Effect of indomethacin on bile acid-phospholipid interactions: implication for small intestinal injury induced by nonsteroidal anti-inflammatory drugs.

The injurious effect of nonsteroidal anti-inflammatory drugs (NSAIDs) in the small intestine was not appreciated until the widespread use of capsule endoscopy. Animal studies found that NSAID-induced small intestinal injury depends on the ability of these drugs to be secreted into the bile. Because the individual toxicity of amphiphilic bile acids and NSAIDs directly correlates with their interactions with phospholipid membranes, we propose that the presence of both NSAIDs and bile acids alters their individual physicochemical properties and enhances the disruptive effect on cell membranes and overall cytotoxicity. We utilized in vitro gastric AGS and intestinal IEC-6 cells and found that combinations of bile acid, deoxycholic acid (DC), taurodeoxycholic acid, glycodeoxycholic acid, and the NSAID indomethacin (Indo) significantly increased cell plasma membrane permeability and became more cytotoxic than these agents alone. We confirmed this finding by measuring liposome permeability and intramembrane packing in synthetic model membranes exposed to DC, Indo, or combinations of both agents. By measuring physicochemical parameters, such as fluorescence resonance energy transfer and membrane surface charge, we found that Indo associated with phosphatidylcholine and promoted the molecular aggregation of DC and potential formation of larger and isolated bile acid complexes within either biomembranes or bile acid-lipid mixed micelles, which leads to membrane disruption. In this study, we demonstrated increased cytotoxicity of combinations of bile acid and NSAID and provided a molecular mechanism for the observed toxicity. This mechanism potentially contributes to the NSAID-induced injury in the small bowel.

Aldosterone-induced Sgk1 relieves Dot1a-Af9-mediated transcriptional repression of epithelial Na+ channel alpha.

Aldosterone plays a major role in the regulation of salt balance and the pathophysiology of cardiovascular and renal diseases. Many aldosterone-regulated genes--including that encoding the epithelial Na+ channel (ENaC), a key arbiter of Na+ transport in the kidney and other epithelia--have been identified, but the mechanisms by which the hormone modifies chromatin structure and thus transcription remain unknown. We previously described the basal repression of ENaCalpha by a complex containing the histone H3 Lys79 methyltransferase disruptor of telomeric silencing alternative splice variant a (Dot1a) and the putative transcription factor ALL1-fused gene from chromosome 9 (Af9) as well as the release of this repression by aldosterone treatment. Here we provide evidence from renal collecting duct cells and serum- and glucocorticoid-induced kinase-1 (Sgk1) WT and knockout mice that Sgk1 phosphorylated Af9, thereby impairing the Dot1a-Af9 interaction and leading to targeted histone H3 Lys79 hypomethylation at the ENaCalpha promoter and derepression of ENaCalpha transcription. Thus, Af9 is a physiologic target of Sgk1, and Sgk1 negatively regulates the Dot1a-Af9 repressor complex that controls transcription of ENaCalpha and likely other aldosterone-induced genes.

A study of xlcaax-1: Patterns of localization, possible function and usefulness as a developmental marker

