Early assembly step of a retroviral envelope glycoprotein: analysis using a dominant negative assay.

As for most integral membrane proteins, the intracellular transport of retroviral envelope glycoproteins depends on proper folding and oligomeric assembly in the ER. In this study, we considered the hypothesis that a panel of 22 transport-defective mutants of the human T cell leukemia virus type 1 envelope glycoprotein might be defective in ER assembly. Upon cell cotransfection with wild-type envelope, however, the vast majority of these transport-defective mutants (21 of 22) exerted a specific trans-dominant negative effect. This effect was due to random dimerization of the mutated and wild-type glycoproteins that prevented the intracellular transport of the latter. This unexpected result suggests that association of glycoprotein monomers precedes the completion of folding. The only mutation that impaired this early assembly was located at the NH2 terminus of the protein. COOH-terminally truncated, soluble forms of the glycoprotein were also trans-dominant negative provided that their NH2 terminus was intact. The leucine zipper-like domain, although involved in oligomerization of the envelope glycoproteins at the cell surface, did not contribute to their intracellular assembly. We propose that, at a step subsequent to translation, but preceding complete folding of the monomers, glycoproteins assemble via their NH2-terminal domains, which, in turn, permits their cooperative folding.

Oligonucleotide delivery by a cationic derivative of the polyene antibiotic amphotericin B. II: study of the interactions of the oligonucleotide/cationic vector complexes with lipid monolayers and lipid unilamellar vesicles.

We report a study of the behavior of oligodeoxyribonucleotide (ODN)/amphotericin B3-(N'-dimethylamino)propylamide (AMA) complexes, in the presence of lipid monolayers and large unilamellar vesicles. This study follows the recent discovery of the capacity of AMA, as a new cationic vector, to enhance ODN cellular uptake and efficacy. It aims at investigating the internalization mode of a nucleic acid by AMA. A first study at the air-water interface of AMA and AMA/ODN by surface pressure measurement shows that only free AMA would adsorb at the air-water interface. Second, in the presence of zwitterionic phospholipid- and sterol-containing mixture, ODN-AMA interactions in solution would be higher than lipid-AMA interactions at the interface. In monolayer or with large unilamellar vesicles, AMA monomers adsorb mainly at the phospholipid interface. These results favor a crossing mechanism through AMA channel formation, despite the size of ODN.

Oligonucleotide delivery by a cationic derivative of the polyene antibiotic amphotericin B. I: interaction oligonucleotide/vector as studied by optical spectroscopy and electron microscopy.

Antisense strategy requires efficient systems for the delivery of oligodeoxyribonucleotides (ODN) into target cells. Cationic amphiphiles have shown good efficiency in vitro and a lot of attention is currently paid to their interaction with nucleic acids. In the present study, this interaction was, for the first time, analysed at the molecular level, taking advantage of the spectroscopic properties of the positively charged chiral polyene molecule amphotericin B 3-dimethylaminopropyl amide (AMA), the efficiency of which, as delivery system, has been demonstrated [Garcia et al., Pharmacol. Ther. (2000), in press]. By UV-visible absorption and circular dichroism (CD) we studied its self-association properties in pure water, saline and RPMI medium. Drastic changes were observed upon ODN addition, stronger in pure water than in media of high ionic strength. At low AMA concentration (<10(-6) M), the strong increase of the CD signal, characteristic of self-association, indicated condensation of AMA on the ODN molecules. At a higher concentration (10(-4) M), and for a nucleic acid negative charge/AMA positive charge ratio higher than 1, spectra were interpreted as a reorganisation of free self-associated AMA species into smaller ones 'decorating' the nucleic acid molecule. Electron microscopy data were interpreted according to this scheme.

Flow (shear stress)-induced endothelium-dependent dilation is altered in mice lacking the gene encoding for dystrophin.

