The Toll pathway is required in the epidermis for muscle development in the Drosophila embryo

BACKGROUND & AIMS: Hydrophobic bile acids have been implicated in the pathogenesis of cholestatic liver injury. The hypothesis that hydrophobic bile acid toxicity is mediated by oxidant stress in an in vivo rat model was tested in this study. METHODS: A dose-response study of bolus intravenous (i.v.) taurochenodeoxycholic acid (TCDC) in rats was conducted. Rats were then pretreated with parenteral alpha-tocopherol, and its effect on i.v. TCDC toxicity was evaluated by liver blood tests and by assessing mitochondrial lipid peroxidation. RESULTS: Four hours after an i.v. bolus of TCDC (10 mumol/100 g weight), serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels peaked, hepatic mitochondria showed evidence of increased lipid peroxidation, and serum bile acid analysis was consistent with a cholestatic injury. Liver histology at 4 hours showed hepatocellular necrosis and swelling and mild portal tract inflammation. Treatment with parenteral alpha-tocopherol was associated with a 60%-70% reduction in AST and ALT levels, improved histology, and a 60% reduction in mitochondrial lipid peroxidation in rats receiving TCDC. CONCLUSIONS: These data show that hepatocyte injury and oxidant damage to mitochondria caused by i.v. TCDC can be significantly reduced by pretreatment with the antioxidant vitamin E. These in vivo findings support the role for oxidant stress in the pathogenesis of bile acid hepatic toxicity.     

The GURKE gene is required for normal organization of the apical region in the Arabidopsis embryo

Dicot plant embryos undergo a transition from radial to bilateral symmetry. In Arabidopsis, this change reflects patterning within the apical region, resulting in the formation of the cotyledon and shoot meristem primordia. Mutations in the GURKE gene give seedlings with highly reduced or no cotyledons. Both strong and weak gurke alleles confer this phenotypic variability although strong alleles often eliminate the entire apex and sometimes also part of the hypocotyl. The root and the root meristem as well as the radial pattern of concentric tissue layers are essentially normal. The mutant seedling phenotype can be traced back to the triangular/early-heart stage of embryogenesis when abnormal cell divisions occur within the apical region such that no or only rudimentary cotyledon primordia are established. The postembryonic development of gurke seedlings was examined in culture. In weak alleles, apical growth gave rise to abnormal leaves and stem-like structures and, eventually, abnormal flowers. In strong alleles, the apical region often failed to grow but occasionally produced fused leaf-like structures with no dorso-ventral polarity and a totally unorganized vascular system while no stems developed. The observations suggest that the GURKE gene is involved primarily in the organization of the apical region in the embryo and may also play a role during postembryonic development.     

The role of N-glycosylation for functional expression of the human platelet-activating factor receptor. Glycosylation is required for efficient membrane trafficking

Streptococcus pneumoniae has been shown to utilize the platelet activating factor receptor for binding and invasion of host cells (Cundell, D. R., Gerard, N. P., Gerard, C., Idanpaan-Heikkila, I., and Tuomanen, E. I. (1995) Nature, in press). Because bacterial binding is in part carbohydrate dependent, and the human platelet-activating factor (PAF) receptor bears a single N-linked glycosylation sequence in the second extracellular loop, we undertook studies to determine the role of this epitope in PAF receptor function. Binding of pneumococci to COS cells transfected with the human PAF receptor is greatly reduced for a receptor mutant that bears no N-linked glycosylation site. Immunohistochemical and binding analyses show decreased expression of the non-glycosylated molecule on the cell membrane relative to the wild type receptor; however, metabolic labeling and immunopurification indicate it is synthesized intracellularly at a level similar to the native molecule. A mutant receptor encoding a functional glycosylation site at the NH2 terminus is better expressed at the cell surface compared with the non-glycosylated form, indicating that trafficking to the cell surface is facilitated by glycosylation, but its location is relatively unimportant. The binding affinity for PAF is not significantly effected by the presence or location of the carbohydrate, and variations in cell surface expression have little influence on signal transduction, as the non-glycosylated PAF receptor is equally effective for activation of phospholipase C as the native molecule. These data are supportive of pneumococcal binding on protein moiety(ies) of the PAF receptor and indicate that N-glycosylation facilitates expression of the protein on the cell membrane.     

