Co-expression in CHO cells of two muscle proteins involved in excitation-contraction coupling

Summary Ryanodine receptors and dihydropyridine receptors are located opposite each other at the junctions between sarcoplasmic reticulum and either the surface membrane or the transverse tubules in skeletal muscle. Ryanodine receptors are the calcium release channels of the sarcoplasmic reticulum and their cytoplasmic domains form the feet, connecting sarcoplasmic reticulum to transverse tubules. Dihydropyridine receptors are L-type calcium channels that act as the voltage sensors of excitation-contraction coupling: they sense surface membrane and tranverse tubule depolarization and induce opening of the sarcoplasmic reticulum release channels. In skeletal muscle, ryanodine receptors are arranged in extensive arrays and dihydropyridine receptors are grouped into tetrads, which in turn are associated with the four subunits of ryanodine receptors. The disposition allows for a direct interaction between the two sets of molecules.CHO cells were stably transformed with plasmids for skeletal muscle ryanodine receptors and either the skeletal dihydropyridine receptor, or a skeletal-cardiac dihydropyridine receptor chimera (CSk3) which can functionally substitute for the skeletal dihydropyridine receptor, in addition to plasmids for the ₂, and subunits. RNA blot hybridization gave positive results for all components. Immunoblots, ryanodine binding, electron microscopy and exposure to caffeine show that the expressed ryanodine receptors forms functional tetrameric channels, which are correctly inserted into the endoplasmic reticulum membrane, and form extensive arrays with the same spacings as in skeletal muscle. Since formation of arrays does not require coexpression of dihydropyridine receptors, we conclude that self-aggregation is an independent property of ryanodine receptors. All dihydropyridine receptor-expressing clones show high affinity binding for dihydropyridine and immunolabelling with antibodies against dihydropyridine receptor. The presence of calcium currents with fast kinetics and immunolabelling for dihydropyridine receptors in the surface membrane of CSk3 clones indicate that CSk3-dihydropyridine receptors are appropriately targeted to the cell's plasmalemma. The expressed skeletal-type dihydropyridine receptors, however, remain mostly located within perinuclear membranes. In cells coexpressing functional dihydropyridine receptors and ryanodine receptors, no junctions between feet-bearing endoplasmic reticulum elements and surface membrane are formed, and dihydropyridine receptors do not assemble into tetrads. A separation between dihydropyridine receptors and ryanodine receptors is not unique to CHO cells, but is found also in cardiac muscle, in muscles of invertebrates and, under certain conditions, in skeletal muscle. We suggest that failure to form junctions in co-transfected CHO cell may be due to lack of an essential protein necessary either for the initial docking of the endoplasmic reticulum to the surface membrane or for maintaining the interaction between dihydropyridine receptors and ryanodine receptors. We also conclude that formation of tetrads requires a close interaction between dihydropyridine receptors and ryanodine receptors.     

Prevalence of hepatitis-C virus RNA in serum and throat washings of children with chronic hepatitis

Serum samples from 46 children with chronic and probably transfusion acquired hepatitis were tested for the presence of hepatitis C virus (HCV) RNA by a “nested” polymerase chain reaction (PCR) assay, to judge a possible risk of HCV transmission from these patients. In 73% of the samples, viral RNA was detected, indicating a high virus prevalence in this patient group. High titers of HCV-RNA were observed in some sera as shown by the detection of virus in some samples even at dilutions of 10^?3. Comparison of simultaneously obtained PCR results and ALT values revealed no significant correlation between virus presence in serum and higher ALT levels. It was, however, shown that unusually high ALT values may reflect a high titer of viral RNA in serum. To investigate the prevalence of viral RNA in saliva, which could be a vehicle of virus transmission, 35 throat washing samples from the HCV-infected children were screened by PCR. Using three different sample preparation procedures, 20% of the throat washings were found to be positive for HCV-RNA. This indicates a prevalence of virus in this fluid lower than that reported previously. © 1994 Wiley-Liss, Inc.     