BACKGROUND: Dystrophin has a key role in striated muscle mechanotransduction of physical forces. Although cytoskeletal elements play a major role in the mechanotransduction of pressure and flow in vascular cells, the role of dystrophin in vascular function has not yet been investigated. Thus, we studied endothelial and muscular responses of arteries isolated from mice lacking dystrophin (mdx mice). METHODS AND RESULTS: Carotid and mesenteric resistance arteries 120 micrometer in diameter were isolated and mounted in vitro in an arteriograph to control intraluminal pressure and flow. Blood pressure was not affected by the absence of dystrophin. Pressure-induced (myogenic), phenylephrine-induced, and KCl-induced forms of tone were unchanged. Flow (shear stress)-induced dilation in arteries isolated from mdx mice was decreased by 50% to 60%, whereas dilation to acetylcholine or sodium nitroprusside was unaffected. NG-nitro-L-arginine methyl ester-sensitive flow dilation was also decreased in arteries from mdx mice. Thus, the absence of dystrophin was associated with a defect in signal transduction of shear stress. Dystrophin was present in vascular endothelial and smooth muscle cells, as shown by immunolocalization, and localized at the level of the plasma membrane, as seen by confocal microscopy of perfused isolated arteries. CONCLUSIONS: -This is the first functional study of arteries lacking the gene for dystrophin. Vascular reactivity was normal, with the exception of flow-induced dilation. Thus, dystrophin could play a specific role in shear-stress mechanotransduction in arterial endothelial cells. Organ damage in such diseases as Duchenne dystrophy might be aggravated by such a defective arterial response to flow.

Mildly oxidized LDL induces activation of platelet-derived growth factor beta-receptor pathway.

BACKGROUND: Mildly oxidized LDL (moxLDL) is thought to play a role in atherogenesis. MoxLDL induces derivatization of cell proteins and triggers a variety of intracellular signaling. We aimed to investigate whether moxLDL-induced protein derivatization may influence the activity of platelet-derived growth factor receptor beta (PDGFRbeta), a tyrosine kinase receptor of major importance in vascular biology and atherogenesis. METHODS AND RESULTS: In cultured rabbit arterial smooth muscle cells, moxLDL induces activation of the PDGFRbeta signaling pathway, as shown by PDGFRbeta tyrosine phosphorylation on Western blot and coimmunoprecipitation of SH2-containing proteins. The cellular events involved in the moxLDL-induced PDGFRbeta activation can be summarized as follows. Oxidized lipids from moxLDL trigger two phases of PDGFRbeta activation involving two separate mechanisms, as shown by experiments on cultured cells (in situ) and on immunopurified PDGFRbeta (in vitro): (1) the first phase may be mediated by 4-hydroxynonenal, which induces PDGFRbeta adduct formation and subsequent PDGFRbeta activation (antioxidant-insensitive step); (2) the second phase involves ceramide-mediated generation of H(2)O(2) (these steps being inhibited by tosylphenylalanylchloromethylketone, an inhibitor of ceramide formation, and by antioxidant BHT, exogenous catalase, or overexpressed human catalase). Because 4-hydroxynonenal-PDGFRbeta adducts are also detected in atherosclerotic aortas, it is suggested that this novel mechanism of moxLDL-induced PDGFRbeta activation may occur during atherogenesis. CONCLUSIONS: MoxLDL acts as a local autoparacrine mediator in the vascular wall, and PDGFRbeta acts as a sensor for both oxidized lipids and oxidative stress. This constitutes a novel mechanism of PDGFRbeta activation in atherosclerotic areas.

NSPc1, a novel mammalian Polycomb gene, is expressed in neural crest-derived structures of the peripheral nervous system.

Anterior-posterior (A-P) patterning is a key element in early embryonic development. Polycomb group (PcG) genes act as transcriptional repressors to regulate A-P patterning by either directly or indirectly controlling the coordinated expression of the HOM/Hox homeobox (Curr. Opin. Genet. Dev. 7 (1997) 488; Trends Genet. 13 (1997) 167). We describe the isolation and characterization of a novel mammalian PcG gene, termed Nervous System Polycomb-1 (NSPc1). Human and mouse NSPc1 genes encode proteins with an N-terminal RING finger domain and share homology with Drosophila melanogaster lethal(3)73Ah and the mammalian Mel18 and Bmi1 genes. Transcripts are observed at 10 dpc in the otic vesicle, urogenital bud and dorsal root ganglia. At 11.5 dpc, transcripts are present in a subset of neural crest cell derivatives of the peripheral nervous system, and in the neural tube. NSPc1 expression is ubiquitous in adult tissue.

HTLV-1 structural proteins.

HTLV-1 structural proteins do not appear to ensure virus transmission as efficiently as most other retrovirus structural proteins do, whereas all other retroviruses can be transmitted via either free virions or cell-to-cell contacts, infection by HTLV-1 by free virions is very inefficient, and effective infection requires the presence of HTLV-1 infected cells. This characteristic feature of HTLV-1 provides a unique tool which can be used to analyse retrovirus cellular transmission in the absence of simultaneous cell-free infection. Here we summarise what is known about HTLV-1 structural proteins and identify the questions about these proteins which remain to be answered.

