Laser Fluorescence Microscopy Research Articles

Glucose stimulates calcium-activated chloride secretion in small intestinal cells

The sodium-coupled glucose transporter-1 (SGLT1)-based oral rehydration solution (ORS) used in the management of acute diarrhea does not substantially reduce stool output, despite the fact that glucose stimulates the absorption of sodium and water. To explain this phenomenon, we investigated the possibility that glucose might also stimulate anion secretion. Transepithelial electrical measurements and isotope flux measurements in Ussing chambers were used to study the effect of glucose on active chloride and fluid secretion in mouse small intestinal cells and human Caco-2 cells. Confocal fluorescence laser microscopy and immunohistochemistry measured intracellular changes in calcium, sodium-glucose linked transporter, and calcium-activated chloride channel (anoctamin 1) expression. In addition to enhancing active sodium absorption, glucose increased intracellular calcium and stimulated electrogenic chloride secretion. Calcium imaging studies showed increased intracellular calcium when intestinal cells were exposed to glucose. Niflumic acid, but not glibenclamide, inhibited glucose-stimulated chloride secretion in mouse small intestines and in Caco-2 cells. Glucose-stimulated chloride secretion was not seen in ileal tissues incubated with the intracellular calcium chelater BAPTA-AM and the sodium-potassium-2 chloride cotransporter 1 (NKCC1) blocker bumetanide. These observations establish that glucose not only stimulates active Na absorption, a well-established phenomenon, but also induces a Ca-activated chloride secretion. This may explain the failure of glucose-based ORS to markedly reduce stool output in acute diarrhea. These results have immediate potential to improve the treatment outcomes for acute and/or chronic diarrheal diseases by replacing glucose with compounds that do not stimulate chloride secretion.

Intra-lymph node injection of biodegradable polymer particles.

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g. size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.

Intra-lymph Node Injection of Biodegradable Polymer Particles

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g. size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.

Seasonal climate signals (1990–2008) in a modern Soreq Cave stalagmite as revealed by high-resolution geochemical analysis

High-resolution isotopic and geochemical analyses in a modern (1990–2008) Soreq Cave stalagmite are compared to instrumental records of rainfall and dripwater from the cave, with the aim of determining how seasonal-resolution climate information is transmitted to speleothem geochemistry. In situ, micron-scale analysis of oxygen isotope ratios (δ18O) and trace elements by ion microprobe in combination with a continuous, linear traverse of trace element concentrations by laser-ablation ICP-MS (LA-ICP-MS) allow the definition of geochemical pathways within the cave. Fluorescent banding, imaged by confocal laser fluorescent microscopy (CLFM), as well as δ18O and trace element variations is used to define 18 annual growth increments. Reduced intensity of fluorescent banding and a change in trace element variability reflect the decrease in average rainfall from 628mm/yr (1990–1998) to 433mm/yr (1999–2008). During the wetter period before 1998, Pearson (r-value) and Spearman (ρ-value) correlation coefficients are >0.5 for ion microprobe analyses of the element pairings Sr–Y, Y–P, and Mn–Si. After the transition to the drier period in 1999, a different set of geochemical pairings have r- and ρ-values >0.5, including Mg–δ18O, Mg–Sr, and Sr–Ba. Principal component analysis of data from the adjacent LA-ICP-MS traverse identifies two primary underlying modes of trace element variability. Based on the ion microprobe correlations and principal component analyses, we suggest that a greater seasonal influx of particulate material into the cave during the wetter period (1990–1998) brought about greater P, Cu, Sr, Na, and U variability in the stalagmite. The co-variability of δ18O, Mg, Sr, and Ba is characteristic of the trace element pattern from the drier period (1999–2008) of growth when particulate transport is reduced. These findings support a two-reservoir model of Soreq Cave dripwaters. One reservoir displays a well-mixed “baseline” with a decadal residence time that supplies water to the cave year-round, probably from fine pores or grain-boundary films in the vadose zone. The second reservoir is seasonal rainwater enriched in organic acids, colloids, and small particles and is rapidly transmitted to the cave. Finally, the similar patterns of fluorescence intensity, P, and Cu concentrations support the hypothesis that fluorescent bands in Soreq Cave speleothems are caused by the influx of organic colloids.

Method of confocal laser fluorescence microscopy for the control of bone marrow cells

The paper shows the use of confocal laser microscopy for the assessing of viability of mesenchymal bone marrow stromal cells of a rabbit with the resolution of 400 nanometers. A three-dimensional structure of cells is obtained by this method.

