phytoplankton) emits a wide variety of benzenoid compounds to the atmosphere at substantial rates. Controlled environment experiments show that plants are able to alter their metabolism to produce and release many benzenoids under stress conditions. The functions of these compounds remain unclear but may be related to chemical communication and protection against stress. We estimate the total global secondary organic aerosol potential from biogenic benzenoids to be similar to that from anthropogenic benzenoids (~10 Tg y(-1)), pointing to the importance of these natural emissions in atmospheric physics and chemistry.

Green leaf volatiles and oxygenated metabolite emission bursts from mesquite branches following light-dark transitions.

Green leaf volatiles (GLVs) are a diverse group of fatty acid-derived compounds emitted by all plants and are involved in a wide variety of developmental and stress-related biological functions. Recently, GLV emission bursts from leaves were reported following light-dark transitions and hypothesized to be related to the stress response while acetaldehyde bursts were hypothesized to be due to the 'pyruvate overflow' mechanism. In this study, branch emissions of GLVs and a group of oxygenated metabolites (acetaldehyde, ethanol, acetic acid, and acetone) derived from the pyruvate dehydrogenase (PDH) bypass pathway were quantified from mesquite plants following light-dark transitions using a coupled GC-MS, PTR-MS, and photosynthesis system. Within the first minute after darkening following a light period, large emission bursts of both C(5) and C(6) GLVs dominated by (Z)-3-hexen-1-yl acetate together with the PDH bypass metabolites are reported for the first time. We found that branches exposed to CO(2)-free air lacked significant GLV and PDH bypass bursts while O(2)-free atmospheres eliminated the GLV burst but stimulated the PDH bypass burst. A positive relationship was observed between photosynthetic activity prior to darkening and the magnitude of the GLV and PDH bursts. Photosynthesis under (13)CO(2) resulted in bursts with extensive labeling of acetaldehyde, ethanol, and the acetate but not the C(6)-alcohol moiety of (Z)-3-hexen-1-yl acetate. Our observations are consistent with (1) the "pyruvate overflow" mechanism with a fast turnover time (<1 h) as part of the PDH bypass pathway, which may contribute to the acetyl-CoA used for the acetate moiety of (Z)-3-hexen-1-yl acetate, and (2) a pool of fatty acids with a slow turnover time (>3 h) responsible for the C(6) alcohol moiety of (Z)-3-hexen-1-yl acetate via the 13-lipoxygenase pathway. We conclude that our non-invasive method may provide a new valuable in vivo tool for studies of acetyl-CoA and fatty acid metabolism in plants at a variety of spatial scales.

Efficient atmospheric cleansing of oxidized organic trace gases by vegetation.

The biosphere is the major source and sink of nonmethane volatile organic compounds (VOCs) in the atmosphere. Gas-phase chemical reactions initiate the removal of these compounds from the atmosphere, which ultimately proceeds via deposition at the surface or direct oxidation to carbon monoxide or carbon dioxide. We performed ecosystem-scale flux measurements that show that the removal of oxygenated VOC via dry deposition is substantially larger than is currently assumed for deciduous ecosystems. Laboratory experiments indicate efficient enzymatic conversion and potential up-regulation of various stress-related genes, leading to enhanced uptake rates as a response to ozone and methyl vinyl ketone exposure or mechanical wounding. A revised scheme for the uptake of oxygenated VOCs, incorporated into a global chemistry-transport model, predicts appreciable regional changes in annual dry deposition fluxes.

SirT1-null mice develop tumors at normal rates but are poorly protected by resveratrol.

The function of the class III histone deacetylase, Sir2, in promoting lifespan extension is well established in small model organisms. By analogy, SirT1, the mammalian orthologue of Sir2, is a candidate gene to slow down aging and forestall the onset of age-associated diseases. We have used SirT1-null mice to study the function of SirT1 in susceptibility to tumorigenesis. The number of intestinal polyps induced in mice carrying the Apc(min) mutation was unaffected by the SirT1 genotype although the average polyp size was slightly smaller in the SirT1-null animals. Similarly, the presence or absence of SirT1 had no effect on incidence and tumor load of skin papillomas induced by the classical two-stage carcinogenesis protocol. We found that resveratrol topically applied to the skin profoundly reduced tumorigenesis. This chemoprotective effect was significantly reduced but not ablated in SirT1-null mice, suggesting that part of the protection afforded by resveratrol requires the SirT1-encoded protein. Thus, our results suggest that SirT1 does not behave like a classical tumor-suppressor gene but the antitumor activity of resveratrol is mediated at least in part by SirT1.