Effect of dietary phenolic compounds on apoptosis of human cultured endothelial cells induced by oxidized LDL.

1. Oxidized low density lipoproteins (LDL) are toxic to cultured endothelial cells. Mildly oxidized LDL, characterized by relatively low levels of TBARS and only minor modifications of apoB, were obtained by using 2 experimental model systems of oxidation, namely oxidation by u.v. radiation or ferrylmyoglobin (a two electron oxidation product from the reaction of metmyoglobin with H2O2). 2. Toxic concentrations of mildly oxidized LDL induce apoptosis (programmed cell death) of cultured endothelial cells, as shown by typical morphological features, by the in situ TUNEL procedure and by DNA fragmentation revealed on gel electrophoresis. This apoptosis is calcium-dependent and subsequent to the intense and sustained cytosolic [Ca2+]i peak elicited by oxidized LDL. 3. Five naturally occurring phenolic compounds present in food and beverages were able to prevent, in a concentration-dependent manner, the apoptosis of endothelial cells induced by oxidized LDL. Among the compounds tested, caffeic acid was the most effective. Under the conditions used, the protective effect of caffeic acid (IC50 8.3+/-2.1 micromol l[-1]) in the prevention of apoptosis induced by oxidized LDL was significantly higher than that of the other compounds tested (IC50s were 12.4+/-3.2, 14.1+/-4.1, 20.4+/-4.4 and 72.6+/-9.2 micromol l(-1) for ferulic, protocatechuic, ellagic and p-coumaric acids, respectively). 4. The anti-apoptotic effect of caffeic acid results from the addition of two effects, (i) the antioxidant effect which prevents LDL oxidation and subsequent toxicity ('indirect' protective effect); (ii) a 'direct' cytoprotective effect, acting at the cellular level. 5. Effective concentrations of caffeic acid acted at the cellular level by blocking the intense and sustained cytosolic [Ca2+]i rise elicited by oxidized LDL. 6. In conclusion, phenolic acids (caffeic and ferulic acids being the most potent of the compounds tested under the conditions used) exhibit a potent cytoprotective effect of cultured endothelial cells against oxidized LDL. In addition to antioxidant effect delaying LDL oxidation, caffeic acid acts as a cytoprotective agent, probably by blocking the intracellular signalling triggered by oxidized LDL and culminating in the sustained calcium rise which is involved in oxidized LDL-induced apoptosis.

Arterial occlusive disease after radiotherapy: a report of fourteen cases.

Fourteen cases of arterial occlusion or severe narrowing following radiotherapy are studied in order to assess the possible etiological role of such therapy in arterial lesion. Surgical results are also discussed in terms of long-term efficacy. The average time of occurrence after radiotherapy was 8 years post-radiotherapy. This series includes 7 supra-aortic trunk stenoses and 7 abdominal aorta trunk stenoses. The doses received in the volumes irradiated ranged from 47 to 70 Gy with standard fractionation. Association of atherosclerotic risk factors (smoking, hyperlipidemia, diabetes, high blood pressure) was present in 12 patients, but stenoses were usually confined to irradiated areas, and at times occurred in uncommon sites. Surgical management included 11 by-passes, 2 endarterectomies and one percutaneous transluminal angioplasty. All patients experienced immediate and satisfactory functional improvements. Three patients were re-operated on because of the re-occlusion of the by-pass (2 cases) and graft infection (1 case). On the whole, stenoses in previously irradiated areas showed no particular difficulties for surgical treatment. It was concluded that radiotherapy seems to be a definite risk factor for arterial occlusion or narrowing, especially in association with atherosclerotic risk factors.

Dose-dependent blood flow velocity changes in the Basal cerebral arteries following low-dose ketamine.

The effects of low-dose ketamine on blood flow velocity and pulsatility index (PI) in the basal cerebral arteries were studied in two groups of healthy volunteers (group A: 0.25 mg/kg, n = 10; group B: 0.5 mg/kg, n = 10) by means of a 2 MHz pulsed transcranial Doppler ultrasonic system (TCD) for an observation period of 70 min. In addition, mean arterial pressure (MAP), heart rate (HR), end-tidal CO2 (petCO2), and oxygen saturation (saO2) were recorded. The mean blood flow velocity (Vmean) increased significantly by 28% in group A and by 68% in group B within 2-9 min. In group B, the PI was reduced by 32% for a period of 2-7 min (p < 0.05). MAP (group A: -20%; group B: -26%) and HR (group A: -39%; group B: -54%) increased significantly (p < 0.05). Vmean and PI were significantly different between groups A and B, suggesting a dose-dependent stimulation of cerebral hemodynamics. The flow velocity increases cannot be entirely explained by systemic hemodynamic changes since there was no intergroup difference with respect to MAP and HR. No significant differences were observed with regard to petCO2 and saO2. With the assumption that the diameter of the insonated vessel is not changed by ketamine, the dose-dependent increase in Vmean with a concomitant decrease in PI may be interpreted as indicating an increase in cerebral blood flow.

