Ultrastructural analysis of primary human urethral epithelial cell cultures infected with Neisseria gonorrhoeae

In men with gonococcal urethritis, the urethral epithelial cell is a site of infection. To study the pathogenesis of gonorrhea in this cell type, we have developed a method to culture primary human urethral epithelial cells obtained at the time of urologic surgery. Fluorescent analysis demonstrated that 100% of the cells stained for keratin. Microscopic analyses indicated that these epithelial cells arrayed in a pattern similar to that seen in urethral epithelium. Using immunoelectron and confocal microscopy, we compared the infection process seen in primary cells with events occurring during natural infection of the same cell type in men with gonococcal urethritis. Immunoelectron microscopy studies of cells infected with Neisseria gonorrhoeae 1291 Opa+ P+ showed adherence of organisms to the epithelial cell membrane, pedestal formation with evidence of intimate association between the gonococcal and the epithelial cell membranes, and intracellular gonococci present in vacuoles. Confocal studies of primary urethral epithelial cells showed actin polymerization upon infection. Polyclonal antibodies to the asialoglycoprotein receptor (ASGP-R) demonstrated the presence of this receptor on infected cells in the primary urethral cell culture. In situ hybridization using a fluorescent-labeled probe specific to the ASGP-R mRNA demonstrated this message in uninfected and infected cells. These features were identical to those seen in urethral epithelial cells in exudates from males with gonorrhea. Infection of primary urethral cells in culture mimics events seen in natural infection and will allow detailed molecular analysis of gonococcal pathogenesis in a human epithelial cell which is commonly infected.     

Characterization of the enzymatic and nonenzymatic reaction of 13-oxooctadecadienoic acid with glutathione

The enzymatic oxygenation of linoleic acid leads to the production of 13-hydroxyoctadecadienoic acid (13-HODE). Subsequent dehydrogenation of 13-HODE by the NAD(+)-dependent 13-HODE dehydrogenase results in the formation of the 2,4-dienone 13-oxooctadecadienoic acid (13-OXO). These oxidized derivatives of linoleic acid have been shown to be involved in several cellular regulatory processes. In the present study, we have examined the enzymatic and nonenzymatic reaction of 13-OXO with glutathione (GSH) and N-acetylcysteine (N-AcCySH). Nonenzymatic reaction rates were determined spectrophotometrically and exhibited a pH optimum of 9.0 which is consistent with attack of a thiolate anion. Product formation was evaluated by reverse-phase HPLC which showed formation of one major product upon reaction with either GSH or N-AcCySH. The HPLC-purified products were examined by FAB MS as well as one- and two-dimensional NMR. The products, with either GSH or N-AcCySH, were found to consist of an equal mixture of two diastereomers arising from addition of a thiolate to the 9 position of 13-OXO. Using GSH as the thiol, the reaction was also shown to be catalyzed by rat glutathione transferase 8-8. In the case of the enzymatic reaction there is stereoselective product formation. Furthermore, submicromolar concentrations of the 13-OXO-GSH conjugate were shown to significantly inhibit glutathione transferase activity in HT-29 homogenates. These investigations provide insight into the potential metabolic disposition of linoleate oxygenation products.     

Effects of administration of the chemoprotective agent oltipraz on CYP1A and CYP2B in rat liver and rat hepatocytes in culture

The success of oltipraz (OPZ) [5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione] as a chemoprotective agent against aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in the rat is thought to depend principally on its ability to enhance detoxication by inducing phase II enzymes, especially glutathione transferases. However, in primary cultures of human hepatocytes, we recently demonstrated that OPZ also has an important inhibitory effect on the major cytochromes P450 (CYPs) of human hepatic AFB1 metabolism. This has prompted a detailed study of the effect of OPZ on some CYPs involved in metabolism of AFB1 in the rat. Primary cultures of rat hepatocytes behaved similarly to human hepatocytes and responded to OPZ by inhibition of ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-depentylase (PROD) activities mainly associated, respectively, with CYP1A and CYP2B. A time-course shows that this inhibition is largely reversible, with EROD and PROD activities reaching a minimum at 12 h and tending towards control values within 24 h. As is to be expected, the incubation of isolated microsomes with OPZ also inhibits CYP1A and 2B. The effect of OPZ on CYP1A is not a phenomenon limited to cells in culture, but also occurs in vivo. Using the whole animal, we were able to demonstrate that OPZ also transiently inhibited CYP1A activity in a rat given caffeine, by measuring the amounts of methylxanthines found in the serum. However, microsomes isolated from rats, that had been treated with OPZ in vivo, show no such inhibition, presumably because, since OPZ is a reversible inhibitor, it dissociates and is lost during the course of conventional procedures of microsomal preparation. This explains some earlier failures in studies of isolated microsomes to observe the inhibition of CYPs by OPZ. In addition to inhibiting their enzymatic activity, OPZ is also an inducer of CYP1A and 2B as shown by the increased levels of their mRNAs and of caffeine metabolism in vivo after 24 h or more. It is concluded that the mechanism of chemoprotection by OPZ, of toxic chemical metabolism in the rat, is complex and involves competitive inhibition of activation succeeded by induction of the enzymes of both activation and detoxication.     

Silent versus symptomatic myocardial ischemia: The role of psychological and medical factors.