Carbon isotope analysis of acetaldehyde emitted from leaves following mechanical stress and anoxia.

Although the emission of acetaldehyde from plants into the atmosphere following biotic and abiotic stresses may significantly impact air quality and climate, its metabolic origin(s) remains uncertain. We investigated the pathway(s) responsible for the production of acetaldehyde in plants by studying variations in the stable carbon isotope composition of acetaldehyde emitted during leaf anoxia or following mechanical stress. Under an anoxic environment, C3 leaves produced acetaldehyde during ethanolic fermentation with a similar carbon isotopic composition to C3 bulk biomass. In contrast, the initial emission burst following mechanical wounding was 5-12 per thousand more depleted in (13)C than emissions under anoxia. Due to a large kinetic isotope effect during pyruvate decarboxylation catalysed by pyruvate dehydrogenase, acetyl-CoA and its biosynthetic products such as fatty acids are also depleted in (13)C relative to bulk biomass. It is well known that leaf wounding stimulates the release of large quantities of fatty acids from membranes, as well as the accumulation of reactive oxygen species (ROS). We suggest that, following leaf wounding, acetaldehyde depleted in (13)C is produced from fatty acid peroxidation reactions initiated by the accumulation of ROS. However, a variety of other pathways could also explain our results, including the conversion of acetyl-CoA to acetaldehyde by the esterase activity of aldehyde dehydrogenase.

The Sirt1 deacetylase modulates the insulin-like growth factor signaling pathway in mammals.

The lifespan of the nematode, Caenorhabditis elegans, can be extended by mutations affecting components of the insulin-like growth factor (IGF) signaling cascade or by overexpression of SIR2, an NAD+-dependent protein deacetylase. The mammalian homologue of SIR2, Sirt1, has been shown to modulate the activity of FoxO, a transcription factor that is downstream of the IGF signaling system. These results suggest that Sirt1 ought to affect the IGF pathway. We report here evidence that this is the case in mice. The loss of Sirt1 protein in mice results in increased expression of the IGF binding protein IGFBP1, a secreted modulator of IGF function. A number of the anatomical characteristics of Sirt1-null mice closely resemble those of transgenic mice overexpressing IGFBP1. Our data suggest that Sirt1 is part of a regulatory loop that limits the production of IGFBP1 thereby modulating IGF signaling.

Reexpression of a cluster of silenced transgenes is associated with their rearrangement.

Irreversible inactivation or silencing of tumor suppressor genes occurs frequently in the development of cancer. A similar process of silencing can occur after the integration of transfected or microinjected genes into the genomes of recipient cells. The inactivation of transfected genes seems particularly efficient in cells with stem cell characteristics. We have been studying the inactivation of genes transfected into cultured P19 embryonal carcinoma cells and found that the CpG-rich sequence comprising the coding region of the lacZ reporter gene becomes extensively methylated after integration into the genome. 5-Aza-2'-deoxycytidine (5AdC), an inhibitor of DNA methylation, induced the reexpression of silent transgenes in one clone of P19 cells studied in detail. However, the reexpressed genes remained heavily methylated over the lacZ coding sequence. We used pulsed-field gel electrophoresis to analyze the structure of the transgenic locus in the parental and in 5AdC-treated cells and found that, in each of the cells reexpressing the transgene, the cluster of transgenes had been rearranged. Each clone had undergone a different rearrangement that appeared to involve recombination within the tandemly repeated copies of the transgene. Our data seem consistent with the idea that 5AdC induces efficient DNA recombination between tandemly repeated genes and that the reexpression of silenced genes induced by 5AdC might be triggered by the chromatin reorganization at the site of DNA recombination.

DNA methylation pattern of a tandemly repeated LacZ transgene indicates that most copies are silent.