Construction of an infectious chimeric classical swine fever virus containing the 5'UTR of bovine viral diarrhea virus, and its application as a universal internal positive control in real-time RT-PCR

RT-PCR is used widely as a diagnostic method to detect and differentiate pestiviruses. The construction of two chimeric classical swine fever virus (CSFV) recombinants based on a marker virus constructed previously [J. Virol. 72 (1998) 5318-5322] is described. These viruses, termed vA187CAT_5UTRBVD and vA187CAT_IRESBVD, contain the entire 5' untranslated region (5'UTR) or the internal ribosome entry site (IRES) of bovine viral diarrhea virus (BVDV), respectively. Both chimeric viruses proved to be infectious in cell culture. Hence, the 5'UTR as well as the IRES element only of BVDV can substitute for the corresponding genome region of CSFV. Next, two sets of primers and corresponding dual-labeled TaqMan probes were designed; one detecting specifically a conserved but CSFV-specific area within the 5'UTR of wild-type CSFV, the other one targeting the CAT gene inserted in vA187CAT_5UTRBVD. The two primer/probe sets were combined in a closed-tube multiplex one-step RT-PCR. To monitor the entire extraction and detection process limited amounts of vA187CAT_5UTRBVD were added directly to clinical samples before RNA extraction. The multiplex RT-PCR proved to be as sensitive as the single primer/probe set method, but allowed the validation of each sample tested individually, based on the detection of the CAT marker gene. vA187CAT_5UTRBVD was also used successfully for foot-and-mouth disease virus (FMDV) TaqMan RT-PCR. Therefore, it is considered a universal internal positive control for RT-PCR assays to exclude loss of RNA during extraction, or failure of amplification due to inhibitory substances present in the sample.     

Blockade of vascular endothelial cell growth factor receptor signaling is sufficient to completely prevent retinal neovascularization

Retinal vasculogenesis and ischemic retinopathies provide good model systems for study of vascular development and neovascularization (NV), respectively. Vascular endothelial cell growth factor (VEGF) has been implicated in the pathogenesis of retinal vasculogenesis and in the development of retinal NV in ischemic retinopathies. However, insulin-like growth factor-I and possibly other growth factors also participate in the development of retinal NV and intraocular injections of VEGF antagonists only partially inhibit retinal NV. One possible conclusion from these studies is that it is necessary to block other growth factors in addition to VEGF to achieve complete inhibition of retinal NV. We recently demonstrated that a partially selective kinase inhibitor, PKC412, that blocks phosphorylation by VEGF and platelet-derived growth factor (PDGF) receptors and several isoforms of protein kinase C (PKC), completely inhibits retinal NV. In this study, we have used three additional selective kinase inhibitors with different selectivity profiles to explore the signaling pathways involved in retinal NV. PTK787, a drug that blocks phosphorylation by VEGF and PDGF receptors, but not PKC, completely inhibited retinal NV in murine oxygen-induced ischemic retinopathy and partially inhibited retinal vascularization during development. CGP 57148 and CGP 53716, two drugs that block phosphorylation by PDGF receptors, but not VEGF receptors, had no significant effect on retinal NV. These data and our previously published study suggest that regardless of contributions by other growth factors, VEGF signaling plays a critical role in the pathogenesis of retinal NV. Inhibition of VEGF receptor kinase activity completely blocks retinal NV and is an excellent target for treatment of proliferative diabetic retinopathy and other ischemic retinopathies.     

Pharmakologisch-aktive polymere, 21. Orientierende Untersuchungen zur körperverteilung und Ausscheidung von poly[2-(methylsulfinyl)ethylacrylat]en bei der Ratte

Poly[2-(methylsulfinyl)ethyl acrylate] ( 1 ) was synthesized as well as derivatives ¹⁴C-labelled in side groups ( 6 ) or ¹⁴C-labelled in the main chain ( 11 ). Polymer 11 with the ¹⁴C-labelled main chain was fractionated by precipitation. The η-M-relation determined by measurements of unlabelled polymers in the ultracentrifuge for comparison was used to establish the viscosimetrically determined molecular weights of the labelled fractions. After intravenous application of aqueous solutions of the polymer in rats the excretion rate up to 72 h after treatment was ascertained to ca. 50%; the concentration in the blood serum was found to be strikingly high. A tendency to reinforced storage in organs of high phagocytose activity by growing molecular weight was observed. The tumor affinity was — if at all — low.     

