Information transfer between large and small two-dimensional polyacrylamide gel electrophoresis.

To determine the feasibility of data transfer, an interlaboratory comparison was conducted on colon carcinoma cell line (DLD-1) proteins resolved by two-dimensional polyacrylamide gel electrophoresis either on small (6 x 7 cm) or large (16x18 cm) gels. The gels were silver-stained and scanned by laser densitometry, and the image obtained was analyzed using Melanie software. The number of spots detected was 1337+/-161 vs. 2382+/-176 for small vs. large format gels, respectively. After gel calibration using landmarks determined using pl and Mr markers, large- and small-format gels were matched and 712+/-36 proteins were found on both types of gels. Having performed accurate gel matching it was possible to acquire additional information after accessing a 2-D PAGE reference database (http://www.expasy.ch/ cgibin/map2/def?DLD1_HUMAN). Thus, the difference in gel size is not an obstacle for data transfer. This will facilitate exchanges between laboratories or consultation concerning existing databases.

Proteomic analysis of cytokine induced proteins in human intestinal epithelial cells: implications for inflammatory bowel diseases.

A role for cytokine regulated proteins in epithelial cells has been suggested in the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to identify such cytokine regulated targets using a proteomic functional approach. Protein patterns from (35)S-radiolabeled homogenates of cultured colon epithelial cells were compared before and after exposure to interferon-gamma, interleukin-1beta and interleukin-6. Proteins were separated by two-dimensional polyacrylamide gel electrophoresis. Both autoradiographies and silver stained gels were analyzed. Proteins showing differential expression were identified by tryptic in-gel digestion and mass spectrometry. Metabolism related proteins were also investigated by Western blot analysis. Tryptophanyl-tRNA synthetase, indoleamine-2,3-dioxygenase, heterogeneous nuclear ribonucleoprotein JKTBP, interferon-induced 35kDa protein, proteasome subunit LMP2 and arginosuccinate synthetase were identified as cytokine modulated proteins in vitro. Using purified epithelial cells from patients, overexpression of indoleamine-2,3-dioxygenase, an enzyme involved in tryptophan metabolism, was confirmed in Crohn's disease as well as in ulcerative colitis, as compared to normal mucosa. No such difference was found in diverticulitis. Potentially, this observation opens new avenues in the treatment of IBD.

A large deletion in the adRP gene PRPF31: evidence that haploinsufficiency is the cause of disease.

PURPOSE: To report a large deletion that encompasses more than 90% of PRPF31 gene and two other neighboring genes in their entirety in an adRP pedigree that appears to show only the typical clinical features of retinitis pigmentosa. METHODS: To identify PRPF31 mutation in a dominant RP family (ADRP2) previously linked to the RP11 locus, the 14 exons of PRPF31 were screened for mutations by direct sequencing. To investigate the possibility of a large deletion, microsatellite markers near PRPF31 gene were analyzed by non-denaturing PAGE. RESULTS: Initial screening of PRPF31 gene in the ADRP2 family did not reveal an obvious mutation. A large deletion was however suspected due to lack of heterozygosity for nearly all PRPF31 intragenic single nucleotide polymorphysm (SNPs). In order to estimate the size of the deletion, SNPs and microsatellite markers spanning and flanking PRPF31 were analyzed in the entire ADRP2 family. Haplotype analysis with the above markers suggested a deletion of approximately 30 kb that included the putative promoter region of a novel gene OSCAR, the entire genomic content of genes NDUFA3, TFPT and more than 90% of PRPF31 gene. Sequence analysis of the region flanking the potential deletion showed a high presence of Alu elements implicating Alu mediated recombination as the mechanism responsible for this event. CONCLUSIONS: This mutation provides evidence that haploinsufficiency rather than aberrant function of mutated proteins is the cause of disease in these adRP patients with mutations in PRPF31 gene.

