Population response of reintroduced bighorn sheep after observed commingling with domestic sheep

Bighorn sheep (Ovis canadensis) often die from respiratory disease after commingling with domestic sheep. From 2000 to 2009, we observed commingling between domestic and reintroduced bighorn sheep in 3 populations in UT, USA. We investigated how commingling affected survival of radio-collared female bighorns that were released initially (founder) and those that were subsequently released (augmented). We predicted that the proportion of young surviving to their first winter and population growth would be lower after observed commingling with domestic sheep. We observed groups of bighorns year-round on 2,712 occasions and commingling between domestic sheep and bighorns in 6 instances. On Mount Timpanogos, survival rates were best modeled as constant for females (n?=?57) before and after observed commingling with domestic sheep. Survival rates of female bighorns, however, decreased significantly in Rock Canyon (n?=?21) and on Mount Nebo (n?=?22) for founder, but not augmented bighorns after observed commingling with domestic sheep. Also, the proportion of young surviving to their first winter was almost 3 times lower and population growth was reduced for bighorns after observed commingling with domestic sheep in Rock Canyon and on Mount Nebo. Commingling between domestic and bighorn sheep reduced population parameters in 2 of 3 bighorn populations we studied; however, on Mount Timpanogos, interactions between those 2 species were not fatal for radio-collared female bighorns. Wildlife biologists should manage for spatial separation of these 2 species and consider the location of hobby farms and trailing operations of domestic sheep near release sites for bighorns.     

Solubilisation of ferricytochrome c in methanol using a crown ether: absorption, circular dichroism and EPR spectral properties

Ferricytochrome c is normally insoluble in methanol, but its solution is facilitated by complexation with 18-crown-6. Absorption, circular dichroism and EPR spectroscopy indicate that the solubilised protein in MeOH exists in at least three conformational states, all different from the native state in neutral aqueous solution. In two states the haem iron (III) is low spin and in one state it is high spin, but it seems likely that all three forms are globular. The proportion in the high spin form increases at increasing crown ether concentration and on ageing the protein solution. The protein appears to return to its native conformation when it is restored to an aqueous environment.     

A smart membrane based on an antigen-responsive hydrogel

Hydrogel membranes have been fabricated that incorporate antibody/antigen moieties. The permeability of large solutes through these membranes is dependent on the presence of soluble antigen that can compete with the internal interactions between antibody and antigen leading to an increase in gel mesh size. Specifically, the membrane's structure is based on a dextran backbone grafted with a fluorescein isothiocyanate (FITC) antigen and a sheep anti-FITC IgG antibody. The backbone is covalently cross-linked by conjugated divinyl sulfone (DVS) groups. The gel structure is additionally stabilized by affinity crosslinks formed by biospecific interactions between the bound IgG and FITC. FTIR spectra of the gel are consistent with formation of covalent bonds between cysteine groups in the IgG and DVS groups in the dextran. Results obtained using isothermal titration calorimetry (ITC) confirmed the competitive interaction binding between IgG-FITC-dextran and free sodium fluorescein at pH 5.0. Scanning electron microscopy (SEM) of samples prepared using cryofixation and cryofracturing techniques showed that observed changes in permeability correlate with free fluorescein-dependent structural changes in the gel. Three-dimensional images obtained from confocal laser scanning microscopy show that these changes occur throughout the gel and indicate that SEM results are not artifacts of sample preparation. The permeability of these gels, as shown by blue-dextran (12 kDa) diffusion, increases in response to the presence of free fluorescein of the external medium, which causes competitive displacement of the affinity cross-links. Sequential addition and removal of sodium fluorescein showed that these permeability changes are reversible.     