The Pgk-1,2-lacZ transgene consists of the ubiquitously-expressed Pgk-1 promoter driving expression of the E. coli lacZ reporter gene. We studied the expression of this transgene in a mouse strain carrying 8-9 tandem copies of this construct. When inherited through the male germ line, the transgene was expressed in all tissues examined but when inherited through the female germ line, the transgene became irreversibly inactivated. The lacZ region is a CpG-rich island that was essentially entirely methylated in all copies of the silent, maternally-inherited transgene. At the active transgenic locus, all but one of the copies were entirely methylated. This one unmethylated copy was adjacent to the cellular DNA and was presumed to be the expressed transgene copy. These results suggest that the tandem repeats of transgenes become silenced by a mechanism associated with DNAmethylation and that proximity to the cellular genome may be important in maintaining expression against the spread of inactivation from the adjacent silent transgenes.

Absence of p53-dependent cell cycle regulation in pluripotent mouse cell lines.

We examined the expression of p53 in three lines of pluripotent embryonal carcinoma (EC) and ES cells. p53 mRNA and protein levels were constitutively high in two lines but absent from one. In the P19 line of EC cells neither p53 protein nor mRNA was detected. The first intron of the p53 gene in these cells had been invaded by a murine leukemia virus and there was extensive hypermethylation of the p53 gene accompanying its inactivation. In all three cell lines, irradiation resulted in arrest of the cells in the G2 but not in the G1 phase of the cell cycle despite the induction of p21cip1 in the cell lines expressing p53. Thus, the chromosomal stability of EC and ES cells appears to be not dependent on the p53 protein and we interpret our results to suggest that these cells may require the deletion of p53 dependent cell cycle regulation in order to become immortalized.

A role for RNA processing in regulating expression from transfected genes.

We have examined the expression of cloned genes following their stable integration into the genome of pluripotent embryonal carcinoma stem cells. Transfected genes integrate into the genome as tandem arrays. Expression of reporter genes from these tandem arrays in embryonal carcinoma cells is inefficient probably because genes are subject to repeat-induced gene silencing. We found that expression of reporter genes was significantly enhanced if co-transfected with cloned fragments derived from the murine Pgk-1 gene. The enhanced expression required (a) that the Pgk-1 fragment carries an active promoter, (b) that the promoter drives transcription through a region of more than 12 kbp, and (c) that this transcribed region contains both introns and exons. Reporter gene activity did not require specific Pgk-1 DNA sequences suggesting that the coupled processes of transcription and RNA processing conferred activity on neighboring genes probably by influencing local chromatin structure. Consistent with this idea, the effect of the Pgk-1 gene could be mimicked by exposing cells to butyrate or trichostatin A, inhibitors of histone deacetylase. Thus, the effect of the co-transfected Pgk-1 gene is to inhibit the process of gene inactivation possibly by functioning like an insulator or boundary element in the chromatin.

Genes transfected into embryonal carcinoma stem cells are both lost and inactivated at high frequency.

Embryonal carcinoma (EC) cells can be efficiently transfected with cloned DNAs but there is a strong tendency for expression from transfected genes to be lost from stably transformed cells. To investigate the mechanism responsible for this loss of expression, we transfected P19 EC cells with a gene encoding the E. coli beta-galactosidase and examined expression of this gene in clonal populations of cells. Cells that carry and express the beta-galactosidase gene give rise to cells that do not express at a rate of about 0.02 events per cell per cell division. These non-expressing cells were of two types, some had lost the transfected genes while others had inactivated them. In those cells that retained but inactivated the transfected genes, the inactive state was stable and suppression was at the level of transcription initiation but not associated with increased DNA methylation. Because transfected DNAs integrate into the genome as tandem arrays, the gene loss and inactivation seen in EC cells may be analogous to the repeat-induced gene inactivation seen in lower eukaryotes.

Cells differentiating into neuroectoderm undergo apoptosis in the absence of functional retinoblastoma family proteins.

The retinoblastoma (RB) protein is present at low levels in early mouse embryos and in pluripotent P19 embryonal carcinoma cells; however, the levels of RB rise dramatically in neuroectoderm formed both in embryos and in differentiating cultures of P19 cells. To investigate the effect of inactivating RB and related proteins p107 and p130, we transfected P19 cells with genes encoding mutated versions of the adenovirus E1A protein that bind RB and related proteins. When these E1A-expressing P19 cells were induced to differentiate into neuroectoderm, there was a striking rise in the expression of c-fos and extensive cell death. The ultrastructural and biochemical characteristics of the dying cells were indicative of apoptosis. The dying cells were those committed to the neural lineages because neurons and astrocytes were lost from differentiating cultures. Cell death was dependent on the ability of the E1A protein to bind RB and related proteins. Our results suggest that proteins of the RB family are essential for the development of the neural lineages and that the absence of functional RB activity triggers apoptosis of differentiating neuroectodermal cells.

