scholarly journals An olive pollen protein with allergenic activity, Ole e 10, defines a novel family of carbohydrate-binding modules and is potentially implicated in pollen germination

2005 ◽  
Vol 390 (1) ◽  
pp. 77-84 ◽  
Author(s):  
Patricia Barral ◽  
Cinthya Suárez ◽  
Eva Batanero ◽  
Carlos Alfonso ◽  
Juan de Dios Alché ◽  
...  
Keyword(s):  
Pollen Tube ◽  
Laser Scanning ◽  
Plant Cell Wall ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Carbohydrate Binding ◽  
Type I ◽  
Sequence Alignments ◽  
Carbohydrate Binding Modules ◽  
Olive Pollen

CBMs (carbohydrate-binding modules) are the most common non-catalytic modules associated with enzymes active in plant cell-wall hydrolysis. They have been frequently identified by amino acid sequence alignments, but only a few have been experimentally established to have a carbohydrate-binding activity. A small olive pollen protein, Ole e 10 (10 kDa), has been described as a major inducer of type I allergy in humans. In the present study, the ability of Ole e 10 to bind several polysaccharides has been analysed by affinity gel electrophoresis, which demonstrated that the protein bound 1,3-β-glucans preferentially. Analytical ultracentrifugation studies confirmed binding to laminarin, at a protein/ligand ratio of 1:1. The interaction of Ole e 10 with laminarin induced a conformational change in the protein, as detected by CD and fluorescence analyses, and an increase of 3.6 °C in the thermal denaturation temperature of Ole e 10 in the presence of the glycan. These results, and the absence of alignment of the sequence of Ole e 10 with that of any classified CBM, indicate that this pollen protein defines a novel family of CBMs, which we propose to name CBM43. Immunolocalization of Ole e 10 in mature and germinating pollen by transmission electron microscopy and confocal laser scanning microscopy demonstrated the co-localization of Ole e 10 and callose (1,3-β-glucan) in the growing pollen tube, suggesting a role for this protein in the metabolism of carbohydrates and in pollen tube wall re-formation during germination.

scholarly journals Quantifying cellulose accessibility during enzyme-mediated deconstruction using 2 fluorescence-tagged carbohydrate-binding modules

2019 ◽  
Vol 116 (45) ◽  
pp. 22545-22551 ◽  
Author(s):  
Vera Novy ◽  
Kevin Aïssa ◽  
Fredrik Nielsen ◽  
Suzana K. Straus ◽  
Peter Ciesielski ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Degradation Mechanism ◽  
Length Distribution ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Distribution Analysis ◽  
Cellulose Accessibility ◽  
Carbohydrate Binding Modules

Two fluorescence-tagged carbohydrate-binding modules (CBMs), which specifically bind to crystalline (CBM2a-RRedX) and paracrystalline (CBM17-FITC) cellulose, were used to differentiate the supramolecular cellulose structures in bleached softwood Kraft fibers during enzyme-mediated hydrolysis. Differences in CBM adsorption were elucidated using confocal laser scanning microscopy (CLSM), and the structural changes occurring during enzyme-mediated deconstruction were quantified via the relative fluorescence intensities of the respective probes. It was apparent that a high degree of order (i.e., crystalline cellulose) occurred at the cellulose fiber surface, which was interspersed by zones of lower structural organization and increased cellulose accessibility. Quantitative image analysis, supported by 13C NMR, scanning electron microscopy (SEM) imaging, and fiber length distribution analysis, showed that enzymatic degradation predominates at these zones during the initial phase of the reaction, resulting in rapid fiber fragmentation and an increase in cellulose surface crystallinity. By applying this method to elucidate the differences in the enzyme-mediated deconstruction mechanisms, this work further demonstrated that drying decreased the accessibility of enzymes to these disorganized zones, resulting in a delayed onset of degradation and fragmentation. The use of fluorescence-tagged CBMs with specific recognition sites provided a quantitative way to elucidate supramolecular substructures of cellulose and their impact on enzyme accessibility. By designing a quantitative method to analyze the cellulose ultrastructure and accessibility, this study gives insights into the degradation mechanism of cellulosic substrates.

Localization of angiotensin II receptor subtypes AT1 and AT2 in the pancreas of rodents

1997 ◽  
Vol 153 (2) ◽  
pp. 269-274 ◽  
Author(s):  
P S Leung ◽  
H C Chan ◽  
L X M Fu ◽  
P Y D Wong
Keyword(s):  
Angiotensin Ii ◽  
Laser Scanning ◽  
Renin Angiotensin System ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Receptor Subtypes ◽  
Similar Distribution ◽  
Type I ◽  
Angiotensin Ii Receptor ◽  
Angiotensin Ii Receptor Subtypes