Abstract 5531: A bioluminescent cell viability assay optimized for 3D microtissues.

Abstract It has been well demonstrated that the morphology, gene expression, and overall biological response of cells assessed in 3-dimensional (3D) culture models are frequently more physiologically relevant than that of cells studied in standard 2D culture formats. In spite of rapidly growing interest, one hindrance to the use of 3D models for drug discovery and other research efforts is the lack of convenient and effective assays explicitly validated for application to 3D microtissues. Here we report on a bioluminescent ATP-detection assay comprised of both an improved formulation and an optimized protocol specifically designed to measure the viability of cells grown in 3D culture. The ability of the 3D-optimized reagent to effectively lyse cells from throughout the full thickness of 3D microtissues, including cancer cell spheroids, was clearly demonstrated by use of confocal laser fluorescent microscopy in conjunction with a fluorogenic, membrane-impermeant DNA-binding dye. Importantly, the assay's ability to accurately report the ATP content, and therefore viability, of various 3D microtissues (including a range of cancer cell line spheroids) was demonstrated by comparison to a trichloroacetic acid ATP extraction method. In scaffold-free as well as scaffold-dependent 3D models, recovery and detection of greater than 80% of ATP present was routinely observed. In contrast, another frequently used ATP-detection assay was found to have greatly reduced lytic and ATP detection properties (<50% recovery). This new, homogeneous 3D-assay format requires minimal sample manipulation, a brief incubation period (generally less than 30 min), and exhibits excellent sensitivity. It should be amenable to both low- and high-throughput applications and accurately report drug sensitivities of cells assayed in both 2D and 3D culture models. Citation Format: Michael P. Valley, Chad Zimprich, James J. Cali, Dan F. Lazar. A bioluminescent cell viability assay optimized for 3D microtissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5531. doi:10.1158/1538-7445.AM2013-5531

Phosphodiesterase type 1, calcitonin gene-related peptide and vasoactive intestinal polypeptide are involved in the control of human vaginal arterial vessels

ObjectiveThe vagina makes a major contribution to the normal female sexual response cycle. An increase in vaginal blood flow is considered a key event in the mechanism of sexual arousal. Recent research has focused mainly on the cyclic GMP pathway and phosphodiesterase type 5 (PDE5, cyclic GMP specific PDE) in the control of vaginal vascular smooth muscle, whereas only little is known on the role of other key proteins and mediators of cyclic nucleotide mediated signaling in this process. The aim of the present study was to evaluate in the human vagina, by means of immunohistochemistry, the expression and distribution of phosphodiesterase type 1 (PDE1, known to hydrolize both cyclic AMP and cyclic GMP) in relation to calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and protein gene product 9.5 (PGP 9.5). Study designSections of human vagina (full wall specimens) were incubated with antibodies directed against PDE1, CGRP, VIP, PGP 9.5 and alpha-actin, followed by exposure to fluorochrome-labelled secondary antibodies. Visualization was commenced by means of laser fluorescence microscopy. ResultsMicroscopic examination revealed a dense meshwork of PGP 9.5-positive nerve fibers innervating the sections of vaginal wall. Small vessels interspersing the tissue presented dense staining for PDE1 in their smooth musculature. Blood vessels were seen surrounded by PDE1-immunoreactive longitudinal smooth muscle fibers. The vessels were also found innervated by PGP-positive varicose nerve fibers characterized by the expression of CGRP. Some fibers presented immunosignals specific for VIP. ConclusionKey mediators of the cyclic AMP and cyclic GMP pathways are co-localized in nerves seen in close proximity to vascular smooth muscle expressing PDE1. These findings suggest that both signaling cascades are involved in the control of vaginal blood flow.

Single-Molecule Lactonization of Octadecylrhodamine B at a Liquid–Liquid Interface

The total internal reflection laser fluorescence microscope method was used to observe the lateral diffusion of a single octadecylrhodamine B (C(18)RB) molecule at the toluene-water interface. The interfacial diffusion constant of single fluorescent cation C(18)RB(+) was obtained from the maximum residence time in a small observation area with pH <2. For pH >3, the maximum residence time was remarkably shortened, indicating that single fluorescent zwitterion C(18)RB(±) rapidly converted to the nonfluorescent lactone at the interface. The lactonization rate was completed within 0.13 ms at the toluene-water interface but slowed to 67 ms at an interface saturated with dimyristoylphosphatidylcholine.