Distinct leaf developmental and gene expression responses to light quantity depend on blue-photoreceptor or plastid-derived signals,and can occur in the absence of phototropins

Leaf palisade cell development and the composition of chloroplasts respond to the fluence rate of light to maximise photosynthetic light capture while minimising photodamage. The underlying light sensory mechanisms are probably multiple and remain only partially understood. Phototropins (PHOT1 and PHOT2) are blue light receptors regulating responses which are light quantity-dependent and which include the control of leaf expansion. Here we show that genes for proteins in the reaction centres show long-term responses in wild type plants, and single blue photoreceptor mutants, to light fluence rate consistent with regulation by photosynthetic redox signals. Using contrasting intensities of white or broad-band red or blue light, we observe that increased fluence rate results in thicker leaves and greater number of palisade cells, but the anticlinal elongation of those cells is specifically responsive to the fluence rate of blue light. This palisade cell elongation response is still quantitatively normal in fully light-exposed regions of phot1 phot2 double mutants under increased fluence rate of white light. Plants grown at high light display elevated expression of RBCS (for the Rubisco small subunit) which, together with expected down-regulation of LHCB1 (for the photosynthetic antenna primarily of photosystem II), is also observed in phot double mutants. We conclude that an unknown blue light photoreceptor, or combination thereof, controls the development of a typical palisade cell morphology, but phototropins are not essential for either this response or acclimation-related gene expression changes. Together with previous evidence, our data further demonstrate that photosynthetic (chloroplast-derived) signals play a central role in the majority of acclimation responses.     

Global profiling of proteolysis during rupture of Plasmodium falciparum from the host erythrocyte

The obligate intracellular parasite pathogen Plasmodium falciparum is the causative agent of malaria, a disease that results in nearly one million deaths per year. A key step in disease pathology in the human host is the parasite-mediated rupture of red blood cells, a process that requires extensive proteolysis of a number of host and parasite proteins. However, only a relatively small number of specific proteolytic processing events have been characterized. Here we describe the application of the Protein Topography and Migration Analysis Platform (PROTOMAP) (Dix, M. M., Simon, G. M., and Cravatt, B. F. (2008) Global mapping of the topography and magnitude of proteolytic events in apoptosis. Cell 134, 679-691; Simon, G. M., Dix, M. M., and Cravatt, B. F. (2009) Comparative assessment of large-scale proteomic studies of apoptotic proteolysis. ACS Chem. Biol. 4, 401-408) technology to globally profile proteolytic events occurring over the last 6-8 h of the intraerythrocytic cycle of P. falciparum. Using this method, we were able to generate peptographs for a large number of proteins at 6 h prior to rupture as well as at the point of rupture and in purified merozoites after exit from the host cell. These peptographs allowed assessment of proteolytic processing as well as changes in both protein localization and overall stage-specific expression of a large number of parasite proteins. Furthermore, by using a highly selective inhibitor of the cysteine protease dipeptidyl aminopeptidase 3 (DPAP3) that has been shown to be a key regulator of host cell rupture, we were able to identify specific substrates whose processing may be of particular importance to the process of host cell rupture. These results provide the first global map of the proteolytic processing events that take place as the human malarial parasite extracts itself from the host red blood cell. These data also provide insight into the biochemical events that take place during host cell rupture and are likely to be valuable for the study of proteases that could potentially be targeted for therapeutic gain.     

Arabidopsis cue mutants with defective plastids are impaired primarily in the photocontrol of expression of photosynthesis-associated nuclear genes

Plant photoreceptors detect light cues and initiate responses ranging from chloroplast differentiation to the control of morphogenesis and flowering. The photocontrol of photosynthesis-related nuclear genes appears closely related to retrograde plastid signals by which the status of the organelle controls the expression of nuclear genes. However, what specific role, if any, plastid-originated signals play in light responses is poorly understood: it has in the past been proposed that plastid signals play a role in all responses to high fluence far-red light perceived by the light-labile phytochrome A, irrespective of whether they involve photosynthesis-related genes. To explore this further, we have re-examined the phenotype of three cue (cab-underexpressed) Arabidopsis mutants, defective in chloroplast development. The mutants have underdeveloped etioplasts, with increasing impairments in cue6, cue8 and cue3. The mutants show only small defects in photocontrol of hypocotyl elongation and cotyledon opening under prolonged far-red or red light, and normal photocontrol under blue. On the other hand, the expression of photosynthesis-associated nuclear genes is much more impaired in the mutants in the dark and following red or far-red light short treatments or continuous light, than that of those phytochrome-dependent genes tested which are not associated with photosynthesis. Furthermore, red/far-red photoreversible responses involving photosynthesis-related genes (induction of Lhcb1–cab promoter activity, and photoreversible extent of greening) mediated by phytochrome B and other photo-stable phytochromes, both show a reduction in the cue mutants, which correlates with the etioplast defect. Our evidence demonstrates that plastid-derived signals need to be operational in order for the phytochrome control of photosynthetic nuclear genes to occur.     