Morphogenesis is the process by which the 3-dimensional structure of the developing embryo takes shape. We are studying xlcaax-1, a gene whose product can be used as a molecular marker for several morphogenetic events. We report here the cellular and subcellular localization of the xlcaax-1 protein during development of Xenopus laevis. Whole mount immunocytochemistry and immunoperoxidase staining of tissue sections showed that during development the xlcaax-1 protein accumulation was coincident with the differentiation of the epidermis, pronephros and mesonephros. In the pronephros and mesonephros the xlcaax-1 protein was localized to the basolateral membrane of differentiated tubule epithelial cells. Thus, the xlcaax-1 protein served as a marker for tubule formation and polarization during Xenopus kidney development. Xlcaax-1 may also be used as a marker for the functional differentiation of the epidermis and the epidermally derived portions of the lens and some cranial nerves. The xlcaax-1 protein was most abundant in kidney and immunogold EM analysis showed that the xlcaax-1 protein was highly enriched in the basal infoldings of the basolateral membrane of the epithelial cells in adult kidney distal tubules. The xlcaax-1 protein was also localized in other ion transporting epithelia. The localization pattern and preliminary functional assays of xlcaax-1 suggest that the protein may function in association with an ion transport channel or pump.^ Cell migration and cell-cell interactions play important roles in numerous processes during morphogenesis. One of these is the formation of the pronephric (wolffian) duct (PD), which connects the pronephros to the cloaca. It is currently accepted that in most amphibians the pronephric duct is formed by active migration of the pronephric duct rudiment (PDR) cells along a pre-determined pathway. However, there is evidence that in Xenopus, the PD may be formed entirely by in situ segregation of cells out of the lateral mesoderm. In this study, we showed, using PDR ablation and X. laevis - X. borealis chimeras, that PD elongation in Xenopus required both active cell migration and an induced recruitment of cells from the posterior lateral plate mesoderm. We also showed that PDR cell migration was limited to only a few stages during development and that this temporal control is due, at least in part, to changes in the competence of the PD pathway to support cell migration. In addition, our data suggested that an alkaline phosphatase (APase) adhesion gradient may be involved in determining this competence. ^

Loss of β1-integrin-deficient cells during the development of endoderm-derived epithelia

Beta1-integrins (beta1) represent cell surface receptors which mediate cell-matrix and cell-cell interactions. Fässler and Meyer described chimeric mice containing transgenic cells that express the LacZ gene instead of the beta1 gene. They observed beta1-negative cells in all germ layers at embryonic day E 8.5. Later in development, using a glucose phosphate isomerase assay of homogenized tissue samples, high levels of transgenic cells were found in skeletal muscle and gut, low levels in lung, heart, and kidney and none in the liver and spleen (Fässler and Meyer 1995). In order to study which cell types require beta1 during development of the primitive gut including its derivatives, chimeric fetuses containing 15 to 25% transgenic cells were obtained at days E 14.5 and E 15.5. They were LacZ (beta-galactosidase) stained "en bloc" and cross-sectioned head to tail. In esophagus, trachea, lung, stomach, hindgut, and the future urinary bladder, we observed various mesoderm-derived beta1-negative cells (e.g. fibroblasts, chondrocytes, endothelial cells, and smooth muscle cells) but no beta1-negative epithelial cells. Since the epithelia of lung, esophagus, trachea, stomach, hindgut, and urinary bladder are derived from the endodermal gut tube, we hypothesize that beta1 is essential for the development and/or survival of the epithelia of the fore- and hindgut and its derivatives.

Congenital nasolacrimal duct fistula in Brown Swiss cattle

BACKGROUND An increased incidence of nasolacrimal duct fistula in the offspring of dam J and three of her sons (bulls A, B and C) prompted a study to investigate the prevalence and clinical manifestation of this anomaly. The dam J, bull B, 255 direct offspring of bulls A, B, and C and eight other direct and indirect offspring of cow J were examined. The periocular region of each animal was examined for unilateral or bilateral nasolacrimal duct fistula and the location, appearance and size of the lesions. RESULTS Of 265 cattle examined, 54 had unilateral (n = 24) or bilateral fistula (n = 30). The prevalence of affected offspring differed significantly among the three bulls. The fistulae were located medial to the medial canthus of the eye and were 1 to 10 mm (median, 1 mm) in height and 1 to 12 mm (median, 2 mm) in length. The shape of the opening was circular in 58, oval in 23 and slit-like in three. One other animal had a large opening with an atypical shape and another had an abnormal medial canthus with several fistulous openings. Seventy openings were pigmented and 52 were hairless. The fistulae were clinically significant in 12 animals. CONCLUSIONS The findings suggest a hereditary cause of nasolacrimal duct fistula in Brown Swiss cattle.

