Identification of the membrane protein of porcine epidemic diarrhea virus

Sequence information on the genome of porcine epidemic diarrhea virus (PEDV) has only recently been determined. In contrast, very little is known about the viral proteins. In the present report we have identified the membrane glycoprotein (M) of PEDV by use of rabbit anti-peptide sera and transient expression of the cloned M gene in Vero cells and by expression in the baculovirus system. The native M protein of PEDV is incorporated into virions, is N-glycosylated, and migrates with a relative mobility (Mr) of 27 k in polyacrylamide gels. In contrast, the M protein synthesized by recombinant baculoviruses migrates with a Mr of 23 k, that is, with identical mobility as the deglycosylated product of PEDV. Thus, it appears that M protein specified by the recombinant baculovirus is poorly, if at all, glycosylated. Using monoclonal antibodies and rabbit antipeptide sera specific for the N and C termini of the M protein, we were able to show that a 19 k band detected in PEDV-infected cells but not in virions represented a fragment of M from which the C terminus had been cleaved off. Finally, by electron microscopy and immunogold labelling, the relative orientation of M within the virion envelope was determined as NexoCcyt. In conclusion, all of these data strongly support the hypothesis that PEDV should be classified with the group I coronaviruses.     

Alveoläre Hypoventilation und künstliche obstruktive Ventilationsstörung

Zusammenfassung Es wird über Versuche berichtet, in denen bei freier und artefiziell behinderter Ausatmung Beziehungen zwischen Atemzeitvolumen und alveolärem CO₂-Druck aufgestellt wurden. Unter Stenoseatmung werden für gleiche Änderungen des Atemzeitvolumens größere Differenzen des alveolären CO₂-Druckes benötigt. Die Verschiebungen werden als Folge der bei erschwerter Atmung erhöhten Atemarbeit gedeutet und nicht auf eine Änderung der Erregbarkeit des Atemzentrums zurückgeführt. Die alveoläre Hypoventilation bei obstruktiven Ventilationsstörungen kann daher — so wird weiter gefolgert — ihren Ursprung in der vermehrten Atemarbeit haben, ohne daß zwingend eine andere pathogenetisch wirksame Ursache diskutiert werden muß. Auf die Bedeutung von Veränderungen des Atemwiderstandes bei der experimentellen Prüfung atemwirksamer Pharmaka wird hingewiesen.Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Determination of Dissolved Oxygen in Heterogenous Systems Particularly in Emulsions and Oily Liquids

The content of dissolved oxygen was determined by four independent methods in a series of non-aqueous or heterogenous systems. The Lex-O₂-Content Analyzer represents a fast and simple apparatus that employs a coulometric oxygen assay with Hersch cell detection. A comparison of the results with different methods demonstrates the reliability of the Lex-O₂ in the determination of oxygen dissolved in heterogeneous or non-aqueous systems. Therefore, this apparatus can be recommended for the measurement of oxygen in oxygenator or perfusion fluids, as well as in blood substitutes or other oxygen transporting systems.     

Impact of donor extracellular vesicle release on recipient cell “cross-dressing” following clinical liver and kidney transplantation

In several murine models of transplantation, the “cross-dressing” of recipient antigen presenting cells (APCs) with intact donor major histocompatibility complex (MHC) derived from allograft-released small extracellular vesicles (sEVs) has been recently described as a key mechanism in eliciting and sustaining alloimmune responses. Investigation of these processes in clinical organ transplantation has, however, been hampered by the lack of sensitivity of conventional instruments and assays. We have employed advanced imaging flow cytometry (iFCM) to explore the kinetics of allograft sEV release and the extent to which donor sEVs might induce cross-dressing following liver and kidney transplantation. We report for the first time that recipient APC cross-dressing can be transiently detected in the circulation shortly after liver, but not kidney, transplantation in association with the release of HLA-bearing allograft-derived sEVs. In liver transplant recipients the majority of circulating cells exhibiting donor HLA are indeed cross-dressed cells and not passenger leukocytes. In keeping with experimental animal data, the downstream functional consequences of the transfer of circulating sEVs harvested from human transplant recipients varies depending on the type of transplant and time posttransplant. sEVs released shortly after liver, but not kidney, transplantation exhibit immunoinhibitory effects that could influence liver allograft immunogenicity.     

Belt-Fuqua operation for penile hypospadias repair

Between 1983 and 1993, 41 patients underwent a first-stage Belt-Fuqua operation for penile hypospadias repair and 39 completed the second stage. Minor complications were observed after the first stage. The primary success rate following the second stage was 82%. Major complications noted after the second stage consisted mainly of fistula formation. The surgical technique is described and alternative methods are discussed.     

Nitric oxide synthase activity is inducible in rat,but not rabbit alveolar macrophages,with a concomitant reduction in arginase activity