[Carney's triad: a new case associated with adrenal cortex adenoma]. / Triade de Carney: un nouveau cas associé à un adénome cortico-surrénalien.

Carney's triad is a combination of at least 2 of 3 tumor sites: multiple gastric leiomyoblastoma, pulmonary chondroma and paraganglioma, most often extra-adrenal and secreting. Thirty-three cases have been published; we report a new case in a 20 year-old woman, with multiple gastric leiomyoblastomas, liver metastasis, and bilateral pulmonary chondroma. The research of paraganglioma, in regard to Carney's disease, led to the discovery of corticoadrenal adenoma.

[Gastric volvulus secondary to para-esophageal and sliding hiatal hernias]. / Incarcérations gastriques sur hernies hiatales paraoesophagiennes et par glissement.

Gastric volvulus is not a rare condition and 350 authentic cases have been documented in the adult population. Most often, gastric volvulus was associated with a large paraoesophageal hernia (40%). We report seven new cases of gastric volvulus: 5 were due to a large paraoesophageal hernia, 1 to mixed hiatus hernia, and 1 to a sliding hiatus hernia. We did not observe any cases of acute strangulation with gastric necrosis. The lesions were reversible in the three cases of acute and four cases of chronic gastric volvulus. Surgical treatment included gastric volvulus reduction and repair of hiatus hernia without gastric resection. Surgical treatment of paraoesophageal hiatus hernia is mandatory to reduce the incidence of gastric volvulus. The possibility of gastric volvulus with hiatus hernia must be recognized.

[Disorders of relaxation of the lower esophageal sphincter after resection of mediothoracic esophageal pulsion diverticulum]. / Troubles de la relaxation du sphincter inférieur de l'oesophage apparus après l'exérèse d'un diverticule oesophagien médio-thoracique par pulsion.

A pulsion diverticulum of the mid-thoracic oesophagus was resected because of severe dysphagia. A subdiverticular myotomy was associated for the treatment of non-propagated oesophageal contractions, without oesophageal low-sphincter abnormalities. Three months post-operatively, the dysphagia was still present and manometry showed the absence of lower sphincter relaxation after 45 percent of deglutitions; a lower oesophageal sphincter myotomy was performed, resulting in correction of the dysphagia and of the sphincter relaxation abnormalities. The deficit of the inhibitory nervous mechanism, responsible for abnormalities of lower oesophageal sphincter relaxation, was present in the entire intramural oesophageal plexus, but, was not symptomatic at the level of the IOS prior to resection of the diverticulum.

[Anorectal manometry and ileo-anal anastomosis: pre- and postoperative manometric comparison]. / Manométrie ano-rectale et anastomose iléo-anale: comparaison manométrique pré- et post-opératoire.

The aim of this study was to evaluate the physiological and manometric changes of anorectal function after proctocolectomy and ileal J pouch anastomosis performed in 13 patients. A short muscular rectal cuff was conserved in the first 5 patients, mucosectomy was performed in the next patients (Group M), and the entire rectum and proximal portion of the anal canal (1 mm above the dentate line) were resected without mucosectomy (Group WM) in the most recent patients. With a median postoperative follow-up of 18 months, anorectal continence was perfect in 9 patients, and minimal incontinence were observed in 4 patients (2 patients in Group M, and 2 patients in Group SM). In Group M, the resting lower anal canal pressure was significantly decreased after the operation and was less than the postoperative resting lower anal canal pressure observed in Group SM. The resting upper anal canal pressure in continent patients was inferior to the postoperative values measured in patients with minimal incontinence. In all the groups, no change in squeeze pressure was observed after the operation. During the postoperative period, the rectoanal inhibitory reflex was absence in all patients. The volume necessary for sensation threshold was increased after the operation, in the same number of patients in Group M and SM, and more frequently in continent patients compared with patients with soiling. The absence of muscular rectal cuff and mucosectomy seems to provide best results than rectal cuff preservation. After the operation, a marked decrease in the resting pressure and an increase in the volume necessary to onset of threshold sensation, were associated with the presence of minimal incontinence.

