Functional domains and sub-cellular distribution of the Hedgehog transducing protein Smoothened in Drosophila.
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The Hedgehog signalling pathway is deployed repeatedly during normal animal development and its inappropriate activity is associated with various tumours in human. The serpentine protein Smoothened (Smo) is essential for cells to respond to the Hedeghog (Hh) signal; oncogenic forms of Smo have been isolated from human basal cell carcinomas. Despite similarities with ligand binding G-protein coupled receptors, the molecular basis of Smo activity and its regulation remains unclear. In non-responding cells, Smo is suppressed by the activity of another multipass membrane spanning protein Ptc, which acts as the Hh receptor. In Drosophila, binding of Hh to Ptc has been shown to cause an accumulation of phosphorylated Smo protein and a concomitant stabilisation of the activated form of the Ci transcription factor. Here, we identify domains essential for Smo activity and investigate the sub-cellular distribution of the wild type protein in vivo. We find that deletion of the amino terminus and the juxtamembrane region of the carboxy terminus of the protein result in the loss of normal Smo activity. Using Green Fluorescent Protein (GFP) and horseradish peroxidase fusion proteins we show that Smo accumulates in the plasma membrane of cells in which Ptc activity is abrogated by Hh but is targeted to the degradative pathway in cells where Ptc is active. We further demonstrate that Smo accumulation is likely to be a cause, rather than a consequence, of Hh signal transduction. (+info)
Serratia marcescens internalization and replication in human bladder epithelial cells.
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BACKGROUND: Serratia marcescens, a frequent agent of catheterization-associated bacteriuria, strongly adheres to human bladder epithelial cells in culture. The epithelium normally provides a barrier between lumal organisms and the interstitium; the tight adhesion of bacteria to the epithelial cells can lead to internalization and subsequent lysis. However, internalisation was not shown yet for S. marcescens strains. METHODS: Elektronmicroscopy and the common gentamycin protection assay was used to assess intracellular bacteria. Via site directed mutagenesis, an hemolytic negative isogenic Serratia strain was generated to point out the importance of hemolysin production. RESULTS: We identified an important bacterial factor mediating the internalization of S. marcescens, and lysis of epithelial cells, as the secreted cytolysin ShlA. Microtubule filaments and actin filaments were shown to be involved in internalization. However, cytolysis of eukaryotic cells by ShlA was an interfering factor, and therefore hemolytic-negative mutants were used in subsequent experiments. Isogenic hemolysin-negative mutant strains were still adhesive, but were no longer cytotoxic, did not disrupt the cell culture monolayer, and were no longer internalized by HEp-2 and RT112 bladder epithelial cells under the conditions used for the wild-type strain. After wild-type S. marcescens became intracellular, the infected epithelial cells were lysed by extended vacuolation induced by ShlA. In late stages of vacuolation, highly motile S. marcescens cells were observed in the vacuoles. S. marcescens was also able to replicate in cultured HEp-2 cells, and replication was not dependent on hemolysin production. CONCLUSION: The results reported here showed that the pore-forming toxin ShlA triggers microtubule-dependent invasion and is the main factor inducing lysis of the epithelial cells to release the bacteria, and therefore plays a major role in the development of S. marcescens infections. (+info)
Developmental allometry of pulmonary structure and function in the altricial Australian pelican Pelecanus conspicillatus.
