Calcium Influx Rescues Adenylate Cyclase-Hemolysin from Rapid Cell Membrane Removal and Enables Phagocyte Permeabilization by Toxin Pores

Bordetella adenylate cyclase toxin-hemolysin (CyaA) penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC) domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC− toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P) toxoid, unable to conduct Ca2+ into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca2+ influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca2+ influx promoted by molecules locked in a Ca2+-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux

Fatigue Crack Growth in A 7075 Al-Alloy with Evaluation of Overloading Effects

Measurement of fatigue crack growth (FCG) in an Al 7075-T7351 alloy of a particularly high homogeneity was carried out as a part of the Proficiency Test Programme on fatigue crack growth measurement, organised by General Electric Aviation, USA, in collaboration with the company Exova in France. To pass the test programme successfully, a particular attention was paid to improve and verify DCPD (direct current potential drop) method being used for the crack length measurement. Additionally, an investigation of effects of overloads on fatigue crack growth and retardation was carried out. In the CT specimens used, the difference between actual and calculated crack length at the crack starting point was quite significant, approximately 4.3 mm. However, the difference as a function of crack length was almost constant. Results of FCG rates were very self consistent, likely due to the material homogeneity. Values of the parameters C and m measured at the SVÚM a.s. laboratory were evaluated as the third best precise from all the 16 worldwide participating laboratories. Retardation effects of overloads of the magnitudes 2.7-times and 3.0-times of the maximum load in the constant range fatigue loading were significant. The overloads resulted in substantial crack closure effects, which, however, did not occur immediately, but just after further fatigue crack extension, comparable with plastic zone size

Fatigue Resistance of High-Thickness Al-Alloy Sheets after Water Jet Cutting

Water jet cutting is a useful advanced technology of separating materials with a great industrial potential due to high cutting speed, precision, shape repeatability and possibilities to be performed automatically with a computer control. It is well known, however, that fatigue properties are particularly strongly affected by surface conditions and quality of surface layer. Water jet cutting was applied to Al-alloy sheets of fairly high thickness, namely 50.8 mm. Fatigue resistance of water cut specimens and failure mechanisms were investigated and compared with those studied using a reference batch of specimens manufactured by fine milling. Character of surface damage caused by water jet cutting resulted in a considerable reduction of fatigue strength in comparison with milled surface. The results are discussed considering microscopic analysis of surface and subsurface conditions of fatigue crack initiation

Conditions of Stress Corrosion Crack Growth and Retardation in X70 Steel in Carbonate Environments

X70 steel has been often used for building of high pressure transit pipelines. In spite of multiple pipeline protection being used, namely insulation and cathodic protection, in complicated soil and environmental conditions, some pipeline sections can be exposed to corrosive environments. Seriousness of stress corrosion cracking (SCC) damage in carbonate environments is being sometimes flouted by some operators. The work was aimed at qualitative and quantitative analyses of SCC process and damage mechanisms under static loading with individual unloading cycles in standard carbonate solution at room and elevated temperatures. SCC growth was demonstrated as well as an importance of stress fluctuations affecting the process

Short Fatigue Crack Growth in a Residual Stress Field in a 7075 Al-Alloy after Shot Peening

The paper contains results of an investigation of effects of shot peening on growth of physically short fatigue cracks in a 2.4 mm thick sheet of an aircraft V-95 Al-alloy (a type of a 7075 alloy), clad with a 7072 Al-alloy (Al-Zn1). Using an improved and verified experimental methodology, particularly direct current potential drop (DCPD) method, short fatigue cracks of the length from 0.2 mm to more than 3 mm, most of them between 0.8 - 1.5 mm, were prepared under high cycle fatigue repeated tensile loading of the constant nominal stress range 160 MPa, load asymmetry R 0. Edges of the specimens with already existing short fatigue cracks were then shot peened using two different groups of parameters. Some reference specimens without cracks were shot peened with the same parameters. Microhardness measurement and informative measurement of residual stresses near the peened edges were carried out. Significant retardation of crack growth was observed particularly in case of cracks shorter than 2 mm. The work was completed by a unique measurement of actual crack growth rate throughout the peened area with residual stresses starting with crack length less than 0.2 mm. Different stages of the growth are discussed

Effect of Inclusions on Fatigue Strength of Heat Treated Components

Inclusions significantly affect fatigue properties of quenched and tempered parts. Data about inclusions importance in fatigue crack initiation process were acquired within the framework of a systematical fatigue test programme carried out to verify fatigue properties of CrNiMo(V) steels quenched and tempered to 800 1000 MPa strength. A valuable set of fatigue failures from the high lifetime area was acquired.Microfractographical analysis of these failures enabled to evaluate critically the influence of inclusions, their dimensions, character and morphology on the initiation and propagation of the fatigue cracks in the area of fatigue strength. Comparison of inclusions size "a" according to Murakami with size determined experimentally was made. Limiting influence of small single inclusions in the structure was shown. Initiation of secondary fatigue cracks on inclusions outside of the fracture initiation area was observed. Danger of the fracture initiation after very high lifetime was confirmed