Endothelin receptor expression in human decidua

The endothelins are signalling peptides that act via two receptors, ET(A) and ET(B). In the human endometrium, endothelin receptors have been demonstrated in glands and stroma and have been shown to vary during the course of the menstrual cycle. The present study was undertaken to determine whether or not expression of endothelin receptors changes during pregnancy or after administration of exogenous progestagens. The expression of the receptors was correlated with the appearance of basement membrane components during decidualization of the endometrial stroma. Decidual specimens (n = 15) were obtained during the first trimester of pregnancy and 10 at term. Sixteen pairs of endometrial biopsies were obtained from women with menorrhagia before and after exposure to exogenous progestagens. A total of 15 hysterectomy specimens were used as controls for the expression of stromal basement membrane proteins in the absence of decidualization. Autoradiography was carried out with selective ligands for ET(A) ([125I]-PD 151242) and ET(B) ([125I]-BQ3020). The distribution of ligand binding was then compared with the distribution of laminin alpha2 light chain and collagen IV. ET(A), ET(B), laminin alpha2 light chain, and collagen IV were expressed in stromal decidual cells in the first trimester of pregnancy. ET(B) was also found on endometrial glandular epithelium. Quantitative macro-autoradiography and multiple regression analysis demonstrated a highly significant positive correlation (P < 0.001) between expression of ET(B) and laminin alpha2 light chain. In the third trimester qualitative examination suggested a reduction of ET(A) in the stroma. Progestagen-induced decidua exhibited a similar pattern to that found in first trimester decidua. This study has demonstrated up-regulation of ET(B) during the progesterone- dependent process of decidualization and suggests a paracrine or autocrine role for endothelins in the decidua.      

Chimeric Clostridial Cytotoxins: Identification of the N-Terminal Region Involved in Protein Substrate Recognition

Clostridium sordellii lethal toxin is a member of the family of large clostridial cytotoxins that glucosylate small GTPases. In contrast to Clostridium difficile toxins A and B, which exclusively modify Rho subfamily proteins, C. sordellii lethal toxin also glucosylates Ras subfamily proteins. By deletion analysis and construction of chimeric fusion proteins of C. sordellii lethal toxin and C. difficile toxin B, we localized the enzyme activity of the lethal toxin to the N terminus of the holotoxin and identified the region involved in protein substrate specificity. The toxin fragment of the N-terminal 546 amino acid residues of C. sordellii lethal toxin glucosylated Rho and Ras subfamily proteins, as the holotoxin did. Deletion of a further 30 amino acid residues from the C terminus of this active fragment drastically reduced glucotransferase activity and blocked glucohydrolase activity. Exchange of amino acid residues 364 through 516 of lethal toxin for those in the active toxin B fragment (1 to 546) allowed glucosylation of Ras subfamily proteins. In contrast, the chimera with amino acids 1 to 364 from toxin B, 365 to 468 from lethal toxin, and 469 to 546 from toxin B exhibited markedly reduced modification of Ras subfamily proteins, whereas modification of Rac and Cdc42 was hardly changed. The data indicate that the region of amino acid residues 364 through 516 primarily defines the substrate specificity of C. sordellii lethal toxin.     

Permanent impairment of embryo development by hydrosalpinges

Recent reports suggest a deleterious effect of hydrosalpinges on pregnancy outcome for in-vitro fertilization (IVF) and improvement following surgical treatment. We compared the effect of hydrosalpinx on pregnancy outcome in 286 patients having 348 IVF cycles and followed the development of untransferred embryos for 7 days to determine if hydrosalpinges affect oocyte quality or embryo development. The delivery rate per retrieval was significantly lower for patients with hydrosalpinx, but was restored by surgical treatment to that of patients without hydrosalpinx. However, the implantation rate per embryo transferred and normal blastulation of untransferred embryos, which were significantly decreased in patients with hydrosalpinx, and growth arrest and degeneration of untransferred embryos, which were significantly increased compared to patients without hydrosalpinx, were not restored by surgical treatment of hydrosalpinges. We conclude that surgical treatment of hydrosalpinges decreases early pregnancy loss and improves pregnancy outcome, possibly by diminishing reversible deleterious effects exerted on the endometrium. As we have seen in our laboratory, hydrosalpinges may have a permanent negative influence on ovarian function, follicular development and oocyte quality since implantation of transferred embryos and normal blastulation of untransferred embryos remain low, and in-vitro growth arrest and degeneration remain high despite surgical treatment of hydrosalpinges.      