Toxicity of aged gasoline exhaust particles to normal and diseased airway epithelia

Particulate matter (PM) pollution is a leading cause of premature death, particularly in those with pre-existing lung disease. A causative link between particle properties and adverse health effects remains unestablished mainly due to complex and variable physico-chemical PM parameters. Controlled laboratory experiments are required. Generating atmospherically realistic Aerosols and performing cell-exposure studies at relevant particle-doses are challenging. Here we examine gasoline-exhaust particle toxicity from a Euro-5 passenger car in a uniquely realistic exposure scenario, combining a smog chamber simulating atmospheric ageing, an aerosol enrichment System varying particle number concentration independent of particle chemistry, and an aerosol Deposition chamber physiologically delivering particles on air-liquid interface (ALI) cultures reproducing normal and susceptible health status. Gasoline-exhaust is an important PM source with largely unknown health effects. We investigated acute responses of fully-differentiated normal, distressed (antibiotics treated) normal, and cystic fibrosis human bronchial epithelia (HBE), and a proliferating, single-cell type bronchial epithelial cell-line (BEAS-2B). We show that a single, short-term exposure to realistic doses of atmospherically-aged gasoline-exhaust particles impairs epithelial key-defence mechanisms, rendering it more vulnerable to subsequent hazards. We establish dose-response curves at realistic particle-concentration levels. Significant differences between cell models suggest the use of fully differentiated HBE is most appropriate in future toxicity studies.

The patent nasopalatine duct: a potential cause of unclear pain in the anterior maxilla

OBJECTIVE The aim of this report is to describe symptoms that can suggest the presence of a patent nasopalatine duct and to illustrate three cases. SUMMARY Patent nasopalatine ducts connecting the oral cavity with the nasal cavity are extremely rare. This malformation can be considered a developmental abnormality. Clinically, patent nasopalatine ducts appear as single or double spherical or oval apertures lateral or posterior to the incisive papilla. This type of anatomical malformation can be associated with an unclear pain sensation in the anterior maxillary region, which may be misinterpreted for example as toothache of endodontic origin. However, persisting nasopalatine ducts can also exist as an asymptomatic abnormality with no clinical sign of discomfort. Accordingly, understanding the differential diagnosis of a possible patent nasopalatine duct can prevent a general practitioner from performing unnecessary interventions, such as endodontic treatments, apical surgeries, or tooth extractions.

37,X/38,XY Mosaicism in a Cryptorchid Bengal Cat with Müllerian Duct Remnants

A 23-month-old tomcat was referred to our clinic because of male behavioral problems, cryptorchidism, and an undefined intra-abdominal organ resembling a uterus. Ultrasonography and computed tomography showed 2 fluid-filled tubular structures dorsolaterally to the bladder and connected to the pelvic urethra. The cat was castrated, and the tubular structures were surgically removed. Histology identified them as Müllerian duct remnants. The testes were hypoplastic, the epididymes and deferent ducts were normal. Cytogenetic analyses revealed the presence of a mosaic 37,X/38,XY karyotype which explains the clinical findings.

Radiographic volume analysis as a novel tool to determine nasopalatine duct cyst dimensions and its association with presenting symptoms and postoperative complications

OBJECTIVES The aims of the study were to use cone beam computed tomography (CBCT) images of nasopalatine duct cysts (NPDC) and to calculate the diameter, surface area, and 3D-volume using a custom-made software program. Furthermore, any associations of dimensions of NPDC with age, gender, presence/absence of maxillary incisors/canines (MI/MC), endodontic treatment of MI/MC, presenting symptoms, and postoperative complications were evaluated. MATERIAL AND METHODS The study comprised 40 patients with a histopathologically confirmed NPDC. On preoperative CBCT scans, curves delineating the cystic borders were drawn in all planes and the widest diameter (in millimeter), surface area (in square millimeter), and volume (in cubic millimeter) were calculated. RESULTS The overall mean cyst diameter was 15 mm (range 7-47 mm), the mean cyst surface area 566 mm(2) (84-4,516 mm(2)), and the mean cyst volume 1,735 mm(3) (65-25,350 mm(3)). For 22 randomly allocated cases, a second measurement resulted in a mean absolute aberration of ±4.2 % for the volume, ±2.8 % for the surface, and ±4.9 % for the diameter. A statistically significant association was found for the CBCT determined cyst measurements and the need for preoperative endodontic treatment to MI/MC and for postoperative complications. CONCLUSION In the hands of a single experienced operator, the novel software exhibited high repeatability for measurements of cyst dimensions. Further studies are needed to assess the application of this tool for dimensional analysis of different jaw cysts and lesions including treatment planning. CLINICAL RELEVANCE Accurate radiographic information of the bone volume lost (osteolysis) due to expansion of a cystic lesion in three dimensions could help in personalized treatment planning.