Multiphoton microscopy imaging of developing tooth germs

Traditionally, tooth germ is observed by histological investigation with hematoxylin and eosin stain and information may loss during the process. The purpose of this study is to use multiphoton laser fluorescence microscopy to observe the developing tooth germs of mice for building up the database of the images of tooth germs and compare with those from conventional histological analysis. Tooth germs were isolated from embryonic and newborn mice with age of Embryonic Day 14.5 and Postnatal Days 1, 3, 5, and 7. Comparison of the images of tooth germ sections in multiphoton microscopy with the images of histology was performed for investigating the molar tooth germs. It was found that various signals arose from different structures of tooth germs. Pre-dentin and dentin have strong second-harmonic generation signals, while ameloblasts and enamel tissues were shown with strong autofluorescence signals. In this study, a novel multiphoton microscopy database of images from developing tooth germs in mice was set up. We confirmed that multiphoton laser microscopy is a powerful tool for investigating the development of tooth germ and is worthy for further application in the study of tooth regeneration.

Expression and Distribution of Phosphodiesterase Isoenzymes in the Human Seminal Vesicles

Phosphodiesterase (PDE) isoenzymes have been shown to play a role in the control of human male genital tissues. There are hints from basic research and clinical studies that PDE5 inhibitors may have the ability to retard the male ejaculatory response. While the expression of PDE isoenzymes in the human seminal vesicles (SVs) has been described, the distribution of cyclic adenosine monophosphate (AMP)- and cyclic guanosine monophosphate (GMP)-PDEs has not yet been investigated. The aim of this study was to elucidate the expression and distribution of PDE isoenzymes PDE3A, PDE4 (isoforms A and B), PDE5A, and PDE11A in human SV tissue. Using immunohistochemistry (double-labeling techniques, laser fluorescence microscopy), the occurrence of PDE3A, PDE4A, PDE4B, PDE5A, and PDE11A, the vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), and protein gene product 9.5 (PGP 9.5) was examined in sections of SV. Cytosolic supernatants prepared from isolated human SV tissue were subjected to Western blot analysis using specific anti-PDE antibodies. The expression and distribution by of PDE3A, PDE4A, PDE4B, PDE5A, and PDE11A in the human SV were investigated by means of immunohistochemistry and Western blot analysis. Immunosignals specific for PDE3A were seen in both the smooth muscle and the glandular epithelium, whereas staining for PDE4A, PDE5A, and PDE11A was mainly limited to epithelial cells. Varicose nerve fibers transversing the sections also presented staining for PDE3A. In nerve fibers and nerve endings, PDE4A and PDE4B were found co-localized with VIP; PDE5A-positive nerves also presented immunosignals specific for CGRP. The expression of said PDE isoenzymes was confirmed by Western blotting. The results indicate that cyclic AMP- and cyclic GMP-PDE isoenzymes are involved in the control of secretory activity and efferent neurotransmission in the SV. These findings might be of importance with regard to the identification of new therapeutic avenues to treat premature ejaculation.

Functional states of kinetochores revealed by laser microsurgery and fluorescent speckle microscopy

The impact of mechanical forces on kinetochore motility was investigated using laser microsurgery to detach kinetochores with associated chromatin (K fragment) from meiotic chromosomes in spermatocytes from the crane fly Nephrotoma suturalis. In spermatocytes, elastic tethers connect telomeres of homologues during anaphase A of meiosis I, thus preventing complete disjunction until mid- to late anaphase A. K fragments liberated from tethered arms moved at twice the normal velocity toward their connected poles. To assess functional states of detached and control kinetochores, we loaded cells with fluorescently labeled tubulin for fluorescent speckle microscopy on kinetochore microtubules. Control kinetochores added fluorescent speckles at the kinetochore during anaphase A, whereas kinetochores of K fragments generally did not. In cases in which speckles reappeared in K-fragment K fibers, speckles and K fragments moved poleward at similar velocities. Thus detached kinetochores convert from their normal polymerization (reverse pac-man) state to a different state, in which polymerization is not evident. We suggest that the converted state is "park," in which kinetochores are anchored to plus ends of kinetochore microtubules that shorten exclusively at their polar ends.