Titelbildbeitrag - Bilaterale subkapsuläre Urinome: Ungewöhnliche Ursache einer Page-Niere mit Nierenversagen

Die Page-Niere ist eine relativ seltene, meist reversible Form der sekundären Hypertonie. Im Gegensatz zum Goldblatt-Mechanismus wird der hyperreninämische Hochdruck durch eine Ischämie der Nierenrinde als Folge einer Kompression des Nierenparenchyms durch einen subkapsulären oder perirenalen Prozess, meistens durch Hämatome, verursacht. Seit den anfänglichen Experimenten von Page im Jahr 1939 wurden über 100 Fälle dieses Zustands in der Literatur beschrieben (Dopson SJ et al. Am J Kidney Dis 2009; 54: 334-339). Soweit uns bekannt ist, wurden nur zwei davon durch Urinome verursacht (Patel MR et al. Urology 1984; 23: 585-587; Matlaga BR et al. J Urol 2002; 168: 672). Im 1. Fall musste ein größerer chirurgischer Eingriff (d. h. Nephrektomie) durchgeführt werden, während der 2. Patient mittels retrograder Pyelografie und Setzen eines Harnleiterkatheters behandelt wurde. Hier stellen wir den ungewöhnlichen Fall einer Page-Niere infolge eines beidseitigen subkapsulären Urinoms und akutem Nierenversagen als Komplikation vor. Die Ultraschall- (US) und computertomografischen (CT) Befunde werden beschrieben, wobei auf die sonografisch gesteuerte perkutane Behandlung besonders eingegangen wird.

In-situ revascularisation for secondary aorto-enteric fistulae: the success of silver-coated Dacron is closely linked to a suitable bowel repair.

OBJECTIVES: The purpose of this study was to assess short- and mid-term results of in-situ revascularisation (ISR) using silver-coated Dacron prostheses and bowel repair for management of secondary aorto-enteric fistulae (SAEF). DESIGN: Single-centre retrospective chart review. MATERIAL AND METHODS: This study includes all the patients treated by ISR using silver-coated Dacron for SAEF between 2006 and 2010. Primary end points were mortality and survival rates. Secondary end points were reinfection-free survival and secondary patency rates. RESULTS: Eighteen male patients with SAEF with a median age of 64 years were operated by ISR using silver-coated Dacron during the study period without operative death. The 30-day mortality was 22% and the in-hospital mortality rate was 39%. Indeed, during hospitalisation, a duodenal leak was observed in four patients including three who died. Four others patients died due to multi-system organ failure. Median follow-up was 16 months (range 1-66). The survival rate at 12 months was 55%. One duodenal leak was observed leading to death. The reinfection-free survival and the secondary patency rates at 12 months were 60% and 89%, respectively. CONCLUSION: In-situ revascularisation with silver-coated Dacron provides acceptable results in terms of mortality. This treatment may be useful for simple vascular reconstruction and allow greater attention to bowel repair that is a determinant in short- and mid-term survival.

Wet chemical silver treatment of endotracheal tubes to produce antibacterial surfaces.

Mechanically ventilated patients in hospitals are subjected to an increased risk of acquiring nosocomial pneumonia that sometimes has a lethal outcome. One way to minimize the risk could be to make the surfaces on endotracheal tubes antibacterial. In this study, bacterial growth was inhibited or completely prevented by silver ions wet chemically and deposited onto the tube surface. Through the wet chemical treatment developed here, a surface precipitate was formed containing silver chloride and a silver stearate salt. The identity and morphology of the surface precipitate was studied using x-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, scanning electron microscopy, and x-ray powder diffraction. Leaching of silver ions into solution was examined, and bacterial growth on the treated surfaces was assayed using Pseudomonas aeruginosa wild type (PAO1) bacteria. Furthermore, the minimum inhibitory concentration of silver ions was determined in liquid- and solid-rich growth medium as 23 and 18 microM, respectively, for P. aeruginosa.

Identification of biologic markers of the premature rupture of fetal membranes: proteomic approach.

