Morbidity and mortality factors in key deer (Odocoileus virginianus clavium)

The population health of endangered Key deer (Odocoileus virginianus clavium) was monitored from 10 February 1986 to 28 September 2000 by necropsy of animals that were killed by vehicles, euthanized because of terminal injuries or disease conditions, or found dead. The predominant mortality factor during the period was collision with motor vehicles; however, several infectious diseases were diagnosed, including infections with Arcanobacterium pyogenes, Haemonchus contortus, Salmonella spp., and Mycobacterium avium subsp. paratuberculosis. During the period monitored, the only infectious disease that was thought to have affected population dynamics was haemonchosis. Nevertheless, several of the observed diseases have potential to impact viability of the Key deer population under appropriate environmental conditions.     

Oxidant-induced DNA damage by quartz in alveolar epithelial cells

Respirable quartz has recently been classified as a human carcinogen. Although, studies with quartz using naked DNA as a target suggest that formation of oxyradicals by particles may play a role in the DNA-damaging properties of quartz, it is not known whether this pathway is important for DNA damage in the target cells for quartz carcinogenesis, i.e. alveolar epithelial cells. Therefore, we determined in vitro DNA damage by DQ12 quartz particles in rat and human and alveolar epithelial cells (RLE, A549) using the single cell gel electrophoresis/comet assay. The radical generation capacity of quartz was analysed by electron spin resonance (ESR) and by immunocytochemical analysis of the hydroxyl radical-specific DNA lesion 8-hydroxydeoxyguanosine (8-OHdG) in the epithelial cells. Quartz particles as well as the positive control hydrogen peroxide, caused a dose-dependent increase in DNA strand breaks in both cell lines. DNA damage by quartz was significantly reduced in the presence of the hydroxyl-radical scavengers mannitol or DMSO. The involvement of hydroxyl radicals was further established by ESR measurements and was also demonstrated by the ability of the quartz to induce formation of 8-OHdG. In conclusion, our data show that quartz elicits DNA damage in rat and human alveolar epithelial cells and indicate that these effects are driven by hydroxyl radical-generating properties of the particles.     

Ligand receptor interactions in the Wnt signaling pathway in Drosophila

Secreted Wnt proteins have numerous signaling functions during development, mediated by Frizzled molecules that act as Wnt receptors on the cell surface. In the genome of Drosophila, seven Wnt genes (including wingless; wg), and five frizzled genes have been identified. Relatively little is known about signaling and binding specificities of different Wnt and Frizzled proteins. We have developed an assay to determine the strength of binding between membrane-tethered Wnts and ligand binding domains of the Frizzled receptors. We found a wide spectrum of binding affinities, reflecting known genetic interactions. Most Wnt proteins can bind to multiple Frizzleds and vice versa, suggesting redundancy in vivo. In an extension of these experiments, we tested whether two different subdomains of the Wg protein would by themselves bind to Frizzled and generate a biological response. Whereas these two separate domains are secreted from cells, suggesting that they form independently folded parts of the protein, they were only able to evoke a response when co-transfected, indicating that both are required for function. In addition to the Frizzleds, members of the LRP family (represented by the arrow gene in Drosophila) are also necessary for Wnt signal transduction and have been postulated to act as co-receptors. We have therefore examined whether a soluble form of the Arrow molecule can bind to Wingless and Frizzled, but no interactions were detected.     

Short-term effects of biological and physical forces on aggregate formation in soils with different clay mineralogy