[Pancreatico-pleural fistula in chronic pancreatitis]. / Les fistules wirsungo-pleurales de la pancréatite chronique. A propos de sept observations.

Seven patients presented with a pancreatic pleural fistula in the context of chronic pancreatitis. The diagnosis was made by analysis of the pleural effusion and by endoscopic retrograde pancreatography which opacified the fistula in six cases. Surgical procedure (five resections, one Roux-en-Y drainage and one resection associated with Roux-en-Y drainage) was guided by the morphology of the pancreatic duct and the presence of pseudocysts. All patients were successfully treated by surgery.

[The role of colorectal resection in severe constipation after promontory rectopexy]. / Place de l'exérèse colo-rectale pour constipation sévère après promonto-fixation rectale.

Abdominal rectopexy to the promontory is an effective treatment for total rectal prolapse, internal procidentia and solitary ulcer. This paper is designed to stress the risk of severe constipation following rectopexy, a complication which required surgical resection in 5 cases. This raises the problems of the physiological mechanisms of this complication, the definition of a possible high risk population (young women, patients who were constipated preoperatively?), and an alternative to promontory rectopexy: sacral fixation of the rectum, associated sigmoidectomy, Delorme's operation?

Oncological long-term outcome of 4772 patients with prostate cancer undergoing radical prostatectomy: does the anaesthetic technique matter?

INTRODUCTION: Recent data suggest that using additional neuroaxial anaesthesia during oncological surgery is associated with favourable recurrence-free survival, when compared with general anaesthesia alone. We assessed the impact of adjunctive perioperative spinal anaesthesia and dose of opioids on the oncological long-term outcome of patients following radical prostatectomy. METHODS: We selected patients from our institutional review board-approved database who consecutively underwent radical prostatectomy between 2002 and 2007. Patients were stratified by type of anaesthesia, administered as general anaesthesia alone, or spinal anaesthesia in addition to general anaesthesia. Biochemical recurrence-free survival, metastasis-free survival and overall survival were analysed by a multivariate Cox regression model and by Kaplan-Meier analysis in propensity-score based matched cohorts, adjusted for standard clinico-pathological variables and year of surgery. RESULTS: Overall, 4772 patients were analysed. Regarding the type of anaesthesia no significant difference for biochemical recurrence-free survival, metastasis-free survival and overall survival was analysed by a multivariate Cox regression model (p = 0.5, 0.8 and 0.7). The Kaplan-Meier analyses after propensity-score matched based comparisons revealed no significant difference depending on type of anaesthesia for biochemical recurrence-free survival, metastasis-free survival and overall survival (p = 0.6, 0.1 and 0.4). The same accounted for a propensity-score matched model adjusted for the year of surgery on biochemical recurrence-free survival (p = 0.7). CONCLUSIONS: The oncological outcome after radical prostatectomy was not affected by the adjunctive use of spinal anaesthesia.

[Course of central body temperature in the laminar airflow operating room in various anesthesia procedures]. / Verlauf der Körper-kerntemperatur im Laminar-air-flow-Operationsraum unter verschiedenen Narkoseverfahren.

The oesophageal body temperature of 130 patients was measured pre- and intraoperatively. 92% (n = 116) of the operations (implantation or replacement of hip prostheses) were performed in an operating room having a laminar air flow system with horizontal air flow. 9% (n = 14) of the operations (laparotomies) were performed in a room of identical design without an air circulation system. Three different forms of anesthesia were investigated with regard to their influence on interior body temperature: 1) general anesthesia with a volatile anesthetic (INH); 2) peridural anesthesia with additional general anesthesia (KPDA+ITN); and 3) neuroleptic anesthesia (NLA). A drop in temperature during the operation was found in all patients. In the conventional operating room the mean drop was 0.3 degrees C/h. In the operating room with laminar air flow the INH-patients sustained the greatest decrease in temperature; the mean value in the first hour was 1.1 degrees C/h, and up to 4.6 degrees C/3 h toward the end of the operation. There was a comparable drop in temperature in the first hour in patients anesthetized with KPDA+ITN, but the rate slowed down toward the end of the investigation (2.2 degrees C/3 h). NLA caused a characteristic temperature behavior, with an initial fall in temperature, plateau phase, and subsequent rise (total: -1.0 degrees C/3 h) Temperature regulation was influenced least by NLA in the operating room with laminar air flow; thus, in this context, NLA proved to be a favourable form of anesthesia.