Tuberous sclerosis (TSC) is a human genetic syndrome characterized by the development of benign tumors in a variety of tissues, as well as rare malignancies. Two different genetic loci have been implicated in TSC; one of these loci, the tuberous sclerosis-2 gene (TSC2), encodes an open reading frame with a putative protein product of 1784 amino acids. The putative TSC2 product (tuberin) contains a region of limited homology to the catalytic domain of Rap1GAP. We have generated antisera against the N-terminal and C-terminal portions of tuberin, and these antisera specifically recognize a 180-kDa protein in immunoprecipitation and immunoblotting analyses. A wide variety of human cell lines express the 180-kDa tuberin protein, and subcellular fractionation revealed that most tuberin is found in a membrane/particulate (100,000 x g) fraction. Immunoprecipitates of native tuberin contain an activity that specifically stimulates the intrinsic GTPase activity of Rap1a. These results were confirmed in assays with a C-terminal fragment of tuberin, expressed in bacteria or Sf9 cells. Tuberin did not stimulate the GTPase activity of Rap2, Ha-Ras, Rac, or Rho. These results suggest that the loss of tuberin leads to constitutive activation of Rap1 in tumors of patients with tuberous sclerosis.

Background: Inflammatory bowel disease (IBD) is thought to result from a dysregulated interaction between the host immune system and commensal microflora. Toll-like receptors (TLRs) recognize microbe-associated molecular patterns (MAMPs), but their role in enteropathies in dogs is unknown.Hypothesis: That there is a dysregulation of TLRs recognizing bacterial MAMPs in dogs with IBD. Animals: Sixteen healthy beagles and 12 dogs with steroid-treated (ST) and 23 dogs with food-responsive (FR) diarrhea. Methods: Prospective, observational study. mRNA expression of canine TLR2, 4, and 9 was evaluated by quantitative realtime RT-PCR in duodenal and colonic biopsies obtained before and after standard therapy. Samples from control dogs were taken at necropsy, with additional biopsies of stomach, jejunum, ileum, and mesenteric lymph node in 6 dogs.Results: There were significant differences (P .017) in expression of TLR2, 4, and 9 between the 6 sampled locations in healthy control dogs (lymph node 4 small intestine ! colon). Before therapy, ST expressed more mRNA than control dogs for all 3 receptors (P o .05). There were no significant differences between pretreatment and posttreatment values, even though 32/ 35 dogs improved clinically. No associations were found when comparing receptor mRNA expression with either histology or clinical activity scores.Conclusions and Clinical Importance: Bacteria-responsive TLR2, 4, and 9 are upregulated in duodenal and colonic mucosa in IBD. This might lead to increased inflammation through interaction with the commensal flora. The absence of significant changes after therapy despite clinical improvement might point toward the existence of a genetic predisposition to IBD as described in human IBD.

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. Collagen VII, the major component of cutaneous anchoring fibrils is expressed at a low level by normal human keratinocytes and fibroblasts in vitro. In cocultures of these two cell types, signals from fibroblasts enhance expression of collagen VII by keratinocytes and vice versa . In this study, the effects of a possible mediator of such a stimulation, transforming growth factor-ß (TGF-ß), were investigated . Its effect on the expression and deposition of the highly insoluble collagen VII was assessed in a semiquantitative manner by a newly developed enzyme-linked immunoassay which is based on immunoblotting . In keratinocyte monocultures, 0.5-20 ng/ml of TGF-ß2 induced a dose-dependent stimulation of collagen VII expression as measured per microgram of DNA . The maximal enhancement was about sevenfold compared to con-HE dermo-epidermal junction of the skin represents an epithelial-mesenchymal interface with a unique structure and highly specialized functions (Palade and Farquhar, 1965 ;Bruns, 1969 ; Briggaman and Wheeler, 1975x ;Tidman and Eady, 1984 ;Burgeson, 1987). One ofits main functions is to provide the resistance ofthe skin against shearing forces which requires strong cohesion of the skin layers . This is achieved by several interconnected macromolecular networks which attach the epidermis to the basement membrane and to the underlying dermal connective tissue (Timpl, 1989;Burgeson et al ., 1990 ; Yurchenko and Schittny, 1990) . One of the major structures mediating the attachment is the anchoring fibril network that extends from the basement membrane to the anchoring plaques in thepapillary dermis Keene et al., 1987) . Abnormalities ofthe anchoring fibrils lead to separation of the epidermis from the dermis and to clinical blistering of the skin, as seen in dystrophic epidermolysis bullosa, a group of inherited blistering disorders (Briggaman and Wheeler, 1975b ;Hashimoto et al ., 1976;Tidman and Eady, 1985 ;Heagerty et al ., 1986 ;Bruckner-Tuderman et al., 1989,

SUMMARY1. River ecosystems are threatened by multiple stressors, including habitat degradation, pollution and invasive species. However, freshwater ecologists have largely disregarded the contribution of toxicants to stress in rivers, whereas ecotoxicologists have primarily examined toxicant effects in artificial systems. As a result, there is a paucity of information on the co-occurrence of organic toxicants with other stressors and on the relative importance of toxicants for overall ecological risk in rivers. 2. We used monitoring data for German rivers to analyse the individual and joint occurrence of four stressors: habitat degradation, invasive species, nutrient pollution and organic toxicants. All stressors were examined for ecological risks in terms of whether they exceeded low-and high-risk thresholds derived from published studies and regulatory thresholds. 3. Nutrients and habitat degradation exceeded low and high risk thresholds at c. 85% of the sites and invasive species and organic toxicants at c. 50% of the sites. At least one stressor exceeded thresholds at all sites for which data on all four stressors were available. Toxicity showed weak positive correlations with nutrients and habitat degradation (0.2 < Spearman's q < 0.34, 0.009 < P < 0.08). The risks of ecological effects arising from habitat degradation and invasive species were higher in lowland rivers, particularly for invasive species. 4. Our assessment shows that organic toxicants contribute notably to risks of ecological effects in rivers, to a similar extent as invasive species, although habitat degradation and nutrients are the dominant stressors. Exposure to multiple stressors is the typical situation prevailing in rivers. Consequently, mitigation measures focusing on individual stressors may not be effective at reducing ecological risks. This suggests that integrating concepts and data from freshwater ecology and ecotoxicology is essential to meet the challenge of managing multiple stressors in river ecosystems.