The mechanisms resulting in the binding of primary soil particles into stable aggregates vary with soil parent material, climate, vegetation, and management practices. In this study, we investigated short-term effects of: (i) nutrient addition (Hoagland's solution), (ii) organic carbon (OC) input (wheat residue), (iii) drying and wetting action, and (iv) root growth, with or without dry–wet cycles, on aggregate formation and stabilization in three soils differing in weathering status and clay mineralogy. These soils included a young, slightly weathered temperate soil dominated by 2:1 (illite and chlorite) clay minerals; a moderately weathered soil with mixed [2:1 (vermiculite) and 1:1 (kaolinite)] clay mineralogy and oxides; and a highly weathered tropical soil dominated by 1:1 (kaolinite) clay minerals and oxides. Air-dried soil was dry sieved through a 250 m sieve to break up all macroaggregates and 100 g-subsamples were brought to field capacity and incubated for 42 days. After 14 and 42 days, aggregate stability was measured on field moist and air-dried soil, to determine unstable and stable aggregation respectively. In control treatments (i.e., without nutrient or organic matter addition, without roots and at constant moisture), the formation of unstable and stable macroaggregates (> 250 m) increased in the order: 2:1 clay soil < mixed clay soil < 1:1 clay soil. After 42 days of incubation, nutrient addition significantly increased both unstable and stable macroaggregates in the 2:1 and 1:1 clay soils. In all soils, additional OC input increased both unstable and stable macroaggregate formation. The increase in macroaggregation with OC input was highest for the mixed clay soil and lowest for the 1:1 clay soil. In general, drying and wetting cycles had a positive effect on the formation of macroaggregates. Root growth caused a decrease in unstable macroaggregates in all soils. Larger amounts of macroaggregates were found in the mixed clay and oxides soil when plants were grown under 50% compared to 100% field capacity conditions. We concluded that soils dominated by variable charge clay minerals (1:1 clays and oxides) have higher potential to form stable aggregates when OC concentrations are low. With additional OC inputs, the greatest response in stable macroaggregate formation occurred in soils with mixed mineralogy, which is probably a result of different binding mechanisms occurring: i.e., electrostatic bindings between 2:1 clays, 1:1 clays and oxides (i.e. mineral-mineral bindings), in addition to OM functioning as a binding agent between 2:1 and 1:1 clays.     

Wingless blocks bristle formation and morphogenetic furrow progression in the eye through repression of Daughterless

In the developing eye, wingless activity represses proneural gene expression (and thus interommatidial bristle formation) and positions the morphogenetic furrow by blocking its initiation in the dorsal and ventral regions of the presumptive eye. We provide evidence that wingless mediates both effects, at least in part, through repression of the basic helix-loop-helix protein Daughterless. daughterless is required for high proneural gene expression and furrow progression. Ectopic expression of wingless blocks Daughterless expression in the proneural clusters. This repression, and that of furrow progression, can be mimicked by an activated form of armadillo and blocked by a dominant negative form of pangolin/TCF. Placing daughterless under the control of a heterologous promoter blocks the ability of ectopic wingless to inhibit bristle formation and furrow progression. hedgehog and decapentapleigic could not rescue the wingless furrow progression block, indicating that wingless acts downstream of these genes. In contrast, Atonal and Scute, which are thought to heterodimerize with Daughterless to promote furrow progression and bristle formation, respectively, can block ectopic wingless action. These results are summarized in a model where daughterless is a major, but probably not the only, target of wingless action in the eye.     

delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase,that mediates the first committed step in penicillin biosynthesis,is a cytosolic enzyme

Penicillin biosynthesis by Penicillium chrysogenum is a compartmentalized process. The first catalytic step is mediated by delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACV synthetase), a high molecular mass enzyme that condenses the amino acids L-alpha-aminoadipate, L-cysteine, and L-valine into the tripeptide ACV. ACV synthetase has previously been localized to the vacuole where it is thought to utilize amino acids from the vacuolar pools. We localized ACV synthetase by subcellular fractionation and immuno-electron microscopy under conditions that prevented proteolysis and found it to co-localize with isopenicillin N synthetase in the cytosol, while acyltransferase localizes in microbodies. These data imply that the key enzymatic steps in penicillin biosynthesis are confined to only two compartments, i.e., the cytosol and microbody.     