Transformationsvorgänge in der Frühphase der Entstehung des arteriellen Thrombus

Zusammenfassung In unserer ultrastrukturell durchgeführten Studie wurden Thromben in der Arteria carotis communis von Ratten nach einer zuerst von Meng und Seuter (1977) beschriebenen Methode experimentell erzeugt. Induktion der Thrombusbildung erfolgte in vivo durch Unterkühlung eines kleinen Gefäßabschnittes unter konstantem Druck und kurzfristiger Stase. Eine Änderung des Blutflusses wurde durch einen Silberclip erzeugt. Die geschädigten Gefäßsegmente einschließlich der Thromben bzw. deren Vorstufen wurden nach 5, 10, 30 min und 1, 4 und 24 h nach der Thrombosestimulation entnommen und fixiert. Semidünnschnitte und Ultradünnschnitte wurden im Licht- und Elektronenmikroskop morphologisch untersucht.Den Transformationsvorgängen im Thrombus konnten exakte Zeitmarken zugeordnet werden. Als wichtigstes histopathologisches Merkmal für die Altersbestimmung arterieller Thromben in der Frühphase der Thrombogenese werteten wir die Querstreifung der Fibrinfasern. Diese trat bereits nach 5 min auf, erreichte nach 30 min ein Maximum und verschwand als Folge der zunehmenden Verdichtung der Fasern nach einer Stunde. Nach 4 h sahen wir eine weitgehende Retraktion der Fibrinfasern, die nach 24 h zur Bildung des Fibrinfasergerüstes mit Einmauerung korpuskulärer Elemente führte. Überdies beobachteten wir zwei Thrombocytenaggregate von differenter Struktur. Wir unterschieden ein fibrinarmes Aggregat, in dem die Thrombocyten dichtgepackt und pseudopodienreich erschienen von einem thrombocytenarmen Aggregat mit reichlich interponierten Fibrinfasern. Die nach 5 min im Zentrum des Thrombus auftretende Agglutination der Plättchen im thrombocytenreichen Aggregat führte nach 30 min zur Thrombocytorrhexis und ergab daher einen weiteren Anhalt für die Altersbestimmung des Coagulum. Der entstandene celluläre Abraum stimulierte mononucleäre Zellen und Leukocyten zur Phagocytose. Daher sahen wir nach 4 h eine massive Leukocytose als Folge der frühen Thrombocytorrhexis. Nach 24 h war die viscöse Metamorphose im fibrinreichen und fibrinararmen Aggregat weitgehend abgeschlossen. Innerhalb des beobachteten Zeitraumes entstand eine Verballung und bizarre Deformierung der Erythrocyten, die bereits nach 5 min vom Zentrum des Thrombus ausging und nach 24 h die Peripherie erreichte. Eine Hämolyse der Erythrocyten war nach dieser Zeit noch nicht erkennbar.

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Evolution in the early changes in the establishment of arterial thrombi

Summary Ultrastructural studies of thrombi were carried out on the common carotid artery of the rat using a method first described by Meng and Seuter (1977). Induction of thrombus formation in vivo was achieved by chilling of a small vessel segment under constant pressure and short-termed stasis. Disturbance of the blood flow was produced by a silver clip. The damaged vessel segments with the thrombotic deposits were removed 5, 10, 30 min, and 1, 4 and 24 h after stimulation of thrombosis. They were fixed and samples were studied as semithin and ultrathin sections morphologically using light and electronmicroscopy.In the maturation of thrombi exact time intervals could be determined. The most important histopathological characteristics for age determination of arterial thrombi in the early period of thrombogenesis were the cross stripes of fibrin fibres. They appeared after 5 min, reaching a maximum after 10 min and disappeared as a result of increasing fibre density after 1 h. After 4 h nearly complete retraction of fibrin fibres was found which led after 24 h to the formation of a corresponding frame walling in the corpuscular elements. Apart from this aggregation of thrombocytes, which were of two different types were observed, one showing a fibrin-poor aggregate in which the thrombocytes appeared densely packed with numerous pseudopods, and one showing a thrombocyte poor aggregate with abundant interposed fibrin fibres. Agglutination of platelets which occurred in the thrombocyte-rich aggregate in the centre of the thrombus after 5 min led to thrombocytorrhexis after 30 min. The resulting cellular waste stimulated phagocytosis by mononuclear cells and leucocytes. Because of this a massive leucocytosis was found as a result of the early thrombocytorrhexis after 4 h. After 24 h the viscous metamorphosis in the fibrin-rich and in the fibrin-poor aggregate was largely completed. Clumping and deformation of erythrocytes was observed in the middle of the thrombus after 5 min and at the periphery of the thrombus after 24 h. Haemolysis did not occur within this time interval.

Frau Antoni, Herrn Ing. grad. Derks und Herrn Rieger sei für ausgezeichnete technische Assistenz herzlichst gedankt.