Course Of Fibrillation Research Articles

Physico-chemical stress induced amyloid formation in insulin: Amyloid characterization, cytotoxicity analysis against human neuroblastoma cell lines and its prevention using black seeds (Nigella sativa).

To investigate the aggregation and fibrillation of insulin at low pH and moderate temperature; and to further test the aggregated insulin for its cytotoxicity on human neuroblastoma (SH-SY5Y) cell line and inhibition of the cytotoxicity by black seeds (Nigella sativa) extract. Bovine pancreatic insulin was incubated at pH 2.0, 45 ℃ under stirring condition at 400 r/min for 24 h. Amyloids like structures in the aggregated insulin were characterized using various techniques such as thioflavin T assay (ThT), 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence, circular dichroism (CD) and dynamic light scattering (DLS). Moreover, cytotoxicity of aggregated insulin was monitored on SH-SY5Y cell line in the presence and absence of black seeds extract using standard 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) and reactive oxygen species (ROS) assay kit. Our finding demonstrated that insulin under the mentioned conditions formed amyloid-like structure. ANS binding to aggregated insulin showed increase in fluorescence, suggesting structural change and increase in hydrophobicity in insulin occurring during the fibril formation. DLS measurement revealed progressive increase in hydrodynamic radius of aggregated insulin. Cytotoxicity assays illustrated aggregated insulin induced apoptosis in SH-SY5Y cell through ROS formation. Moreover, LDH measurement showed aggregated insulin triggered membrane damage in SH-SY5Y cell lines. Black seeds extract was found to inhibit amyloid formation and protected the cells against amyloid toxicity. Insulin molded into amyloid like structure at low pH and under stirring conditions. Characterization of insulin aggregates illustrated conformational change in insulin and it experiences α-helix to β-sheet transition during the course of fibrillation. Black seeds extract inhibited amyloid progression of insulin via ROS scavenging and restrained the cytotoxicity caused by insulin fibrils suggesting black seeds containing polyphenols may serve as a lead structure to a novel anti-amyloidogenic drugs.

Inhibition of Insulin Fibrillation by a Non Toxic Peptide NK9

Many proteins form ordered structural aggregates called amyloid fibrils, which are associated with numerous neurodegenerative diseases. Insulin, a largely α-helical protein, undergoes conformational changes under certain stress conditions leading to amyloid fibrils. Fibrillation of insulin poses a problem in its long-term storage, reducing its efficacy in treating type II diabetes. Oligomeric dissociation of insulin into its monomer is the key step for the onset of fibrillation. In the present study, we have used a non-toxic nine residue peptide, NK9 (NIVNVSLVK) constituting mostly with hydrophobic residues, which interferes fibrillation of insulin. The time course of insulin fibrillation at 62°C using Thioflavin T fluorescence shows an increase in the lag time from 120 min without peptide to 236 min with peptide. TEM micrographs show branched insulin fibrils in its absence and less inter-fibril association in its presence. FTIR study shows, loss of α-helical structure of insulin upon incubation, but if the peptide is added, secondary structure is almost fully maintained for 3 h, though lost partially at 4 h. Size exclusion chromatography study confirms the resistance of the peptide towards the oligomeric dissociation of insulin into its monomer in the course of fibrillation. Saturation transfer difference (STD) NMR was used to confirm the binding epitope of NK9, where the hydrophobic residues give signal of resonance transfer being in close contact with insulin. MD simulation in conjunction with PCA analysis reveals how the binding of NK9 governs the interruption of insulin fibrillation. An estimation of probable aggregation site of insulin gives a clue for NK9 activity. The present study is considered to be helpful for architecting new peptide based leads for efficient stopping of aggregation of insulin.

Time course of fibrillation and defibrillation thresholds after an intravenous bolus of amiodarone--an experimental study.

Experimental studies have described an increase in ventricular fibrillation threshold (VFT) by intravenous amiodarone. The aim of this study was to examine the early time course of changes in VFT and defibrillation thresholds (DFT) after an intravenous bolus of amiodarone in an experimental pig model of transient myocardial ischemia. VFT and relative effective ventricular refractory period (ERP) were measured in 15 anaesthetized open-chest pigs after 3 min of regional coronary ischaemia before (time 0) and 2, 15, 30, 60, and 90 min after the intravenous injection of normal saline (group A, n = 5) or amiodarone, 5 mg/kg over 15 s (group B, n = 10). DFT was measured by increasing the strength of DC shocks until defibrillation was accomplished. Amiodarone caused an increase in VFT, starting at 2 min after the infusion (11.4 +/- 8.4 mA versus 9.2 +/- 4.6 mA, P = 0.03), became significant at 15 min (13.7 +/- 6.5 mA, P = 0.009), continued to rise at 30 min (34.2 +/- 28.7 mA, P = 0.03) and reached a plateau at 60 min (50.3 +/- 37.8 mA, P = 0.008). An increase was also observed in the ERP (204 +/- 25 ms at 2 min versus 197 +/- 26 ms at baseline, P = 0.074, 211 +/- 38 ms at 15 min, P = 0.084, 212 +/- 40 ms at 30 min, P = 0.037, 220 +/- 34 ms at 60 min, P = 0.002, and 227 +/- 32 ms at 90 min, P = 0.008). No change was observed in DFT after amiodarone administration. No significant change in VFT, ERP, or DFT occurred in the control group. In this porcine model, the intravenous administration of amiodarone increased VFT and ERP over 60 min after the injection, without effect on DFT.