MINERALOCORTICOID REGULATION OF ION CONDUCTIVE PATHWAYS OF THE CORTICAL COLLECTING DUCT

Mineralocorticoids (DOCA) are known to increase Na('+) absorption and K('+) secretion in the rabbit cortical collecting duct (CCD). However, the mechanism of regulation of the apical and basolateral cell membranes and tight junction ion conductive pathways (G('a), G('b), and G('tj), respectively) by mineralocorticoids are only partially understood. Using electrophysiological techniques and microelectrodes it was demonstrated that the apical cell membrane contained a dominant Ba('2+) sensitive K('+) conductive pathway, G(,K)('a), and an amiloride sensitive Na('+) conductive pathway, G(,Na)('a). The basolateral membrane contained a dominant Cl('-) conductive pathway, G(,Cl)('b), and a significant Ba('2+) sensitive K('+) conductive pathway, G(,K)('b). Upon elevating the mineralocorticoid levels of rabbits with intact adrenal glands it was found that V('te) was significantly increased after 1 day with a further increase after 13-16 days. These results indicated both primary and secondary effects of mineralocorticoid elevation. After 1 day of DOCA treatment, G(,Na)('a), I(,Na)('a) and I(,K)('a) increased by more than 2-fold and were maintained at high levels after 13-16 days of DOCA treatment. Secondary (chronic) effects of mineralocorticoids were evident after 4 days or more of DOCA treatment. These included a significant increase in G(,K)('a) from 4.0 to 10.2 mS.cm('-2) and a hyperpolarization of V('b) by -20 mV after 4 days of treatment. After 13-16 days of DOCA treatment V('b) remained hyperpolarized at -98.1 mV and G('tj) decreased from 5.6 to 4.2 mS.cm('-2). The hyperpolarization of V('b) was due to an increase in electrogenic Na('+) pump activity as the pump current, I(,act)('b), increased significantly from 35.7 to 195.2 (mu)A.cm('-2). Whereas net passive K('+) current across the basolateral membrane, I(,K)('b), was near zero in the control group of animals, i.e., K('+) near equilibrium, I(,K)('b) was approximately -40 (mu)A.cm('-2) in chronic DOCA treated animals. These results demonstrate that the initial effect of mineralocorticoid elevation is to increase G(,Na)('a). The ensuing depolarization of the apical membrane increases the driving force for K('+) exit into the lumen. Between 1 and 4 days of elevation, G(,K)('a) more than doubles in magnitude and at the same time the electrogenic activity of the Na('+) pump increases. This results in a hyperpolarization of V('b) which increases the driving force for K('+) uptake from the bath to the cell through a basolateral membrane conductive pathway. After 13-16 days G('tj) decreases thereby serving to maintain high electrochemical gradients across the epithelium. Therefore, the long term effects of mineralocorticoid elevation on the CCD appear to be adaptive mechanisms that serve to maintain high levels of K('+) secretion and Na('+) absorption. ^

Localization of ion-regulatory epithelia in embryos and hatchlings of two cephalopods