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Quantitative methods have been used to correlate maximal oxygen uptake with lung development in Australian pelicans. These birds produce the largest altricial neonates and become some of the largest birds capable of flight. During post-hatching growth to adults, body mass increases by two orders of magnitude (from 88 g to 8.8 kg). Oxygen consumption rates were measured at rest and during exposure to cold and during exercise. Then the lungs were quantitatively assessed using morphometric techniques. Allometric relationships between body mass (M) and gas exchange parameters (Y) were determined and evaluated by examining the exponents of the equation Y=aM(b). This intraspecific study was compared to interspecific studies of adult birds reported in the literature. Total lung volume scales similarly in juvenile pelicans (b=1.05) as in adult birds (b=1.02). However, surface area of the blood-gas barrier greatly increases (b=1.25), and its harmonic mean thickness does not significantly change (b=0.02), in comparison to exponents from adult birds (b=0.86 and 0.07, respectively). As a result, the diffusing capacity of the blood-gas tissue barrier increases much more during development (b=1.23) than it does in adult birds of different sizes (b=0.79). It increases in parallel to maximal oxygen consumption rate (b=1.28), suggesting that the gas exchange system is either limited by lung development or possibly symmorphic. The capacity of the oxygen delivery system is theoretically sufficient for powered flight well in advance of the bird's need to use it. (+info)
Bis(2-chloroethoxy)methane-induced mitochondrial and myofibrillar damage: short-term time-course study.
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Cardiotoxicity induced by 2-, 3-, 5-, and 12-day dermal administration of 400 and 600 mg/kg/day of bis(2-chloroethoxy)methane to F344/N male and female rats was characterized. The severity and incidence of lesions were similar among males and females and in all three regions of the heart examined (atrium, ventricle, interventricular septum). Damage induced by bis(2-chloroethoxy)methane consisted of time-related development of myofiber vacuolation, necrosis, mononuclear-cell infiltration, fibrosis, and atrial thrombosis. Changes were pronounced at day 2, increased in severity at day 3, appeared to decrease at day 5, and resolved by study-day 16 that corresponded to 12 dosings. Ultrastructural analysis of 2- and 5-day 600 mg/kg/day-treated females elucidated the primary site of damage, the mitochondrion, and two types of vacuolation, one that formed as damaged mitochondria became devoid of cristae and their bounding double membranes became reduced to singleness, and the other manifested as distention of the sarcoplasmic reticulum. After the initial damage induced by bis(2-chloroethoxy)methane, or its metabolite, thiodiglycolic acid, protective mechanisms within the heart were apparently initiated, enabling it to cope with the continued exposure to the toxicant while eliminating some damaged myofibers. (+info)
Subcellular distribution of di-(2-ethylhexyl)phthalate in rat testis.
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Subcellular distribution of di-(2-ethylhexyl)phthalate (DEHP) in the testis was studied by single oral administration of [3,4,5,6-(3)H]-phthalic acid di-(2-ethylhexyl) ester (DEHP-3H) or phthalic acid di-(2-ethyl[1-(3)H]hexyl) ester (3H-DEHP) to 8-week-old male rats. Autoradiographs and electron microscopic autoradiographs were prepared from the testis, liver and kidney at 6 and 24 hr after administration and distribution of radioactive materials in the tissues were observed. In the autoradiographic specimen at 6 hr after administration of DEHP 3H-labeled at phthalic acid moiety (DEHP-3H), many grains were observed in the testis, mainly at the basal area of seminiferous tubules at the stages IX to I of the spermatogenic cycle. Electron microscopic autoradiographs taken at the same time revealed that localization of grains were in the smooth-surfaced endoplasmic reticulum and mitochondria of Sertoli cells. A few grains were also present at the Golgi apparatus and lysosome of Sertoli cells, and at the interfaces between the Sertoli cells or between Sertoli cells and spermatocytes, and in the cytoplasm of spermatocytes. Autoradiographs of the liver revealed grains in the centrilobular hepatocytes, localized at mitochondria, rough-surfaced endoplasmic reticulum and peroxisomes. In the kidney, the radioactivity was localized at the brush border of the tubular cells in the pars recta of proximal tubules. In the 24-hr specimen, the grain density in the seminiferous tubules obviously decreased. On the other hand, by autoradiography with DEHP 3H-labeled at the alcohol (3H-DEHP), only a few grains were observed in autoradiographs of the testes at 6 hr after administration. No grains were noted in autoradiographs of the liver and kidney with 3H-DEHP. The results showed that the phthalic acid ester was splitted rapidly in the body and only the phthalic acid moiety distributed into the cells. (+info)
Differential localization of brain-type and epidermal-type fatty acid binding proteins in the adrenal gland of mice.
