Ultra structural changes occurring in duct ectasia and periductal mastitis and their significance in etiopathogenesis - Fig 2

<p>B1- Low power electro micrographic view of a dilated duct with periductal tissue showing focal denudation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173216#pone.0173216.ref001" target="_blank">1</a>] of lining epithelium with intraluminal secretions[S] containing epithelial fragments [F]. Around the duct there is collagen deposition. [x1100]; B2- The epithelial cells in this show large number of vacuoles [V] and dense bodies [indicated by the arrows]. E- epithelial cell. [x5000]; B3- The duct epithelial cell [E] shown in the electro micrograph has elongated nucleus, intracytoplasmic fibrils [f] and intraluminal projections [indicated by the arrows] of the cytoplasm suggestive of mesenchymal transformation of the epithelial cell. [x3200]; B4- This electro micrograph depicts focal distortion of terminal bar [indicated by the arrow] and widening of the interepithelial junction [W]. E –epithelial cells. [x8000]; B5- This electro micrograph shows lifting of the epithelium from the basal lamina [arrow] and duplication of the basal lamina[D]. [x2550]; B6- The above electro micrograph shows periductal tissue filled with thick and thin collagen fibres indicative of active collagenisation. [x2550]; B7- Dilated lymphatic vessel [Ly] with the endothelial cell [En] in the periductal tissue seen in this electro Micrograph. [x1100].</p

Prolonged Glycation of Hen Egg White Lysozyme Generates Non Amyloidal Structures

<div><p>Glycation causes severe damage to protein structure that could lead to amyloid formation in special cases. Here in this report, we have shown for the first time that hen egg white lysozyme (HEWL) does not undergo amyloid formation even after prolonged glycation in the presence of D-glucose, D-fructose and D-ribose. Cross-linked oligomers were formed in all the cases and ribose was found to be the most potent among the three sugars. Ribose mediated oligomers, however, exhibit Thioflavin T binding properties although microscopic images clearly show amorphous and globular morphology of the aggregates. Our study demonstrates that the structural damage of hen egg white lysozyme due to glycation generates unstructured aggregates.</p> </div

Variation of secondary structural components during glycation of HEWL in the presence of different sugars.

<p>Representative far UV-CD spectra of (a) HEWL-glucose, (b) HEWL-fructose and (c) HEWL-ribose solutions respectively obtained after incubation at pH 7.4 at 37 °C at different time intervals. [HEWL]=20 µM in each case.</p

Synchronous fluorescence characteristics of HEWL during glycation in the presence of different sugars.

<p>(a) Synchronous fluorescence spectra of control and HEWL solutions treated in the presence of glucose, fructose and ribose respectively after 31 days of incubation at 37 °C at pH 7.4; (b) Second derivative plot of synchronous fluorescence spectra of HEWL solutions incubated in the presence of glucose, fructose and ribose respectively obtained after 31 days of incubation at 37 °C at pH 7.4. Protein concentration=10 µM and Δλ=40 nm in each case.</p

Characterization of different AGE products formed during glycation of HEWL in the presence of different sugars using fluorescence spectroscopy.

<p>Histograms represent fluorescence intensity of different HEWL solutions incubated in the presence of glucose, fructose and ribose respectively over a period of 120 days. Formation of different AGE products such as (a) other AGE products (λ_ex=350 nm), (b) pentosidine (λ_ex=335 nm) and (c) malondialdehyde (MDA) (λ_ex=370 nm). [HEWL]=5 µM in each case. Control represents native HEWL incubated in the absence of sugars at pH 7.4 at 37 °C keeping other conditions similar as that of sets in each case.</p

Estimation of β-sheet content of HEWL solutions incubated in the presence of different sugars.

<p>Percentage β-sheet content of different HEWL solutions (HEWL-glucose, HEWL-fructose and HEWL-ribose) obtained after incubation at pH 7.4 at 37 °C estimated using online server DICHROWEB at different time intervals.</p

Determination of the mass of glycated HEWL.

<p>MALDI TOF spectra of different HEWL solutions obtained after an incubation of 31 days at pH 7.4 at 37 °C (a) Native HEWL (14345.30 Da) (b) HEWL-glucose (15401.22 Da) (c) HEWL-fructose (15955.81 Da) (d) HEWL-ribose (16233.48 Da).</p

Identification of attachment of sugar moieties to HEWL: Fuchsin based SDS PAGE.

<p>Representative SDS polyacrylamide gel electrophoresis of different HEWL solutions obtained after incubation at pH 7.4 at 37 °C for 31 days in the presence of different sugars using Fuchsin staining. lane 1: Horseradish peroxidase; lane 2 and 5: HEWL-glucose; lane 3 and 6: HEWL-fructose; lane 4 and 7: HEWL-ribose.</p

Tertiary structural alterations of HEWL solutions during glycation in the presence of three different sugars.

<p>Representative near UV-CD spectra of (a) HEWL-glucose, (b) HEWL-fructose and (c) HEWL-ribose solutions respectively obtained after incubation at pH 7.4 at 37 °C at different time intervals.</p

Densitometric analysis of SDS-PAGE.

<p>Histograms represent relative mean band intensity of different oligomeric species (dimer, trimer and tetramer) with respect to their corresponding monomer at definite time intervals (a) 1 day, (b) 20 days and (c) 31 days respectively.</p