Locomotion of Xenopus epidermis cells in primary culture.

The locomotion of single epidermis cells, grown out from Xenopus laevis tadpole tails has been investigated by time-lapse cinemicrography using phase-contrast and reflection-contrast optics. The cells develop a large, mostly 200-250 nm thick, lamella, which adheres homogeneously to the supporting coverglass and exceeds the projection area of the cell body. From the comparison of RIC-pictures taken at high (1.06) and low (0.62) numerical aperture of illumination (I.N.A.) we deduce that at low I.N.A. the embossment of the medium-facing side of the lamella is visualized. By this method microcolliculi are demonstrated, which form at the edge of the lamellipodium and move backward. They resemble ruffles, but are flatter and no membrane flow towards the perinuclear region is observed. Indirect immunofluorescence reveals an enhanced staining for actin and alpha-actinin in the lamellipodium and in the transition region of cell body and lamella. Tonofilaments do not participate in lamella formation, the relatively few microtubules seem to be oriented in the direction of cytoplasmic flow. Electron micrographs demonstrate the course of fibrils in the cell body and a meshwork of actin filaments and membranous tubules in the lamella. Based on these findings a model for cell locomotion is presented: the motive force is generated by the cell body causing a flow of cytoplasm towards the periphery and extension of the lamella at its edge. The activity of the lamellipodium has to ensure the flat form of the advanced edge; microcolliculi are assumed to represent a small membrane store for the extension of the lamella. The lamellipodium is not involved in the production of motive force. The cell body is anchored to the lamella by radiating fibrils and the fibrillar meshwork is inserted at the 'dorsal' membrane of the lamella and the basal filament cortex of the cell body. This anchorage provides the structural basis for the uptake of lamella material into the cell body in the transition region.

MULTIPLE RECORDING DURING ELECTRICALLY INDUCED ATRIAL FIBRILLATION.

1. Atrial fibrillation was induced in dogs by single electrical shocks. Close bipolar direct electrograms were taken from various parts of the atrium; as many as 22 electrograms and a standard lead II electrocardiogram were recorded simultaneously. Initial phase and recovery phase of fibrillation were analyzed. 2. In the initial phase of atrial fibrillation, local firing consisting of rapid tachysystole of 996 to 3000 per minute was observed near the stimulating point in most experiments. Except for the leads showing local firing, the interval between beats at each recording point tended to be constant, and a time gap between two successive beats was observed. A propagation pattern could be plotted which seemed to start from the origin of local firing; some beats were conducted only locally, and others (every second, third or fourth discharge) were conducted widely. The cause of atrial fibrillation was considered to be local firing of one small area of the atrium, i.e., the unifocal theory of the origin of atrial fibrillation was supported. 3. During recovery from atrial fibrillation, features similar to those seen in the initial phase often appeared, although local firing disappeared. In some experiments, the atrium was separated electrically into a few groups of cells beating independently, and in others a "circus pathway" appeared to exist in the last beat. It seems that electrical separation of the atrium into many parts and circus movements, or multiple re-entry, may develop during the course of fibrillation, and that these phenomena perpetuate atrial fibrillation. 4. During the last beat of atrial fibrillation, the time required for all cells to depolarize was occasionally less than the duration of the normal sinus beat. A similar phenomenon was sometimes observed in the premature beats following atrial fibrillation.

А. Bethe. Ueber die Primitivfibrillen in den Ganglienzellen vom Menschen u. anderen Wirbelthier en.—Morphol. Arb. Bd. 8, 1898, p. 45 — 117

In view of the results obtained by Apathy in the study of nerves. systems of invertebrates (worms) and confirmed by the author's research on the piyavkoyu, results indicating the continuous, created by fibrils, connection of the perceiving and motor nerve pathways, ed. with the help of a special method (this method was subsequently published by A. Bethe in Zeitschr. f. wissensch. Mikroskopie, Bd. 17, 1900, p. 13) studied the nerves. pads center. nervous systems of vertebrates (man, dog, rabbit, frog), having the main purpose to find out the course of fibrils in fibers and cells and their relation to these last.