In Situ Recording of Particle Network Formation in Liquids by Ion Conductivity Measurements

The formation of fractal silica networks from a colloidal initial state was followed in situ by ion conductivity measurements. The underlying effect is a high interfacial lithium ion conductivity arising when silica particles are brought into contact with Li salt-containing liquid electrolytes. The experimental results were modeled using Monte Carlo simulations and tested using confocal fluorescence laser microscopy and ζ-potential measurements.

In vivo diagnosis of murine pancreatic intraepithelial neoplasia and early-stage pancreatic cancer by molecular imaging

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with poor patient outcome often resulting from late diagnosis in advanced stages. To date methods to diagnose early-stage PDAC are limited and in vivo detection of pancreatic intraepithelial neoplasia (PanIN), a preinvasive precursor of PDAC, is impossible. Using a cathepsin-activatable near-infrared probe in combination with flexible confocal fluorescence lasermicroscopy (CFL) in a genetically defined mouse model of PDAC we were able to detect and grade murine PanIN lesions in real time in vivo. Our diagnostic approach is highly sensitive and specific and proved superior to clinically established fluorescein-enhanced imaging. Translation of this endoscopic technique into the clinic should tremendously improve detection of pancreatic neoplasia, thus reforming management of patients at risk for PDAC.

A method for enhancing the ocular penetration of eye drops using nanoparticles of hydrolyzable dye

This report describes a method for enhancing the ocular penetration of eye drops using nanoparticles of hydrolyzable dye, which is similar to a prodrug approach. The entry of eye drops into the ocular globe is restricted predominantly by corneal barrier functions. The barrier functions are epithelial tight junctions as well as a physicochemical property consisting of the opposite characteristics of a lipophilic epithelium and a hydrophilic stroma. We found that using a formulation of nanoparticles of hydrolyzable dye (with particles of 200 nm in diameter on average) attained a greater than tenfold higher (about 50-fold) ocular penetration than that of micron-sized particles. The nanoparticles were prepared by a carrier-free technique; i.e., the reprecipitation method. Confocal laser fluorescence microscopy showed that dyes originating from the nanoparticles surmounted the corneal epithelium barrier, which has tight junctions, and achieved deeper penetration into the cornea. The high penetration rate of the dyes into the cornea was attributed to the size of particles (i.e., nanoparticles) and a transformation of dye polarity from lipophilic to hydrophilic in in vivo hydrolysis reactions. We concluded that utilizing in vivo hydrolysis reactions to alter the physicochemical nature of nanoparticles consisting of hydrolyzable compounds was an effective approach for enhancing the ocular penetration of eye drops.

Anti-Stokes Fluorescence Spectra of Chloroplasts in Parachlorella kessleri and Maize at Room Temperature as Characterized by Near-Infrared Continuous-Wave Laser Fluorescence Microscopy and Absorption Microscopy

Microscopic autofluorescence spectral imaging of chloroplasts in maize mesophyll cells using near-infrared laser excitation has previously shown that a photosystem I spectral component exhibits an intensity similar to that of photosystem II at ~294 K when a continuous-wave laser at 800-820 nm is used. To establish the generality of this phenomenon, chloroplasts in Parachlorella kessleri cells (P. kessleri) were studied. A continuous-wave laser at 785 nm promoted photosystem-I-specific fluorescence in P. kessleri chloroplasts. The difference in chlorophyll fluorescence peak wavelengths between P. kessleri and maize correlated well with those observed at cryogenic temperatures. To further clarify the nature of anti-Stokes fluorescence, we studied chloroplasts in acetone-treated P. kessleri cells (in a medium containing 15% (v/v) acetone) by microscopic fluorescence and absorption spectra on a cell-by-cell basis. A continuous-wave laser at 785 nm led to significant fluorescence from acetone-treated cells, which was attributed to aggregation of chlorophylls. Anti-Stokes fluorescence spectral imaging thus seems to be effective for detection of lowest-energy trap states that are only weakly fluorescent.