This study examined the relationship of psychological, cardiac, and general medical history factors to asymptomatic (silent) versus symptomatic myocardial ischemia among 102 patients who underwent treadmill exercise testing and had perfusion imaging indicative of ischemia. During exercise, 68 patients exhibited silent ischemia, and 34 experienced chest pain. Patients with silent ischemia rated higher than symptomatic patients on anger control, externally oriented thinking, and somatosensory amplification, but did not differ on depression or global alexithymia. Anger control and externally oriented thinking remained independent correlates in multivariate analysis, controlling for demographic and cardiac factors. Groups did not differ on general medical or cardiac variables. Thus, this study suggests that affective and cognitive factors, but not biomedical factors, are associated with silent, as opposed to symptomatic, ischemia during exercise testing. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Generation of 4 ps light pulses from directly modulated V-groove lasers

4 ps light pulses at repetition rates up to 10 MHz with an output power of some milliwatts are generated at 840 nm by directly modulating commercial GaAlAs V-groove lasers by specially designed avalanche generators. The pulses are measured using an ultrafast streak camera and present the fastest light pulses obtained until now by direct modulation of a semiconductor laser. The avalanche generators produce pulses of 125?200 ps FWHM with voltages up to 25 and 38 V, respectively.     

How We Make DNA Origami

DNA origami has attracted substantial attention since its invention ten years ago, due to the seemingly infinite possibilities that it affords for creating customized nanoscale objects. Although the basic concept of DNA origami is easy to understand, using custom DNA origami in practical applications requires detailed know‐how for designing and producing the particles with sufficient quality and for preparing them at appropriate concentrations with the necessary degree of purity in custom environments. Such know‐how is not readily available for newcomers to the field, thus slowing down the rate at which new applications outside the field of DNA nanotechnology may emerge. To foster faster progress, we share in this article the experience in making and preparing DNA origami that we have accumulated over recent years. We discuss design solutions for creating advanced structural motifs including corners and various types of hinges that expand the design space for the more rigid multilayer DNA origami and provide guidelines for preventing undesired aggregation and on how to induce specific oligomerization of multiple DNA origami building blocks. In addition, we provide detailed protocols and discuss the expected results for five key methods that allow efficient and damage‐free preparation of DNA origami. These methods are agarose‐gel purification, filtration through molecular cut‐off membranes, PEG precipitation, size‐exclusion chromatography, and ultracentrifugation‐based sedimentation. The guide for creating advanced design motifs and the detailed protocols with their experimental characterization that we describe here should lower the barrier for researchers to accomplish the full DNA origami production workflow.     

Multilayer polyimide film substrates for interconnections in microsystems

 Currently there is a great interest in flexible substrates for their use in the packaging of semiconductors. They allow high packaging density and permit mechanical motion of the components, if necessary. This paper describes a new substrate material which possesses a high flexibility and which can be used for the realization of interconnection cables and MCM-D. These substrates are not only highly flexible but also mechanically stable, chemically inert and can be produced with the same processes as those for rigid substrates. Received: 14 December 1998 / Accepted: 28 December 1998     

The pathogenesis of gonococcal urethritis in men: confocal and immunoelectron microscopic analysis of urethral exudates from men infected with Neisseria gonorrhoeae

Confocal and immunoelectron microscopic analysis of urethral exudates from 12 men with gonococcal urethritis showed that Neisseria gonorrhoeae can invade urethral epithelial cells. Studies with acridine orange stain demonstrated that the majority of organisms within urethral epithelial cells were viable at the time of fixation. Three-dimensional modeling of an infected epithelial cell using image analysis of 21 digitized confocal sections stained with YOYO-1 and DiIC 18(3) revealed that gonococcal invasion of these cells occurred in a polar fashion, most likely at the epithelial luminal surface. Serial immunoelectron micrographs showed evidence of membrane fusion with pedestal formation between the gonococcus and the epithelial cell, gonococci within vacuoles, and occasional gonococci free in the cytoplasm. Immunoelectron microscopy studies showed ruptured vacuoles at the cell surface releasing organisms. These studies demonstrate that urethral epithelial cells are invaded by gonococci during the course of infection in males.     

Generation of 4 ps light pulses from directly modulated V-groove lasers

4 ps light pulses at repetition rates up to 10 MHz with an output power of some milliwatts are generated at 840 nm by directly modulating commercial GaAlAs V-groove lasers by specially designed avalanche generators. The pulses are measured using an ultrafast streak camera and present the fastest light pulses obtained until now by direct modulation of a semiconductor laser. The avalanche generators produce pulses of 125?200 ps FWHM with voltages up to 25 and 38 V, respectively.     

Institutions vs. ‘first‐nature’ geography: What drives economic growth in Europe's regions?

The debate on whether institutions or geography prevail in driving economic growth has been rife (e.g., Sachs 2003 vs. Rodrik et al. 2004). Most of the empirical analyses delving into this debate have focused on world countries, whose geographical and institutional conditions differ widely. Subnational analyses considering groups of countries with, in principle, more similar institutional and geographical conditions have been limited and tended to highlight that geography is more important than institutions at subnational level. This paper aims to address whether this is the case by investigating how differences in institutional and ‘first‐nature’ geographical conditions have affected economic growth in Europe's regions in the period 1995–2009. In the analysis we use a newly developed dataset including regional quality of government indicators and geographical characteristics and employ two‐stage least squares (2SLS) and instrumental variables‐generalized method of moments (IV‐GMM) estimation techniques with a number of regional historical variables as instruments. Our results indicate that at a regional level in Europe institutions rule. Regional institutional conditions – and, particularly, government effectiveness and the fight against corruption – play an important role in shaping regional economic growth prospects. This does not imply, however, that geography is irrelevant. There is evidence of geographical factors affecting regional growth, although their impact is dwarfed by the overriding influence of institutions.