Transcriptional regulation of cell lineage-specific expression of the murine type I collagen genes and of osteoblast differentiation

The aim of my project is to examine the mechanisms of cell lineage-specific transcriptional regulation of the two type I collagen genes by characterizing critical cis-acting elements and trans-acting factors. I hypothesize that the transcription factors that are involved in the cell lineage-specific expression of these genes may have a larger essential role in cell lineage commitment and differentiation. I first examined the proximal promoters of the proα1(I) and the proα2(I) collagen genes for cell type-specific DNA-protein interactions, using in vitro DNaseI and in vivo DMS footprinting. These experiments demonstrated that the cis-acting elements in these promoters are accessible to ubiquitous DNA-binding proteins in fibroblasts that express these genes, but not in other cells that do not express these genes. I speculate that in type I collagen-expressing cells, cell type-specific enhancer elements facilitate binding of ubiquitous proteins to the proximal promoters of these genes. Subsequently, examination of the upstream promoter of the proα(I) collagen gene by transgenic mice experiments delineated a 117 bp sequence (-1656 to -1540 bp) as the minimum element required for osteoblast-specific expression. This 117 bp element contained two segments that appeared to have different functions: (1) the A-segment, which was necessary to obtain osteoblast-specific expression and (2) the C-segment, which was dispensable for osteoblast-specific expression, but was necessary to obtain high-level expression. In experiments to identify trans-acting factors that bind to the 117 bp element, I have demonstrated that the cell lineage-restricted homeodomain proteins, Dlx2, Dlx5 and mHOX, bound to the A-segment and that the ubiquitous transcription factor, Sp1, bound to the C-segment of this element. These results suggested a model where the binding of cell lineage-restricted proteins to the A-segment and of ubiquitous proteins to the C-segment of the 117 bp element of the proα1 (I) collagen gene activated this gene in osteoblasts. These results, combined with additional evidence that Dlx2, Dlx5 and mHOX are probably involved in osteoblast differentiation, support my hypothesis that the transcription factors involved in osteoblast-specific expression of type I collagen genes may have essential role in osteoblast lineage commitment and differentiation. ^

Ascorbic acid effects on the immune system and type -1 /type -2 cytokine balance in humans

Vitamin C (ascorbic acid--AA) can have a substantial impact on human health by reducing the incidence and/or severity of coryza. Studies also suggest it has immunomodulatory functions in humans. Immune function is controlled by cytokines, such as type-1 cytokines (IFNγ) that promote antiviral immunity and type-2 cytokines (IL-4, IL-10) that promote humoral immunity. Knowing the mechanisms responsible for both antiviral immunity and type-1/type-2 cytokine balance, we sought to identify AA-induced alterations of human peripheral blood mononuclear cells (PBMC) in vivo and in vitro . We hypothesized that AA modulates the immune system, altering both number and function of PBMC. We first described the effect of 14 days of oral (1 gram) AA in healthy subjects. AA increased circulating natural killer (NK) cells, CD25+ and HLA-DR+ T cells, and PMA/ionomycin-stimulated intracellular IFNγ. We subsequently developed models for in vitro use. We determined that AA was toxic in vitro to T cells when used at doses found intracellularly but doses found in plasma from individuals taking 1gm/day AA were nontoxic. The model that most fully reproduced our in vivo intracellular cytokine findings used dehydroascorbic acid and buffers to deliver AA intracellularly. This model generated the largest increase in IFNγ at physiologic plasma concentrations. Previous studies demonstrate that chronic psychological stress is associated with a type-2 cytokine response. We hypothesized that vitamin C could prevent the type-2 cytokine shift associated with stress. In a study of medical students taking 1 g AA or placebo, a significant increase in IFNγ was seen intracellularly in CD4+ and CD8+ cells and in tetanus-stimulated cultures in the AA group only. We also observed increases in IFNγ/IL-4 and IFNγ/IL-10 ratios with AA supplementation, indicating a type-1 shift. Furthermore, we noted increased numbers of NK cells and activated T cells in the peripheral blood in the AA treated group only. Lastly, we investigated the role of the CD40L/CD40 and CD28/B7 costimulatory pathway in these cytokine alterations. AA did not have any effect on either pathway studied. Thus costimulatory pathways are not contributing to AA induced modulation of the type-1/type-2 immune balance. ^