222 EXPRESSION AND DISTRIBUTION OF PHOSPHODIESTERASE (PDE) ISOENZYMES IN THE HUMAN URETHRA

You have accessJournal of UrologyBladder and Urethra: Anatomy, Physiology and Pharmacology1 Apr 2011222 EXPRESSION AND DISTRIBUTION OF PHOSPHODIESTERASE (PDE) ISOENZYMES IN THE HUMAN URETHRA George Kedia, Markus Kuczyk, Petter Hedlund, and Stefan Ückert George KediaGeorge Kedia Hannover, Germany More articles by this author , Markus KuczykMarkus Kuczyk Hannover, Germany More articles by this author , Petter HedlundPetter Hedlund Lund, Sweden More articles by this author , and Stefan ÜckertStefan Ückert Hannover, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.332AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Up untill today, only a very few studies have addressed the physiology/pharmacology of the human urethra (Hassouna M. et al., J. Urol. 129: 1262–1264, 1983; Abdel-Hakim A. et al., J. Urol. 130: 988–991, 1983; Werkström V. et al., BJU Int. 98: 414–423, 2006). An involvement of the nitric oxide (NO)/cyclic GMP pathway in mediating the relaxation of the bladder outlet and urethra during the micturition phase has been supposed. The present study aimed to evaluate in the human male urethra by means of immunohistochemistry the expression and distribution of cyclic AMP and cyclic GMP phosphodiesterase (PDE) isoenzymes in relation to key mediators/proteins of the cyclic GMP and cyclic AMP pathways. METHODS Using conventional immunohistochemistry (double-labelling technique, laser fluorescence microscopy), thin sections (8 μm – 10 μm) of the human male urethra were exposed to anti-PDE1A, -PDE2A, -PDE3A, -PDE4A, -PDE4B, -PDE5A, anti-nNOS (neuronal nitric oxide synthase), -VIP (vasoactive intestinal polypeptide) and -CGRP (calcitonin gene-related peptide) antibodies followed by the application of fluorochrome-labelled secondary antibodies. RESULTS In the smooth muscle (SM) portion of the urethra, immunosignals specific for PDE1A (cyclic AMP/cyclic GMP PDE, calcium/calmodulin-dependent), PDE4A, PDE4B (cyclic AMP PDEs) and PDE5A (cyclic GMP PDE) were registered. SM bundles were seen innervated by varicose nerve fibers characterized by the expression of nNOS. Some of these nerves also presented staining related to VIP or CGRP. No immunoreactivities specific for PDE2A or PDE3A were detected. CONCLUSIONS PDE isoenzymes type 1A, 4A, 4B and 5A are expressed in human urethral smooth muscle. The findings implicate that the cyclic AMP and cyclic GMP pathways work synergistically together in the control of urethral smooth muscle function. This might be of importance with regard to the identification of new pharmacological avenues to facilitate the contraction and relaxation of the human outflow region. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e91 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information George Kedia Hannover, Germany More articles by this author Markus Kuczyk Hannover, Germany More articles by this author Petter Hedlund Lund, Sweden More articles by this author Stefan Ückert Hannover, Germany More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

746 PHOSPHODIESTERASE TYPE 1, THE CALCITONIN-GENE RELATED PEPTIDE AND VASOACTIVE INTESTINAL POLYPEPTIDE ARE INVOLVED IN THE CONTROL OF HUMAN VAGINAL ARTERIAL VESSELS

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research1 Apr 2011746 PHOSPHODIESTERASE TYPE 1, THE CALCITONIN-GENE RELATED PEPTIDE AND VASOACTIVE INTESTINAL POLYPEPTIDE ARE INVOLVED IN THE CONTROL OF HUMAN VAGINAL ARTERIAL VESSELS Stefan Ückert, Matthias Oelke, Markus Kuczyk, and Petter Hedlund Stefan ÜckertStefan Ückert Hannover, Germany More articles by this author , Matthias OelkeMatthias Oelke Hannover, Germany More articles by this author , Markus KuczykMarkus Kuczyk Hannover, Germany More articles by this author , and Petter HedlundPetter Hedlund Milan, Italy More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1771AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The vagina contributes mainly to the normal female sexual response cycle. An increase in vaginal blood flow is considered a key event in the mechanism of sexual arousal. Recent research has focused mainly on the cyclic GMP pathway and the phosphodiesterase type 5 (PDE5, cyclic GMP-specific PDE) in the control of vaginal vascular smooth muscle. In contrast, only little is known on the role of other key proteins and mediators of the cyclic nucleotide mediated signaling in this process. The aim of the present study was to evaluate in the human vagina, by means of immunohistochemistry, the expression and distribution of the phosphodiesterase type 1 (PDE1, known to hydrolize both cyclic AMP and cyclic GMP) in relation to the calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), and protein gene product 9.5 (PGP 9.5). METHODS Cryostat sections of the human vagina (full wall specimens) were incubated with antibodies directed against PDE1, CGRP, VIP, PGP 9.5 and alpha-actin, followed by exposure to fluorochrome-labelled secondary antibodies. Visualization was commenced by means of laser fluorescence microscopy. RESULTS Microscopical examination revealed a dense meshwork of PGP 9.5-positive nerve fibers innervating the sections of the vaginal wall. Small vessels interspersing the tissue presented dense staining for PDE1 in their smooth musculature while no signals were observed in the non-vascular smooth muscle. The vessels were found innervated by PGP-positive varicose nerve fibers characterized by the expression of CGRP. Some fibers also presented immunosignals specific for VIP. CONCLUSIONS Key mediators of the cyclic AMP and cyclic GMP pathways are co-localized in nerves seen in close proximity to vascular smooth muscle expressing PDE1. These findings give hint that both signaling cascades are involved in the control of vaginal blood flow. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e300 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Stefan Ückert Hannover, Germany More articles by this author Matthias Oelke Hannover, Germany More articles by this author Markus Kuczyk Hannover, Germany More articles by this author Petter Hedlund Milan, Italy More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Submicron Polymer Particles Containing Fluorescent Semiconductor Nanocrystals Cdse/Zns for Bioassays