In obstetrics, premature rupture of the membranes (PROM) is a frequent observation which is responsible for many premature deliveries. PROM is also associated with an increased risk of fetal and maternal infections. Early diagnosis is mandatory in order to decrease such complications. Despite that current biological tests allowing the diagnosis of PROM are both sensitive and specific, contamination of the samples by maternal blood can induce false positive results. Therefore, in order to identify new potential markers of PROM (present only in amniotic blood, and absent in maternal blood), proteomic studies were undertaken on samples collected from six women at terms (pairs of maternal plasma and amniotic fluid) as well as on four samples of amniotic fluid collected from other women at the 17(th) week of gestation. All samples (N = 16) were analyzed by two-dimensional (2-D) high-resolution electrophoresis, followed by sensitive silver staining. The gel images were studied using bioinformatic tools. Analyses were focused on regions corresponding to pI between 4.5 and 7 and to molecular masses between 20 and 50 kDa. In this area, 646 +/- 113 spots were detected, and 27 spots appeared to be present on the gels of amniotic fluid, but were absent on those of maternal plasma. Nine out of these 27 spots were also observed on the gels of the four samples of amniotic fluids collected at the 17(th) week of pregnancy. Five of these 9 spots were unambiguously detected on preparative 2-D gels stained by Coomassie blue, and were identified by mass spectrometry analyses. Three spots corresponded to fragments of plasma proteins, and 2 appeared to be fragments of proteins not known to be present in plasma. These 2 proteins were agrin (SWISS-PROT: O00468) and perlecan (SWISS-PROT: P98160). Our results show that proteomics is a valuable approach to identify new potential biological markers for future PROM diagnosis.

Un partenariat public-privé rigide ou flexible ? Théorie et application aux concessions routières à péage

In this article, we explore the contractual design of toll road concession contracts. We highlight the fact that contracting parties try to sign not only complete rigid contracts in order to avoid renegotiations but also flexible contracts in order to adapt contractual framework to contingencies and to create incentives for cooperative behavior. This gives rise to multiple toll adjustment provisions and to a tradeoff between rigid and flexible contracts. Such a tradeoff is formalized using an incomplete contract framework - including ex post maladaptation and renegotiation costs - and propositions are tested using an original database of 71 concession contracts. Our results suggest an important role for economic efficiency concerns, as well as politics, in designing such public-private contracts. Codes JEL : D23, H11, H54, L14, L9.

Monoclonal antibody against carcinoembryonic antigen (CEA) identifies two new forms of crossreacting antigens of molecular weight 90,000 and 160,000 in normal granulocytes.

Two new forms of non-specific crossreacting antigens (NCAs) were identified in the Nonidet P40 (NP-40) extracts of normal granulocytes by precipitation with the monoclonal antibody (MAb) 192 directed against carcinoembryonic antigen (CEA) and already known to crossreact with the perchloric acid soluble NCA-55. The NP-40 soluble NCAs recognized by MAb 192 have apparent mol. wts of 90,000 and 160,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both NCAs appear to consist of a single monomeric polypeptide chain, since they have the same electrophoretic mobility in SDS-PAGE under reduced and non-reduced conditions. When granulocytes were extracted with perchloric acid instead of NP-40, only the 55,000 mol. wt antigen, corresponding to the previously described NCA-55, was precipitated by MAb 192. Furthermore, it was shown that NCA-55 is not a degradation product of NCA-90 or NCA-160 due to the perchloric acid treatment because exposure to perchloric acid of NCA preparations purified from NP-40 extracts did not change their apparent mol. wts in SDS-PAGE. It was also shown that NCA-160 is not a granulocytic form of CEA because it was not precipitated by the MAb 35 reacting exclusively with CEA. Immunocytochemical studies of granulocytes and macrophages showed that MAb 192 stained both types of cells whereas MAb 47 stained only the granulocytes and MAb 35 none of these cells. In granulocytes both MAbs reacted with antigens associated with granules and also present at the periphery of the nucleus as well as in the Golgi apparatus. The NCA-90 identified by MAb 192 was found by sequential immunodepletion to be antigenically distinct from the NCA-95 precipitated by MAb 47. The epitope recognized by MAb 192 on CEA and NCA molecules appears to be on the peptidic moiety because the antigens deglycosylated by the enzyme Endo F were still precipitated by this MAb. Taken together, the results indicate that MAb 192 identifies two novel forms of NCA (NCA-90 and NCA-160) in NP-40 extracts of granulocytes, which are distinct from CEA and the previously described NCA-55 and NCA-95 identified by MAbs 192 and 47, respectively, in perchloric acid extracts of granulocytes.