Abstract Previous studies have demonstrated the existence of several key components of the renin–angiotensin system in the pancreas. In the present study, the localization of angiotensin II receptor subtypes, type I (AT1) and type II (AT2), in the mouse and the rat pancreas was studied by immunocytochemistry using specific antipeptide antibodies against the second extracellular loops of AT1 and AT2 receptors in conjunction with confocal laser scanning microscopy. In the mouse, immunoreactivity for AT1 and AT2 was observed predominantly in the endothelia of the blood vessels and the epithelia of the pancreatic ductal system. Similar distribution of immunoreactivity for AT1 and AT2 was also observed. However, the intensity of immunoreactivity for AT1 and AT2 was stronger in the rat than that found in the mouse pancreas. Much weaker immunostaining for both AT1 and AT2, as compared with that found in ductal regions, was also found in the acini of the rodent pancreas. Together with the previous findings, the present results suggest that AT1 and/or AT2 receptors may play a role in regulating pancreatic functions in the rodent. Journal of Endocrinology (1997) 153, 269–274

scholarly journals Water transport and the distribution of aquaporin-1 in pulmonary air spaces

1997 ◽  
Vol 83 (3) ◽  
pp. 1002-1016 ◽  
Author(s):  
R. M. Effros ◽  
C. Darin ◽  
E. R. Jacobs ◽  
R. A. Rogers ◽  
G. Krenz ◽  
...  
Keyword(s):  
Pulmonary Artery ◽  
Water Transport ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Pulmonary Vasculature ◽  
Confocal Laser ◽  
Alveolar Type ◽  
Type I ◽  
Pulmonary Endothelium ◽  
Aquaporin 1

Effros, R. M., C. Darin, E. R. Jacobs, R. A. Rogers, G. Krenz, and E. E. Schneeberger. Water transport and the distribution of aquaporin-1 in pulmonary air spaces. J. Appl. Physiol. 83(3): 1002–1016, 1997.—Recent evidence suggests that water transport between the pulmonary vasculature and air spaces can be inhibited by HgCl2, an agent that inhibits water channels (aquaporin-1 and -5) of cell membranes. In the present study of isolated rat lungs, clearances of labeled (3HOH) and unlabeled water were compared after instillation of hypotonic or hypertonic solutions into the air spaces or injection of a hypotonic bolus into the pulmonary artery. The clearance of 3HOH between the air spaces and perfusate after intratracheal instillation and from the vasculature to the tissues after pulmonary arterial injections was invariably greater than that of unlabeled water, indicating that osmotically driven transport of water is limited by permeability of the tissue barriers rather than the rate of perfusion. Exposure to 0.5 mM HgCl2 in the perfusate and air-space solution reduced the product of the filtration coefficient and surface area ( P f S) of water from the air spaces to the perfusate by 28% after instillation of water into the trachea. In contrast, perfusion of 0.5 mM HgCl2 in air-filled lungs reduced P f Sof the endothelium by 86% after injections into the pulmonary artery, suggesting that much of the action of this inhibitor is on the endothelial surfaces. Confocal laser scanning microscopy demonstrated that aquaporin-1 is on mouse pulmonary endothelium. No aquaporin-1 was found on alveolar type I cells with immunogold transmission electron microscopy, but small amounts were present on some type II cells.

scholarly journals In Vitro Studies on Mg-Zn-Sn-Based Alloys Developed as a New Kind of Biodegradable Metal

Materials ◽  
2021 ◽  
Vol 14 (7) ◽  
pp. 1606
Author(s):  
Yafeng Wen ◽  
Qingshan Liu ◽  
Weikang Zhao ◽  
Qiming Yang ◽  
Jingfeng Wang ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Corrosion Resistance ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Cell Counting ◽  
Confocal Laser ◽  
Second Phase ◽  
Dead Cell ◽  
Type I ◽  
Alizarin Red

Mg-Zn-Sn-based alloys are widely used in the industrial field because of their low-cost, high-strength and heat-resistant characteristics. However, their application in the biomedical field has been rarely reported. In the present study, biodegradable Mg-1Zn-1Sn and Mg-1Zn-1Sn-0.2Sr alloys were fabricated. Their microstructure, surface characteristics, mechanical properties and bio-corrosion properties were carried out using an optical microscope (OM), X-ray diffraction (XRD), electron microscopy (SEM), mechanical testing, electrochemical and immersion test. The cell viability and morphology were studied by cell counting kit-8 (CCK-8) assay, live/dead cell assay, confocal laser scanning microscopy (CLSM) and SEM. The osteogenic activity was systematically investigated by alkaline phosphatase (ALP) assay, Alizarin Red S (ARS) staining, immunofluorescence staining and quantitative real time-polymerase chain reaction (qRT-PCR). The results showed that a small amount of strontium (Sr) (0.2 wt.%) significantly enhanced the corrosion resistance of the Mg-1Zn-1Sn alloy by grain refinement and decreasing the corrosion current density. Meanwhile, the mechanical properties were also improved via the second phase strengthening. Both Mg-1Zn-1Sn and Mg-1Zn-1Sn-0.2Sr alloys showed excellent biocompatibility, significantly promoted cell proliferation, adhesion and spreading. Particularly, significant increases in ALP activity, ARS staining, type I collagen (COL-I) expression as well as the expressions of three osteogenesis-related genes (runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (Bglap)) were observed for the Mg-1Zn-1Sn-0.2Sr group. In summary, this study demonstrated that Mg-Zn-Sn-based alloy has great application potential in orthopedics and Sr is an ideal alloying element of Mg-Zn-Sn-based alloy, which optimizes its corrosion resistance, mechanical properties and osteoinductive activity.