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In immuno-light and -electron microscopy, brain-type fatty acid binding protein (B-FABP) is localized in the sustentacular cells enclosing the chromaffin cells in the adrenal medulla. This represents another new feature commonly shared by the sustentacular cells and ganglionic satellite cells, the latter of which has already been reported to localize this molecule, and suggests a common feature in lipid metabolism shared by the two cells enclosing peripheral neurons and paraneurons. On the other hand, epidermal-type fatty acid binding protein (E-FABP) is localized in two discrete cells in the adrenal gland: the one is a subpopulation of intra-adrenal macrophages which are intensely immunoreactive for F4/80, a marker of macrophages, and are rich in pleomorphic lysosomes. Because of their direct apposition to adjacent cortical endocrine cells and medullary chromaffin cells, the macrophages may be involved not only in phagocytosis of degenerating adrenal cells but also in exertion of some yet unknown effects on the endocrine function of the cortical and medullary cells via humoral factors such as cytokines which have recently been known to be secreted by macrophages. The other is a population of cells having scanty perikaryal cytoplasm poor in organneles and several thinny extended processes in the cortex and exhibiting weak immunoreactivity for E-FABP. The possible natures of these cells immunoreactive for E-FABP are discussed in view of a subpopulation of endothelial cells or the dendritic cells of antigen-presenting property. (+info)
Genetic deletion of mouse platelet glycoprotein Ibbeta produces a Bernard-Soulier phenotype with increased alpha-granule size.
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Here we report the characterization of a mouse model of the Bernard-Soulier syndrome generated by a targeted disruption of the gene encoding the glycoprotein (GP) Ibbeta subunit of the GP Ib-IX complex. Similar to a Bernard-Soulier model generated by disruption of the mouse GP Ibalpha subunit, GP Ibbeta(Null) mice display macrothrombocytopenia and a severe bleeding phenotype. When examined by transmission electron microscopy, the large platelets produced by a GP Ibbeta(Null) genotype revealed alpha-granules with increased size as compared with the alpha-granules from control mouse platelets. Data are presented linking the overexpression of a septin protein, SEPT5, to the presence of larger alpha-granules in the GP Ibbeta(Null) platelet. The SEPT5 gene resides approximately 250 nucleotides 5' to the GP Ibbeta gene and has been associated with modulating exocytosis from neurons and platelets as part of a presynaptic protein complex. Fusion mRNA transcripts present in megakaryocytes can contain both the SEPT5 and GP Ibbeta coding sequences as a result in an imperfect polyadenylation signal within the 3' end of both the human and mouse SEPT5 genes. We observed a 2- to 3-fold increase in SEPT5 protein levels in platelets from GP Ibbeta(Null) mice. These results implicate SEPT5 levels in the maintenance of normal alpha-granule size and may explain the variant granules associated with human GP Ibbeta mutations and the Bernard-Soulier syndrome. (+info)
Laminin (LN) alpha2 chain deficiency in humans and mice leads to severe forms of congenital muscular dystrophy (CMD). Here, we investigated whether LNalpha1 chain in mice can compensate for the absence of LNalpha2 chain and prevent the development of muscular dystrophy. We generated mice expressing a LNalpha1 chain transgene in skeletal muscle of LNalpha2 chain deficient mice. LNalpha1 is not normally expressed in muscle, but the transgenically produced LNalpha1 chain was incorporated into muscle basement membranes, and normalized the compensatory changes of expression of certain other laminin chains (alpha4, beta2). In 4-month-old mice, LNalpha1 chain could fully prevent the development of muscular dystrophy in several muscles, and partially in others. The LNalpha1 chain transgene not only reversed the appearance of histopathological features of the disease to a remarkable degree, but also greatly improved health and longevity of the mice. Correction of LNalpha2 chain deficiency by LNalpha1 chain may serve as a paradigm for gene therapy of CMD in patients. (+info)