Silver-assisted laser desorption ionization for high spatial resolution imaging mass spectrometry of olefins from thin tissue sections.

Silver has been demonstrated to be a powerful cationization agent in mass spectrometry (MS) for various olefinic species such as cholesterol and fatty acids. This work explores the utility of metallic silver sputtering on tissue sections for high resolution imaging mass spectrometry (IMS) of olefins by laser desorption ionization (LDI). For this purpose, sputtered silver coating thickness was optimized on an assorted selection of mouse and rat tissues including brain, kidney, liver, and testis. For mouse brain tissue section, the thickness was adjusted to 23 ± 2 nm of silver to prevent ion suppression effects associated with a higher cholesterol and lipid content. On all other tissues, a thickness of at 16 ± 2 nm provided the best desorption/ionization efficiency. Characterization of the species by MS/MS showed a wide variety of olefinic compounds allowing the IMS of different lipid classes including cholesterol, arachidonic acid, docosahexaenoic acid, and triacylglyceride 52:3. A range of spatial resolutions for IMS were investigated from 150 μm down to the high resolution cellular range at 5 μm. The applicability of direct on-tissue silver sputtering to LDI-IMS of cholesterol and other olefinic compounds presents a novel approach to improve the amount of information that can be obtained from tissue sections. This IMS strategy is thus of interest for providing new biological insights on the role of cholesterol and other olefins in physiological pathways or disease.

Microvascular changes in late-life schizophrenia and mood disorders: stereological assessment of capillary diameters in anterior cingulate cortex.

AIMS: Previous neuroimaging reports described morphological and functional abnormalities in anterior cingulate cortex (ACC) in schizophrenia and mood disorders. In earlier neuropathological studies, microvascular changes that could affect brain perfusion in these disorders have rarely been studied. Here, we analysed morphological parameters of capillaries in this area in elderly cases affected by these psychiatric disorders. METHODS: We analysed microvessel diameters in the dorsal and subgenual parts of the ACC in eight patients with schizophrenia, 10 patients with sporadic bipolar disorder, eight patients with sporadic major depression, and seven age- and gender-matched control cases on sections stained with modified Gallyas silver impregnation using a stereological counting approach. All individuals were drug-naïve or had received psychotropic medication for less than 6 months, and had no history of substance abuse. Statistical analysis included Kruskal-Wallis group comparisons with Bonferroni correction as well as multivariate regression models. RESULTS: Mean capillary diameter was significantly decreased in the dorsal and subgenual parts of areas 24 in bipolar and unipolar depression cases, both in layers III and V, whereas schizophrenia patients were comparable with controls. These differences persisted when controlling for age, local neuronal densities, and cortical thickness. In addition, cortical thickness was significantly smaller in both layers in schizophrenia patients. CONCLUSIONS: Our findings indicate that capillary diameters in bipolar and unipolar depression but not in schizophrenia are reduced in ACC. The significance of these findings is discussed in the light of the cytoarchitecture, brain metabolism and perfusion changes observed in ACC in mood disorders.