Effects of thyroid state on the expression of hepatic thyroid hormone transporters in rats

Liver uptake of thyroxine (T4) is mediated by transporters and is rate limiting for hepatic 3,3',5-triiodothyronine (T3) production. We investigated whether hepatic mRNA for T4 transporters is regulated by thyroid state using Xenopus laevis oocytes as an expression system. Because X. laevis oocytes show high endogenous uptake of T4, T4 sulfamate (T4NS) was used as an alternative ligand for the hepatic T4 transporters. Oocytes were injected with 23 ng liver mRNA from euthyroid, hypothyroid, or hyperthyroid rats, and after 3-4 days uptake was determined by incubation of injected and uninjected oocytes for 1 h at 25 degrees C or for 4 h at 18 degrees C with 10 nM [125I]T4NS. Expression of type I deiodinase (D1), which is regulated by thyroid state, was studied in the oocytes as an internal control. Uptake of T4NS showed similar approximately fourfold increases after injection of liver mRNA from euthyroid, hypothyroid, or hyperthyroid rats. A similar lack of effect of thyroid state was observed using reverse T3 as ligand. In contrast, D1 activity induced by liver mRNA from hyperthyroid and hypothyroid rats in the oocytes was 2.4-fold higher and 2.7-fold lower, respectively, compared with euthyroid rats. Studies have shown that uptake of iodothyronines in rat liver is mediated in part by several organic anion transporters, such as the Na+/taurocholate-cotransporting polypeptide (rNTCP) and the Na-independent organic anion-transporting polypeptide (rOATP1). Therefore, the effects of thyroid state on rNTCP, rOATP1, and D1 mRNA levels in rat liver were also determined. Northern analysis showed no differences in rNTCP or rOATP1 mRNA levels between hyperthyroid and hypothyroid rats, whereas D1 mRNA levels varied widely as expected. These results suggest little effect of thyroid state on the levels of mRNA coding for T4 transporters in rat liver, including rNTCP and rOATP1. However, they do not exclude regulation of hepatic T4 transporters by thyroid hormone at the translational and posttranslational level.     

Role of the Bullous Pemphigoid Antigen 180 (BP180) in the Assembly of Hemidesmosomes and Cell Adhesion—Reexpression of BP180 in Generalized Atrophic Benign Epidermolysis Bullosa Keratinocytes

Bullous pemphigoid antigen 180 (BP180) is a transmembrane component of hemidesmosomes (HD), cell–substrate attachment complexes in stratified and complex epithelia. To determine the role of BP180 in the assembly of HD and cell adhesion, using SV40 virions we have immortalized BP180-deficient keratinocytes derived from a patient with the inherited skin blistering disorder generalized atrophic benign epidermolysis bullosa (GABEB). The GABEB keratinocytes form HD-like structures, which contain α6β4 integrin and HD1/plectin, but not the bullous pemphigoid antigen 230 (BP230). The expression of integrin subunits by GABEB keratinocytes was comparable to that of an immortalized normal human keratinocyte cell line (NHK), except for α6 and β4, which were less strongly expressed in GABEB cells. In short-term adhesion assays, both GABEB keratinocytes and NHK bound strongly and to a similar extent to laminin-1, laminin-5, fibronectin, and type IV and V collagens, which suggests that BP180 is not involved in promoting the initial adhesion to these ligands. Transfection of GABEB keratinocytes with cDNAs for wild-type or a mutant of BP180 lacking the collagenous extracellular domain resulted in the expression of recombinant BP180 proteins that were correctly polarized at the basal cell surface together with α6β4. In addition, restored synthesis of BP180 affected the subcellular localization of BP230, which was no longer diffusely distributed in the cytoplasm, but was found in HD-like structures. In contrast, a BP180 mutant with a 36-amino-acid deletion from the amino terminus of the cytoplasmic domain failed to localize to HD-like structures. These results demonstrate that a region within the cytoplasmic domain of BP180 is essential for its localization into HD and that BP180 may play a critical role in coordinating the subcellular distribution of BP230.     