A binding site for nuclear receptors is required for the differential expression of the aldolase A fast-twitch muscle promoter in body and head muscles

In hind limb muscles, the aldolase A muscle-specific promoter is specifically expressed in glycolytic fast-twitch fibers. Here, we show that in addition, it is expressed at higher levels in trunk and limb muscles than in neck and head muscles independent of their fiber-type content. We have identified by analysis of transgenic mice a DNA element that is required for this differential expression and, to a lesser extent, for fiber-type specificity. We show that members of the nuclear receptor superfamily bind this element in skeletal muscle nuclear extracts. Interestingly, in gel mobility shift assays, different complexes were formed with this sequence in tongue nuclear extracts compared with limb or trunk muscle nuclear extracts. Therefore, binding of distinct nuclear receptors to a single regulatory sequence appears to be associated with the location-dependent expression of the aldolase A muscle-specific promoter.     

The ftsQ1p gearbox promoter of Escherichia coli is a major sigma S-dependent promoter in the ddlB-ftsA region

Thrombospondin (TSP) 2, and its close relative TSP1, are extracellular proteins whose functions are complex, poorly understood, and controversial. In an attempt to determine the function of TSP2, we disrupted the Thbs2 gene by homologous recombination in embryonic stem cells, and generated TSP2-null mice by blastocyst injection and appropriate breeding of mutant animals. Thbs2-/- mice were produced with the expected Mendelian frequency, appeared overtly normal, and were fertile. However, on closer examination, these mice displayed a wide variety of abnormalities. Collagen fiber patterns in skin were disordered, and abnormally large fibrils with irregular contours were observed by electron microscopy in both skin and tendon. As a functional correlate of these findings, the skin was fragile and had reduced tensile strength, and the tail was unusually flexible. Mutant skin fibroblasts were defective in attachment to a substratum. An increase in total density and in cortical thickness of long bones was documented by histology and quantitative computer tomography. Mutant mice also manifested an abnormal bleeding time, and histologic surveys of mouse tissues, stained with an antibody to von Willebrand factor, showed a significant increase in blood vessels. The basis for the unusual phenotype of the TSP2-null mouse could derive from the structural role that TSP2 might play in collagen fibrillogenesis in skin and tendon. However, it seems likely that some of the diverse manifestations of this genetic disorder result from the ability of TSP2 to modulate the cell surface properties of mesenchymal cells, and thus, to affect cell functions such as adhesion and migration.     

Multiple transitions to non-B-DNA structures occur in the distal regulatory region of the rat prolactin gene

The developmentally regulated rat prolactin (rPRL) gene presents a promising model system toward understanding the biological role of non-B-DNA structural elements. Two predominantly alternating purine-pyrimidine (APP) (dA-dC)n.(dG-dT)n repeats of 58 and 178 base-pairs flank the (A + T)-rich distal regulatory region. We have characterized several transitions to non-B-DNA structures within this region in negatively supercoiled plasmids by utilizing high resolution chemical probing. Each repeat undergoes a full-length conversion to a novel left-handed helical structure via the stepwise nucleation and propagation of discrete "segments". These segments are delimited by out-of-alternation bases that are susceptible to attack by potassium permanganate and thus appear to be significantly unstacked within the left-handed helices. Moreover, the spatial order of successive right- to left-handed DNA transitions within each repeat exhibits a clear polarity toward the distal regulatory region of the rPRL gene. An additional transition involving the long-range unpairing of (A + T)-rich sequences establishes a directional propagation toward the regulatory region. These data demonstrate a complex series of quasi-independent transitions to non-B-DNA structures that impinge upon a known regulatory control region.     

A gp330/megalin-related protein is required in the major epidermis of Caenorhabditis elegans for completion of molting

A genetic analysis of a gp330/megalin-related protein, LRP-1, has been undertaken in Caenorhabditis elegans. Consistent with megalin's being essential for development of mice, likely null mutations reveal that this large member of the low density lipoprotein receptor family is also essential for growth and development of this nematode. The mutations confer a striking defect, an inability to shed and degrade all of the old cuticle at each of the larval molts. The mutations also cause an arrest of growth usually at the molt from the third to the fourth larval stage. Genetic mosaic analysis suggests that the lrp-1 gene functions in the major epidermal syncytium hyp7, a polarized epithelium that secretes cuticle from its apical surface. Staining of whole mounts with specific monoclonal antibodies reveals that the protein is expressed on the apical surface of hyp7. Sterol starvation can phenocopy the lrp-1 mutations, suggesting that LRP-1 is a receptor for sterols that must be endocytosed by hyp7. These observations indicate that LRP-1 is related to megalin not only structurally but also functionally.