Komik, Ernst und Mise en page. Zum Problem der Farblinien in Heinrich Wittenwilers "Der Ring"

Der Aufsatz nähert sich dem Interpretationsproblem des ,Ring', eines seit Forschungsbeginn ausserordentlich konträr diskutierten Textes, aus wissenschaftsanalytischer Perspektive. Er führt die Zersplitterung und die Extremisierung der Deutungsansätze, die die breite Forschung zu Wittenwilers Werk kennzeichnet, auf einen vergleichsweise simplen Befund zurück: die ungeklärte Frage nach dem Verhältnis von Komik und Didaktik und der Ernsthaftigkeit des lehrhaften Angebots im ,Ring'. Die so auf grundlegendster Ebene offen gebliebene Erfassung des Textes führt in einen interpretatorischen Leerraum, in dem die widersprüchlichsten, oft wissenschaftsideologisch begründeten Positionen Platz finden. Ihre Analyse zeigt, dass sie sich in wiederkehrende Gruppierungen ordnen. Als Schlüsselglied sowohl der Befunddaten als auch der konträren Vernetzungen von Befund und Deutung wird die wechselnd rote und grüne Initialenlinie der Münchner Handschrift identifiziert. Als im Prolog eingeführte Markierung von Ernst auf der einen und Komik auf der anderen Seite ist sie nicht nur visueller Ausdruck des Deutungsproblems des ,Ring', sondern sie hat es wegen der scheinbaren Unstimmigkeit ihrer Zuweisungen auch wesentlich bedingt. Der Aufsatz zeigt ihren bisher übersehenen Aufschluss für das Verständnis des Werks. Nimmt man die Linie als graphisches Verfahren ernst, stellt sie sich als strukturelle Markierung zum Auffinden von Text im diskontinuierlichen Zugriff dar - nicht aber, wie man sie bisher ausnahmslos verstand, als semantische Kommentierung eines laufenden, kontinuierlich zu lesenden Textes. Die strukturierende Funktion des Farbwechsels ist auf zwei Ebenen nachzuweisen: Auf Makroebene trennen seine Zuweisungen vorwiegend narrative und vorwiegend wissensvermittelnde Grosspartien des ,Ring' durch eine jeweilige Grundfarbe. Auf Mikroebene wird diese Grundfarbe durch die jeweils andere Farbe durchbrochen, um formale Einschnitte wie etwa Sprecherwechsel, Ortswechsel, Handlungsneueinsätze, Beginn und Ende eines eingeschalteten Binnentextes oder einer Sentenz anzuzeigen. Dem Benutzer der autornahen Handschrift sollte so in einem ersten Schritt (makrostrukturell) ermöglicht werden, gezielt auf gewünschte Stellen des Textes zuzugreifen und ihn in einem zweiten Schritt (mikrostrukturell) schneller zu erfassen. Nicht in seiner Umsetzung, sehr wohl aber in seiner Funktion steht dieses ungewöhnliche Layout zeitgenössischen Techniken der Buchgliederung durchaus nahe. Auch im Profil der Zusammenstellung seiner Binnentexte rückt der ,Ring' damit in die Nähe der im Spätmittelalter beliebten Sammelhandschriften mit Ziel einer Wissenssumme, die hier narrativ verbundenen wird. Es scheint, als möchte der ,Ring' in einem ungewöhnlichen Experiment beides sein: Kompilation und Werkganzes, Wissenssammlung und Erzählwerk - Texte und Text. Als Ergebnis einer Neuuntersuchung des Münchner codex unicus des ,Ring' weist der Beitrag schliesslich darauf hin, dass auch zahlreiche Markierungszeichen am Spaltenrand formale und strukturelle Texteinschnitte anzeigen und somit die gleiche Funktion haben wie der Farbwechsel auf Mikroebene. Ein kausaler, produktionstechnischer Zusammenhang zwischen diesen Zeichen und der Verteilung des Farbwechsels ist nicht auszuschliessen, zumal beide Verfahren etwa im letzten Fünftel des Textes zunehmend zusammenfallen. Möglicherweise war nur die makrostrukturelle Funktion des Farbwechsel ursprünglich indendiert und mit der Erläuterung im Prolog bezeichnet, während seine mikrostrukturelle Funktion erst Resultat des Abschreibprozesses ist.