Tissue metal concentrations and metal transfer functions of Glaucous Gulls (Larus hyperboreus) from Bjørnøya and Jan Mayen

Heavy metals (Cd, Cu, Fe, Mn and Zn) concentrations were determined in different tissues (muscle, kidney, liver, brain, gonads, heart and feathers) of Glaucous Gulls (Larus hyperboreus) from Bjornoya and Jan Mayen. The age and spatial dependent variations in heavy metals were quantified and interpreted in view of the three chemometric techniques, i.e. non-parametric Mann-Whitney U test, redundancy gradient analysis and detrended correspondence analysis. The Glaucous Gulls from Bjornoya contained significantly higher (p < 0.05) levels of Cd, Cu and Zn than those inhabited Jan Mayen. Adult birds were characterized by greater (p < 0.01) concentration of muscle, hepatic and renal heavy metals in comparison to chicks. Insignificantly higher slope constant Zn/Cd for the liver than for the kidney may reflect insignificant Cd exposure. Estimate of transfer factor (TF) allows us to assess variations in heavy metal concentrations during the individual development of Glaucous Gulls. It may be stated that there is a distinct increase of bioaccumulation of all the studied metals during subsequent stages of the bird life.

Seagrass and associated benthic community data derived from field surveys at Low Isles, Great Barrier Reef, conducted July-August, 1997

The distribution of seagrass and associated benthic communities on the reef and lagoon of Low Isles, Great Barrier Reef, was mapped between the 29 July and 29 August 1997. For this survey, observers walked or free-dived at survey points positioned approximately 50 m apart along a series of transects. Visual estimates of above-ground seagrass biomass and % cover of each benthos and substrate type were recorded at each survey point. A differential handheld global positioning system (GPS) was used to locate each survey point (accuracy ±3m). A total of 349 benthic survey points were examined. To assist with mapping meadow/habitat type boundaries, an additional 177 field points were assessed and a georeferenced 1:12,000 aerial photograph (26th August 1997) was used as a secondary source of information. Bathymetric data (elevation below Mean Sea Level) measured at each point assessed and from Ellison (1997) supplemented information used to determine boundaries, particularly in the subtidal lagoon. 127.8 ±29.6 hectares was mapped. Seagrass and associated benthic community data was derived by haphazardly placing 3 quadrats (0.25m**2) at each survey point. Seagrass above ground biomass (standing crop, grams dry weight (g DW m**-2)) was determined within each quadrat using a non-destructive visual estimates of biomass technique and the seagrass species present identified. In addition, the cover of all benthos was measured within each of the 3 quadrats using a systematic 5 point method. For each quadrat, frequency of occurrence for each benthic category was converted to a percentage of the total number of points (5 per quadrat). Data are presented as the average of the 3 quadrats at each point. Polygons of discrete seagrass meadow/habitat type boundaries were created using the on-screen digitising functions of ArcGIS (ESRI Inc.), differentiated on the basis of colour, texture, and the geomorphic and geographical context. The resulting seagrass and benthic cover data of each survey point and for each seagrass meadow/habitat type was linked to GPS coordinates, saved as an ArcMap point and polygon shapefile, respectively, and projected to Universal Transverse Mercator WGS84 Zone 55 South.

Discrete carbonate chemistry measured during the M87/1 cruise in April 2012

The track of the cruise, and the location of the different stations cover a large range of water masses, many of which take part in the exchange across the Greenland-Scotland Ridge, and of importance for the biogeochemical fluxes in the region. These water masses are of very different origins, which can be observed in the concentration of the different biogeochemical parameters. The concentrations are a result of the combination of the physical and biogeochemical environment in each formation region, and the processes acting on the water masses as they are transported away from the formation areas. The aim of the biogeochemistry measurements was to achieve a better understanding of the strength and variability of the biological carbon pump in the North Atlantic and Nordic Seas.