Adult reserve stem cells and their potential for tissue engineering

Tissue restoration is the process whereby multiple damaged cell types are replaced to restore the histoarchitecture and function to the tissue. Several theories, have been proposed to explain the phenomenon of tissue restoration in amphibians and in animals belonging to higher order. These theories include dedifferentiation of damaged tissues, transdifferentiation of lineage-committed progenitor cells, and activation of reserve, precursor cells. Studies by Young et al. and others demonstrated that connective tissue compartments throughout postnatal individuals contain reserve precursor cells. Subsequent repetitive single cell-cloning and cell-sorting studies revealed that these reserve precursor cells consisted of multiple populations of cells, including, tissue-specific progenitor cells, germ-layer lineage stem cells, and pluripotent stem cells. Tissue-specific progenitor cells display various capacities for differentiation, ranging from unipotency (forming a single cell type) to multipotency (forming multiple cell types). However, all progenitor cells demonstrate a finite life span of 50 to 70 population doublings before programmed cell senescence and cell death occurs. Germ-layer lineage stem cells can form a wider range of cell types than a progenitor cell. An individual germ-layer lineage stem cell can form all cells types within its respective germ-layer lineage (i.e., ectoderm, mesoderm, or endoderm). Pluripotent stem cells can form a wider range of cell types than a single germ-layer lineage stem cell. A single pluripotent stem cell can form cells belonging to all three germ layer lineages. Both germ-layer lineage stem cells and pluripotent stem cells exhibit extended capabilities for self-renewal, far surpassing the limited life span of progenitor cells (50–70 population doublings). The authors propose that the activation of quiescent tissue-specific progenitor cells, germ-layer lineage stem cells, and/or pluripotent stem cells may be a potential explanation, along with dedifferentiation and transdifferentiation, for the process of tissue restoration. Several model systems are currently being investigated to determine the possibilities of using these adult quiescent reserve precursor cells for tissue engineering.     

Determination of d-fenfluramine, d-norfenfluramine and fluoxetine in plasma, brain tissue and brain microdialysate using high-performance liquid chromatography after precolumn derivatization with dansyl chloride

A HPLC method is described for the simultaneous determination of d-fenfluramine (FEN), d-norfenfluramine (NF) and fluoxetine (FLX) using fluorometric detection after precolumn derivatization with dansyl-chloride. The method has limits of quantitation of 200 fmol for FEN and NF, 500 fmol for FLX in brain microdialysate, and 1 pmol for NF and FEN, and 2 pmol for FLX in plasma. Brain tissue standards were linear between 5 and 200 pmol/mg for all three compounds. The inter-assay variability (relative standard deviation) was 6.6%, 6.9% and 9.3% for FEN, 4.6%, 3.7% and 7.9% for NF and 10.4%, 4.9% and 12.2% for FLX, for brain microdialysate (2 pmol/μl), plasma (2 pmol/ μl) and brain tissue (50 pmol/mg), respectively. Intra-assay variability was always lower, typically several times lower than inter-assay variability. Extraction recovery was 108% and 48% for FEN, 105% and 78% for NF and 94% and 45% for FLX, in plasma (2 pmol/μl) and brain tissue (5 pmol/mg), respectively. Due to the stability of the dansyl-chloride derivatives this method is well suited for an autoinjector after manual derivatization with dansyl chloride at room temperature for 4 h.