scholarly journals Nanomechanical Subsurface Characterisation of Cellulosic Fibres

2021 ◽  
Author(s):  
Julia Auernhammer ◽  
Markus Langhans ◽  
Jan-Lukas Schäfer ◽  
Tom Keil ◽  
Tobias Meckel ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Three Dimensions ◽  
Confocal Laser ◽  
Subsurface Imaging ◽  
Microscopy Imaging ◽  
Paper Production ◽  
Carbohydrate Binding Modules ◽  
Cellulosic Fibres

Abstract The mechanical properties of single fibres are highly important in the paper production process to produce and adjust properties for the favoured fields of application. The description of mechanical properties is usually characterised via linearized assumptions and is not resolved locally or spatially in three dimensions. In tensile tests or nanoindentation experiments on cellulosic fibres, only one mechanical parameter, such as elastic modulus or hardness, is usually obtained. To obtain a more detailed mechanical picture of the fibre, it is crucial to determine mechanical properties in depth. To this end, we discuss an atomic force microscopy-based approach to examine the local stiffness as a function of indentation depth via static force-distance curves. This method has been applied to linter fibres (extracted from a finished paper sheet) as well as to natural raw cotton fibres to better understand the influence of the pulp treatment process in paper production on the mechanical properties. Both types of fibres were characterised in dry and wet conditions with respect to alterations in their mechanical properties. Subsurface imaging revealed which wall in the fibre structure protects the fibre against mechanical loading. Via a combined 3D display, a spatially resolved mechanical map of the fibre interior near the surface can be established. Additionally, we labelled fibres with carbohydrate binding modules tagged with fluorescent proteins to compare the AFM results with fluorescence confocal laser scanning microscopy imaging. Nanomechanical subsurface imaging is thus a tool to better understand the mechanical behaviour of cellulosic fibres, which have a complex, hierarchical structure.

scholarly journals Proteins Exported via the PrsD-PrsE Type I Secretion System and the Acidic Exopolysaccharide Are Involved in Biofilm Formation by Rhizobium leguminosarum

2006 ◽  
Vol 188 (12) ◽  
pp. 4474-4486 ◽  
Author(s):  
Daniela M. Russo ◽  
Alan Williams ◽  
Anne Edwards ◽  
Diana M. Posadas ◽  
Christine Finnie ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Laser Scanning ◽  
Rhizobium Leguminosarum ◽  
Cellular Adhesion ◽  
Secretion System ◽  
Laser Scanning Microscopy ◽  
Secreted Proteins ◽  
Confocal Laser ◽  
Type I ◽  
Biofilm Structure

ABSTRACT The type I protein secretion system of Rhizobium leguminosarum bv. viciae encoded by the prsD and prsE genes is responsible for secretion of the exopolysaccharide (EPS)-glycanases PlyA and PlyB. The formation of a ring of biofilm on the surface of the glass in shaken cultures by both the prsD and prsE secretion mutants was greatly affected. Confocal laser scanning microscopy analysis of green-fluorescent-protein-labeled bacteria showed that during growth in minimal medium, R. leguminosarum wild type developed microcolonies, which progress to a characteristic three-dimensional biofilm structure. However, the prsD and prsE secretion mutants were able to form only an immature biofilm structure. A mutant disrupted in the EPS-glycanase plyB gene showed altered timing of biofilm formation, and its structure was atypical. A mutation in an essential gene for EPS synthesis (pssA) or deletion of several other pss genes involved in EPS synthesis completely abolished the ability of R. leguminosarum to develop a biofilm. Extracellular complementation studies of mixed bacterial cultures confirmed the role of the EPS and the modulation of the biofilm structure by the PrsD-PrsE secreted proteins. Protein analysis identified several additional proteins secreted by the PrsD-PrsE secretion system, and N-terminal sequencing revealed peptides homologous to the N termini of proteins from the Rap family (Rhizobium adhering proteins), which could have roles in cellular adhesion in R. leguminosarum. We propose a model for R. leguminosarum in which synthesis of the EPS leads the formation of a biofilm and several PrsD-PrsE secreted proteins are involved in different aspects of biofilm maturation, such as modulation of the EPS length or mediating attachment between bacteria.

3-D imaging of cells using a confocal laser scanning microscope and digital image processing

1988 ◽  
Vol 46 ◽  
pp. 96-97
Author(s):  
Thomas M. Jovin ◽  
Michel Robert-Nicoud ◽  
Donna J. Arndt-Jovin ◽  
Thorsten Schormann
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Scanning Microscope ◽  
Laser Scanning Microscope ◽  
Biochemical Processes ◽  
Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

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