Location of the membrane-docking face on the Ca2+-activated C2 domain of cytosolic phospholipase A2

Docking of C2 domains to target membranes is initiated by the binding of multiple Ca2+ ions to a conserved array of residues imbedded within three otherwise variable Ca2+-binding loops. We have located the membrane-docking surface on the Ca2+-activated C2 domain of cPLA2 by engineering a single cysteine substitution at 16 different locations widely distributed across the domain surface, in each case generating a unique attachment site for a fluorescein probe. The environmental sensitivity of the fluorescein-labeled cysteines enabled identification of a localized region that is perturbed by Ca2+ binding and membrane docking. Ca2+ binding to the domain altered the emission intensity of six fluoresceins in the region containing the Ca2+-binding loops, indicating that Ca2+-triggered environmental changes are localized to this region. Similarly, membrane docking increased the protonation of six fluoresceins within the Ca2+-binding loop region, indicating that these three loops also are directly involved in membrane docking. Furthermore, iodide quenching measurements revealed that membrane docking sequesters three fluorescein labeling positions, Phe35, Asn64, and Tyr96, from collisions with aqueous iodide ion. These sequestered residues are located within the identified membrane-docking region, one in each of the three Ca2+-binding loops. Finally, cysteine substitution alone was sufficient to dramatically reduce membrane affinity only at positions Phe35 and Tyr96, highlighting the importance of these two loop residues in membrane docking. Together, the results indicate that the membrane-docking surface of the C2 domain is localized to the same surface that cooperatively binds a pair of Ca2+ ions, and that the three Ca2+-binding loops themselves provide most or all of the membrane contacts. These and other results further support a general model for the membrane specificity of the C2 domain in which the variable Ca2+-binding loops provide headgroup recognition at a protein-membrane interface stabilized by multiple Ca2+ ions.     

Invasive group A streptococcal disease in Taiwan is not associated with the presence of streptococcal pyrogenic exotoxin genes

We reviewed the clinical features of 44 patients with invasive group A streptococcal (GAS) disease who were treated at two teaching hospitals in southern Taiwan from 1991 to 1994. Genes encoding streptococcal pyrogenic exotoxin types A (speA), B (speB), C (speC), and F (speF) and serotypes of M1, M6, and M12 were determined by polymerase chain reaction to target specific sequences in the 44 isolates recovered from these patients and in 28 isolates recovered from upper respiratory sites in 28 additional patients during the study period. The protease activity of these isolates was tested by using the casein plate method. Of the 44 patients with invasive diseases, 25 (57%) had no obvious underlying diseases, and 14 (32%) had preexisting neoplastic diseases or had previously used steroids. Twenty-five patients (57%) presented with cellulitis or necrotizing fasciitis, 24 (55%) had bacteremia, and eight (18%) had streptococcal toxic shock syndrome (STSS). Eight patients (18%) died of invasive GAS disease; seven had STSS, and seven had underlying diseases. All eight patients died within 48 hours after hospitalization. The presence of speA, speC, or speF was not implicated in any particular clinical syndrome in patients with invasive GAS disease. High-level protease activity and the M1 serotype of the isolates were significantly associated with the clinical signs of STSS and with mortality. M1 serotype and protease activity, as well as host immune status, might play significant roles in the pathogenesis of invasive GAS disease in Taiwan.     

Influence of Deposition Positions on Fretting Behaviors of DLC Coating on Ti-6Al-4V