Alveolar macrophages were obtained by broncho-alveolar lavage of isolated rat and rabbit lungs and cultured (2.5 × 10⁶ cells/dish) for 18 h in the absence or presence of bacterial lipopolysaccharides (LPS) alone or in combination with cytokines. Thereafter, accumulation of ³H-citrulline (NO synthase activity) and ³H-ornithine (arginase activity) were determined.During incubation of rat alveolar macrophages with ³H-arginine clear amounts of ³H-citrulline and ³H-ornithine (3.8 and 4.6% of the added ³H-arginine, respectively) were formed and most of these metabolites appeared in the incubation medium (ratios extra-/intracellular of 17 and 70 for ³H-citrulline and ³H-ornithine, respectively). When rat alveolar macrophages had been cultured with LPS the formation of ³H-citrulline was increased about 30-fold and this was accompanied by a reduction in ³H-ornithine formation of about 60%. The effects of LPS were largely attenuated by dexamethasone (10 mol/1). Inhibition of NO synthase by N^G-monomethyl-l,-arginine (l-NMMA, 100 mol/1) in LPS treated alveolar macrophages reduced the formation ³H-citrulline by more than 90% and restored the ³H-ornithine formation. After culturing in the presence of LPS the ratios extra/intracellular of ³H-citrulline and ³H-ornithine were markedly enhanced and this effect was not dexamethasone sensitive. During incubation of rabbit alveolar macrophages a marked formation of ³H-ornithine (about 5.3% of the added ³H-arginine), but no significant formation of ³H-citrulline could be detected. Pretreatment with LPS tended to enhance the formation of ³H-ornithine (by 50%) without effects on ³H-citrulline. Rabbit-interferon and/or tumor necrosis factor- present together with LPS during the culture period did not result in a significant ³H-citrulline formation. Under all conditions tested, culture media of rabbit alveolar macrophages did not contain significant amounts of nitrite (less than 0.5 nmol) whereas in culture media of untreated rat alveolar macrophages 22 nmol nitrite (per 18 h) were detected, and LPS induced a 3-fold nitrite accumulation, an effect prevented by dexamethasone.In conclusion, in rabbit alveolar macrophages NO synthase activity was not detectable and could also not be induced by LPS and different cytokines, whereas in rat alveolar macrophages NO synthase was readily inducible. Alveolar macrophages of both species showed marked arginase activity. After induction of marked NO synthase activity, ornithine formation was largely reduced possibly by concomitant inhibition of arginase and/or withdrawn of arginine from arginase.     

Ophthalmic findings in GAPO syndrome

BACKGROUND: The main manifestations of GAPO syndrome are growth retardation (G), alopecia (A), pseudoanodontia (P), and optic atrophy (O). CASES: This syndrome has been described in 21 patients from 16 different families. Four cases are from Turkey and have been presented by Sayli and Gül. The purpose of our study is to document the cases from Turkey and discuss the ophthalmological and neuro-ophthalmolgical findings of these and other reported GAPO cases. OBSERVATIONS: All patients in the literature and our 4 cases have severe growth retardation with delayed bone age in infancy, characteristic facial appearance (high and bossed forehead, midface hypoplasia), alopecia or severe hypotrichosis, and pseudoanodontia. Optic atrophy was present in 1 of our cases and in 5 previous cases. Glaucoma was present in 5 cases, including 2 of ours. Buphthalmia and keratopathy secondary to glaucoma were also observed. White eyelashes, seen only in our cases, may be a sign of "early senility." CONCLUSIONS: Optic atrophy is not a constant finding in GAPO syndrome. Glaucoma may accompany the ocular findings. This syndrome has been attributed to either ectodermal dysplasia or the accumulation of extracellular connective tissue matrix, due to an enzyme deficiency involved in its metabolism. Current studies show that an elastin defect and secondary changes in collagen may be important in the pathogenesis of the disease.     

The effects of acetylcholine and propolis extract on corneal epithelial wound healing in rats.

PURPOSE: To investigate the effects of topical acetylcholine and topical administration of propolis, a natural beehive product, on corneal epithelial wound healing. METHODS: The whole corneal epithelium was debrided in 42 eyes of 21 rats by mechanical scraping with a dulled scalpel blade. Animals were divided into three groups. Group 1 received topical 1% water extract of propolis (WEP), group 2 received topical acetylcholine (ACh), and group 3 (control group) received topical phosphate-buffered saline, 6 times a day for 3 days, starting immediately after debridement. The area of the corneal epithelial defect was stained with fluorescein, photographed, and then measured every 12 h. The mean epithelial defect area and the mean percentage of epithelial defect remaining at each follow-up were compared between the groups. RESULTS: The mean epithelial defect area and the mean percentage of epithelial defect remaining at each time were significantly smaller (p < 0.001, p < 0.05, respectively) in the ACh and propolis groups as compared with control groups. There was no statistically significant difference between the propolis or ACh groups at any time (p > 0.05). At 72 h, the mean percentage of defect remaining was 2.58% in the ACh group, 1.3% in the propolis-treated group, and 8.68% in the control group. CONCLUSIONS: This study demonstrated that ACh and propolis facilitated corneal epithelial wound healing of rats. Although the mechanisms of the effect of propolis on wound healing and its clinical use still remain to be determined, ACh may have a place in the treatment of corneal epithelial injuries.     

Dose-dependent alteration of rat cardiac sodium current by isoproterenol: Results from direct measurements on multicellular preparations

Conflicting results have been reported in literature about the influence of-adrenergic stimulation on the fast cardiac sodium current (I _Na ⁺). To elucidate these mechanisms in multicellular preparations we used the loose-patch-clamp technique to evaluate the effect of the-adrenergic agonist isoproterenol 1–1000 nmol/l. Isoproterenol enhancedI _Na+ at all membrane potentials by elevation of the maximal availableI _Na ⁺. Only at the high concentration of 1 mol/l wasI _Na ⁺ slightly depressed after depolarizing conditioning clamps. The most marked increase of the maximal availableI _Na ⁺ was 30 ± 9 % after application of 100 nmol/l isoproterenol. To learn about the mechanisms in view of sodium channel modulation we combined isoproterenol with the sodium channel blocker lidocaine (47 mol/l). Under these circumstances the effects of both drugs were completely independent. This investigation shows clearly that low concentrations of isoproterenol increaseI _Na+ in multicellular preparations by a gating-independent mechanism.