Characterization of Interactions between the Mammalian Polycomb-Group Proteins Enx1/EZH2 and EED Suggests the Existence of Different Mammalian Polycomb-Group Protein Complexes

In Drosophila melanogaster, the Polycomb-group (PcG) and trithorax-group (trxG) genes have been identified as repressors and activators, respectively, of gene expression. Both groups of genes are required for the stable transmission of gene expression patterns to progeny cells throughout development. Several lines of evidence suggest a functional interaction between the PcG and trxG proteins. For example, genetic evidence indicates that the enhancer of zeste [E(z)] gene can be considered both a PcG and a trxG gene. To better understand the molecular interactions in which the E(z) protein is involved, we performed a two-hybrid screen with Enx1/EZH2, a mammalian homolog of E(z), as the target. We report the identification of the human EED protein, which interacts with Enx1/EZH2. EED is the human homolog of eed, a murine PcG gene which has extensive homology with the Drosophila PcG gene extra sex combs (esc). Enx1/EZH2 and EED coimmunoprecipitate, indicating that they also interact in vivo. However, Enx1/EZH2 and EED do not coimmunoprecipitate with other human PcG proteins, such as HPC2 and BMI1. Furthermore, unlike HPC2 and BMI1, which colocalize in nuclear domains of U-2 OS osteosarcoma cells, Enx1/EZH2 and EED do not colocalize with HPC2 or BMI1. Our findings indicate that Enx1/EZH2 and EED are members of a class of PcG proteins that is distinct from previously described human PcG proteins.In Drosophila melanogaster, the genes of the Polycomb group (PcG) and trithorax group (trxG) are part of a cellular memory system, which is responsible for the stable inheritance of gene activity. The PcG and trxG genes have been identified in Drosophila as repressors (PcG) (18, 22, 27, 28, 38) and activators (trxG) (20, 21), respectively, of homeotic gene activity. PcG and trxG genes were originally found in Drosophila, but mammalian homologs have also been identified and appear to function like their Drosophila homologs (reviewed in reference 37). It has been proposed that PcG proteins repress gene expression through the formation of multimeric protein complexes. We have recently shown that the human PcG proteins HPH1 and HPH2 coimmunoprecipitate, cofractionate, and colocalize in nuclear domains with the human PcG proteins BMI1 (2, 12, 33) and HPC2, a recently identified, novel human Polycomb protein (33, 34). Furthermore, we have found that the human RING1 protein coimmunoprecipitates and colocalizes with HPC2 and other PcG proteins, indicating that RING1 is associated with, or is part of, the mammalian PcG complex (33, 35). These results indicate that mammalian PcG proteins form a multimeric protein complex. This observation is in agreement with observations that different PcG proteins, including Pc, bind in overlapping patterns on polytene chromosomes in Drosophila salivary gland cells (4, 10, 29).Interestingly, also the trithorax gene product trx colocalizes with Drosophila PcG proteins at many sites on polytene chromosomes (6, 24). Even more strikingly, binding of the trx protein has been mapped to small DNA fragments that also contain binding sites for PcG proteins, the Polycomb response elements (5, 6). This finding is further substantiated by the observation that GAGA factor, the gene product of the trxG gene trithorax-like (Trl) (13), colocalizes with Pc protein within the close vicinity of a Polycomb response element (41). Furthermore, the PcG gene Enhancer of zeste [E(z)] contains a domain with sequence homology with the activator protein trx (17). This observation is in agreement with genetic data which indicate that E(z) can be considered both a PcG gene and a trxG gene (26). Double mutations of E(z) and trxG genes result in homeotic phenotypes which are similar to the homeotic phenotypes which are also observed in double mutants of trxG genes (26). Finally, polytene chromosome binding of the trx protein is strongly reduced in homozygous E(z) mutants (4), and vice versa, polytene chromosome binding of the E(z) protein is reduced in trx mutants (24). These data suggest functional interactions between activators (trxG proteins) and repressors (PcG proteins) that are important for their mode of action.To start to investigate these puzzling features of the E(z) gene product, we used the two-hybrid system (8, 9) in order to identify proteins that interact with a mammalian homolog of E(z), the Enx1/EZH2 protein (15, 16). Here, we report the identification of the human EED protein, which interacts with Enx1/EZH2. EED is the human homolog of eed, a murine PcG gene (7, 36) which has extensive homology with the Drosophila PcG gene extra sex combs (esc) (14, 32, 39). Whereas Enx1/EZH2 and EED coimmunoprecipitate, they neither coimmunoprecipitate nor colocalize with other human PcG proteins, such as HPC2 and BMI1. Our findings indicate that both Enx1/EZH2 and EED form a class of mammalian PcG proteins that is distinct from previously described human PcG proteins.