The tissue distribution and ontogeny of Na+/K+-ATPase has been examined as an indicator for ion-regulatory epithelia in whole animal sections of embryos and hatchlings of two cephalopod species: the squid Loligo vulgaris and the cuttlefish Sepia officinalis. This is the first report of the immunohistochemical localization of cephalopod Na+/K+-ATPase with the polyclonal antibody alpha (H-300) raised against the human alpha1-subunit of Na+/K+-ATPase. Na+/K+-ATPase immunoreactivity was observed in several tissues (gills, pancreatic appendages, nerves), exclusively located in baso-lateral membranes lining blood sinuses. Furthermore, large single cells in the gill of adult L. vulgaris specimens closely resembled Na+/K+-ATPase-rich cells described in fish. Immunohistochemical observations indicated that the amount and distribution of Na+/K+-ATPase in late cuttlefish embryos was similar to that found in juvenile and adult stages. The ion-regulatory epithelia (e.g., gills, excretory organs) of the squid embryos and paralarvae exhibited less differentiation than adults. Na+/K+-ATPase activities for whole animals were higher in hatchlings of S. officinalis (157.0 ± 32.4 µmol/g FM/h) than in those of L. vulgaris (31.8 ± 3.3 µmol/g FM/h). S. officinalis gills and pancreatic appendages achieved activities of 94.8 ± 18.5 and 421.8 ± 102.3 µmol ATP/g FM/h, respectively. High concentrations of Na+/K+-ATPase in late cephalopod embryos might be important in coping with the challenging abiotic conditions (low pH, high pCO2) that these organisms encounter inside their eggs. Our results also suggest a higher sensitivity of squid vs. cuttlefish embryos to environmental acid-base disturbances.

Bile Acids Induce Glucagon-Like Peptide 2 Secretion with Limited Effects on Intestinal Adaptation in Early Weaned Pigs

Early weaning is a stressful event characterized by a transient period of intestinal atrophy that may be mediated by reduced secretion of glucagon-like peptide (GLP) 2. We tested whether enterally fed bile acids or plant sterols could increase nutrient-dependent GLP-2 secretion and improve intestinal adaptation in weanling pigs. During the first 6 d after weaning, piglets were intragastrically infused once daily with either deionized water -control-, chenodeoxycholic acid -CDC; 60mg/kg body weight-, or b-sitoesterol -BSE; 100 mg/kg body weight-. Infusing CDC increased plasma GLP-2 -P menor que 0.05- but did not affect plasma GLP-1 and feed intake. The intestinal expression of Glp2r -glucagon-like peptide 2 receptor-, Asbt -sodium-dependent bile acid transporter-, Fxr -farnesoid X receptor-, and Tgr5 -guanosine protein?coupled bile acid receptor- genes were not affected by CDC treatment. The intragastric administration of CDC did not alter the weight and length of the intestine, yet increased the activation of caspase-3 in ileal villi -P menor que 0.02- and the expression of Il6 -interleukin 6; P menor que 0.002- in the jejunum. In contrast, infusing BSE did not affect any of the variables that were measured. Our results show that the enteral administration of the bile acid CDC potentiates the nutrient-induced secretion of endogenous GLP-2 in early-weaned pigs. Bile acid?enhanced release of GLP-2, however, did not result in improved intestinal growth, morphology, or inflammation during the postweaning degenerative phase.

Distribution of the coal flow in the mill-duct system of the As Pontes Power Plant using CFD modeling

The efficiency of a Power Plant is affected by the distribution of the pulverized coal within the furnace. The coal, which is pulverized in the mills, is transported and distributed by the primary gas through the mill-ducts to the interior of the furnace. This is done with a double function: dry and enter the coal by different levels for optimizing the combustion in the sense that a complete combustion occurs with homogeneous heat fluxes to the walls. The mill-duct systems of a real Power Plant are very complex and they are not yet well understood. In particular, experimental data concerning the mass flows of coal to the different levels are very difficult to measure. CFD modeling can help to determine them. An Eulerian/Lagrangian approach is used due to the low solid–gas volume ratio.