Intragenic regions of the murine Pgk-1 locus enhance integration of transfected DNAs into genomes of embryonal carcinoma cells.

Introduction of recombinant genes into mammalian cells in culture has been an important procedure in establishing the molecular mechanisms of various cellular processes. The efficiency with which plasmid borne recombinant genes are expressed following stable integration into genomes of embryonal carcinoma cells is low. Using the P19 embryonal carcinoma cells as recipients, we found that constructs carrying the promoter and intragenic regions of the murine Pgk-1 gene were expressed with high efficiency. This elevated expression was associated with increased numbers of copies of the transfected plasmid DNA stably associated with the genomes of recipient cells. The elevated plasmid copy numbers may result from enhanced ligation of transfected plasmids because cotransfected plasmids were also integrated in increased numbers. The enhanced integration and expression of transfected plasmids required active transcription through an intragenic region of Pgk-1, perhaps resulting in more recombinogenic plasmid DNAs.

Unstable integration of transfected DNAs into embryonal carcinoma cells.

Plasmid DNA can be efficiently transfected into embryonal carcinoma cells but it is difficult to isolate clones of cells stably expressing genes present on the transfected plasmids. Even in clonal populations derived from transfected cells, the introduced genes are expressed in some but not all cells. Cotransfection with a region of the Pgk-1 gene results in more efficient, stable cotransformation due to increased numbers of copies of the transfected plasmids integrated into the genomic DNA. The PgK-1 genomic sequences did not allow the plasmid DNA to replicate autonomously but seemed to enhance the ligation of transfected plasmids before their integration into the host genome. Our results suggest a model in which the plasmid DNAs are able to integrate and subsequently excise from the host genome by recombination events enhanced by transcription through the tandemly repeated sequences of the transfected plasmids.

Murine PGK-1 promoter drives widespread but not uniform expression in transgenic mice.

Pgk-1 is an X-linked gene encoding 3-phosphoglycerate kinase, an enzyme necessary in every cell for glycolysis. The regulatory sequences of the Pgk-1 gene were used to drive the E. coli lacZ reporter gene and 2 strains of transgenic animals created with this Pgk-lacZ transgene carried on autosomes. The levels of expression of Pgk-1 varied from one adult tissue to another and the transgene was similarly regulated. However, in situ staining of the beta-galactosidase encoded by the transgene indicated extensive cell-to-cell variability in its level of expression. A reproducible subset of cells stained darkly for the transgene product. Some of these beta-galactosidase positive cells were rapidly proliferating while others appeared to be metabolically very active, suggesting that the Pgk-1 promoter is regulated so as to be more active in cells requiring high levels of glycolysis. Although Pgk-1 is X-linked and subject to X chromosome inactivation, the transgenes were not inactivated in either female somatic or male germ cells. Thus, the Pgk-1 promoter drives transgene expression in all tissues but the levels of expression are not uniform in each cell.

Expression of the Brachyury gene during mesoderm development in differentiating embryonal carcinoma cell cultures.

When aggregated and treated with dimethyl sulfoxide (DMSO), P19 embryonal carcinoma cells differentiate into cell types normally derived from the mesoderm and endoderm including epithelium and cardiac and skeletal muscle. The Brachyury gene is expressed transiently in these differentiating cultures several days before the appearance of markers of the differentiated cell types. The expression of Brachyury is not affected by DMSO but is induced by cell aggregation, which requires extracellular calcium. Expression of Brachyury is also induced by various members of the TGF beta family such as activin and bone morphogenetic proteins. D3 is a mutant clone of P19 cells selected for its failure to differentiate when aggregated in DMSO. Aggregated D3 cells express Brachyury mRNA suggesting that the mutation(s) responsible for the phenotype of D3 cells is downstream of the chain of events initiated by Brachyury expression.