Abstract

The influence of diamond-like carbon (DLC) coating positions—coated flat, coated cylinder, and self-mated coated surface tribopairs—on the fretting behaviors of Ti-6Al-4V were investigated using a fretting wear test rig with a cylinder-on-flat contact. The results indicated that, for tests without coating (Ti-6Al-4V–Ti-6Al-4V contact), the friction (Q_max/P) was high (0.8–1.2), wear volumes were large (0.08–0.1?mm³) under a large displacement amplitude of ±40 µm and small (close to 0) under a small displacement amplitude of ±20 µm, and the wear debris was composed of Ti-6Al-4V flakes and oxidized particles. For tests with the DLC coating, under low load conditions, the DLC coating was not removed or was only partially removed, Q_max/P was low (≤0.2), and the wear volumes were small. Under high load conditions, the coating was entirely removed, Q_max/P was high (0.6–0.8), and the wear volumes were similar to those in tests without coating. The wear debris was composed of DLC particles, Ti-6Al-4V flakes, and oxidized particles. The DLC coating was damaged more severely when deposited on a flat surface than when deposited on a cylindrical surface. The DLC coating was damaged more severely when sliding against a DLC-coated countersurface than when sliding against the Ti-6Al-4V alloy.     

Tribological duality of Ti<Subscript>3</Subscript>SiC<Subscript>2</Subscript>

We report here on the friction behavior of fine- and coarse-grained Ti₃SiC₂ against steel and Si₃N₄ balls. Two successive friction regimes have been identified for both grain sizes and both counterparts. First, Type I regime is characterized by a relatively low (0.1–0.15) friction coefficient, and very little wear. Sliding occurs between a tribofilm on the ball and the Ti₃SiC₂ plane when against steel. Then, a Type II regime often follows, with increased friction coefficients (0.4–0.5) and significant wear. Compacted wear debris seems to act as a third body resulting in abrasion of the ball, even in the case of Si₃N₄. The transition between the two regimes occurs at different times, depending on various factors such as grain size, type of pin, and normal load applied. Some experiments under vacuum showed that the atmosphere plays also a major role. The reason for this evolution is not fully clear at that time, but its understanding is of major technological importance given the unusual good properties of this material.     

On energy arguments applied to the hydrodynamic impact force

The hydrodynamic impact problem is investigated within the framework of potential-flow theory. The vertical load acting on the rigid body is derived based on either momentum or energy conservation, and using the concept of added mass together with a homogeneous Dirichlet condition for the potential on the free surface as usually done to model an impact problem. It is demonstrated that the use of this simplified dynamic free-surface condition, instead of the fully nonlinear one, has a direct influence on the computation of the loads. In particular, the equivalence of momentum and energy analysis is in general not recovered. The situation is then highlighted by performing an asymptotic analysis of the two-dimensional blunt-body asymmetric impact problem. The asymptotic solution is given explicitly and validated through comparisons with experimental results. The energy distribution is then studied. It is shown that the contradiction between momentum and energy analysis can be removed, provided that the flux of energy through the jets is taken into account in the energy balance. If the simplified free-surface condition is indeed valid in the far-field, nonlinear terms must be retained near the body, in the spray-root domains. To leading order, the energy distribution during the gravity-free inertia stage does not depend on the blunt-body shape. The general analysis based on momentum or energy conservation suggests that this result also applies for arbitrary body shape as soon as a homogeneous Dirichlet condition can be applied as a dynamical free-surface boundary condition. In this case, and for a constant vertical impact velocity, half the work performed by the body would seem to be transferred to the fluid as kinetic energy within the spray.     

Alzheimer's disease lesions: from morphology to cell biology

Four different approaches to Alzheimer disease changes have been successively applied, and allowed a permanent feed forward-feed back enrichment of knowledge: morphologists described neurofibrillary tangles, senile plaques, amyloid angiopathy; with the help of immunohistochemical and biochemical techniques, they recognised A beta- and tau-associated pathologies; this, in turn, allowed more precise analysis of the lesions, and permitted recognising new ones such as neuropil threads; molecular genetics and molecular biology provided new insights, allowing the discovery of additional pathologic proteins, the relevance of which to physiology and pathology of the nervous system has now to be settled down. The increasingly intricate complex of lesions of Alzheimer syndrome is reviewed. A more comprehensive understanding is urgently needed for initiating efficient therapeutic researches. It will require together continuing a multidisciplinary approach, and a renewal of research in neuropathology.