This study aimed to design a panel of uniform particulate biochemical reagents and to test them in specific bioassays. These reagents are polymer particles of different sizes doped with semiconductor nanocrystals and conjugated with either full-size antibodies or recombinant mini-antibodies (4D5 scFv fragment) designed by genetic engineering approaches. A panel of highly fluorescent polymer particles (150-800 nm) were formed by embedding CdSe/ZnS nanocrystals (quantum dots) into preformed polyacrolein and poly(acrolein-co-styrene) particles. Morphology, content and fluorescence characteristics of the prepared materials were studied by laser correlation spectroscopy, spectrophotometry, optical and fluorescent microscopy and fluorimetry. The obtained fluorescent particles sensitized by anti-Yersinia pestis antibodies were used for rapid agglutination glass test suitable for screening analysis of Y. pestis antigen and for microtiter particle agglutination, which, owing to its speed and simplicity, is very beneficial for diagnostic detection of Y. pestis antigen. Recombinant 4D5 scFv antibodies designed and conjugated with polymer particles containing quantum dots provide multipoint highly specific binding with cancer marker HER2/neu on the surface of SKOV-3 cell.

Vitamin E Microspheres Embedded Within a Biocompatible Film for Planar Delivery

AbstractWe demonstrate a new one‐batch approach to the fabrication of a biocompatible Ca‐alginate film with embedded vitamin E‐loaded microspheres that could be used for planar dermal drug delivery. Stable vitamin E microspheres, coated with gum acacia, are produced by ultrasonic treatment of a two‐phase liquid system. The Fourier transform infrared spectroscopy indicates an interaction between biopolymer functional groups induced by ultrasonication. Confocal laser fluorescence microscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM) demonstrate a homogeneous microsphere distribution within the Ca‐alginate polymer film. The kinetics of in vitro vitamin E release found for the polymer film with entrapped microspheres was much more sustained (100% in 96 h) compared to the polymer film with vitamin E embedded in the free state (100% in 5 h). The novelty of the proposed research involves the ultrasonic fabrication of loaded microspheres and formation of biodegradable coating directly doped with microspheres.

Semisynthesis of a Protein with Cholesterol at the C-Terminal, Targeted to the Cell Membrane of Live Cells

Various proteins are modified post-translationally to localize them at the cell membrane. Among them, hedgehog-family proteins are modified by cholesterol at the C-terminal. In this study, green fluorescent protein (GFP) modified with cholesterol (GFP-Chol) at the C-terminal was prepared semisynthetically and investigated. This semi-synthesis was performed using the following native chemical ligation: GFP-Cα-thioester was prepared using the intein-mediated thioester exchange reaction and was ligated to Cys-NH-diethylene glycol-NHCO-cholesterol in the presence of a detergent. After removal of the detergent, the GFP-Chol was applied to mouse live cells. Confocal laser fluorescent microscopy confirmed localization of GFP-Chol at the cell membrane. The findings suggest that modifying proteins with cholesterol at the C-terminal is useful for targeting the proteins to the cell membrane of live cells.