Granzyme B-induced mitochondrial ROS are required for apoptosis.

Caspases and the cytotoxic lymphocyte protease granzyme B (GB) induce reactive oxygen species (ROS) formation, loss of transmembrane potential and mitochondrial outer membrane permeabilization (MOMP). Whether ROS are required for GB-mediated apoptosis and how GB induces ROS is unclear. Here, we found that GB induces cell death in an ROS-dependent manner, independently of caspases and MOMP. GB triggers ROS increase in target cell by directly attacking the mitochondria to cleave NDUFV1, NDUFS1 and NDUFS2 subunits of the NADH: ubiquinone oxidoreductase complex I inside mitochondria. This leads to mitocentric ROS production, loss of complex I and III activity, disorganization of the respiratory chain, impaired mitochondrial respiration and loss of the mitochondrial cristae junctions. Furthermore, we have also found that GB-induced mitocentric ROS are necessary for optimal apoptogenic factor release, rapid DNA fragmentation and lysosomal rupture. Interestingly, scavenging the ROS delays and reduces many of the features of GB-induced death. Consequently, GB-induced ROS significantly promote apoptosis.

Functional tests for the characterization of surfactant protein B (SP-B) and a fluorescent SP-B analog.

Surfactant protein B (SP-B) enhances lipid insertion into the alveolar air/liquid interface upon inhalation. The aim of this study was (i) to apply a palette of tests for a detailed biochemical and biophysical characterization of SP-B and (ii) to use these tests to compare native SP-B with a fluorescent (Bodipy) SP-B analog. The method of labeling was fast and resulted in a covalent fluorophore-protein bond. The ability of both proteins to spread a surfactant film on top of a buffer surface was determined in a spreading tray using the Wilhelmy plate technique to allow detection of alterations in surface tension and calculation of spreading velocities. In a captive bubble surfactometer surface tensions of spread films were measured. Similar biophysical properties were found for both native and Bodipy-labeled SP-B. It is concluded that the combination of tests used allows detection of small differences in structure and activity between the two proteins.

Effect of streptolysin O on the microelasticity of human platelets analyzed by atomic force microscopy.

Atomic force microscopy (AFM) has been shown to be a suitable tool to probe biophysical properties of cells and cell fragments. We analysed biophysical alterations of human platelets by AFM using streptolysin O (SLO) as a model for pore forming proteins. Permeabilization of platelet membrane by SLO was confirmed by transmission electron and confocal microscopy. Using force volume imaging combined with FIEL analysis we were able to show dynamically the increase in the elasticity of platelets during the pore formation by SLO and could correlate the viscoelasticity to the morphology of platelets. Stabilizing the actin cytoskeleton by phalloidin resulted in partial restoration of the elasticity indicating that loss of stability in platelets by SLO is mediated by alterations of both plasma membrane and cytoskeleton.

Hydrophobic lung surfactant proteins B and C remain associated with surface film during dynamic cyclic area changes.

The biophysical activity of lung surfactant depends, to a large extent, on the presence of the hydrophobic surfactant proteins B (SP-B) and C (SP-C). The role of these proteins in lipid adsorption and lipid squeeze-out under dynamic conditions simulating breathing is not yet clear. Therefore, the aim of this study was to investigate the interaction of spread hydrophobic surfactant proteins with phospholipids in a captive-bubble surfactometer during rapid cyclic area changes (6 cycles/min). We found that SP-B and SP-C facilitated the rapid transport of lipids into the air-water interface in a concentration-dependent manner (threshold concentration > or = 0.05:0.5 mol% SP-B/SP-C). Successive rapid cyclic area changes did not affect the concentration-dependent lipid adsorption process, suggesting that SP-B and SP-C remained associated with the surface film.

A spreading technique for forming film in a captive bubble.

Mechanisms underlying the surface properties of lung surfactant are extensively studied in in vitro systems such as the captive-bubble surfactometer (CBS), the pulsating-bubble surfactometer, and the Wilhelmy balance. Among these systems, the CBS is advantageous when a leakproof system and high cycling rates are required. However, widespread application of the CBS to mechanistic studies of dynamic surfactant protein-phospholipid interactions of spread film and to comparative studies between spread and adsorbed film is hampered because spreading of film is difficult. In addition, when film is formed by adsorption, the amount of material required is fairly large. We have developed an easy spreading technique that allows routine formation of film by spreading of small amounts of surfactant components at the air-water interface of an air bubble in a CBS. The technique is reliable, precise, and accurate, and the biophysical activity of film formed by spreading is similar to that of film formed by adsorption. This method will be useful for mechanistic studies of surfactant components under dynamic conditions and for comparative studies of spread films and adsorbed films.

Bacterial succession within a biofilm in water supply lines of dental air-water syringes.

Biofilms have been implicated as reservoirs for bacterial contamination of water delivered by dental air-water syringes. A 6-month study was done of bacterial colonization and biofilm formation in plastic water supply lines connected to dental air-water syringes. Changes in biofilm flora were observed by both scanning electron microscopy and bacteriologic culture. By day 7, many rod- and spiral-shaped bacteria had colonized the ridged surface of the luminal wall of the tubing, as revealed by scanning electron microscopy. By day 30, individual microcolonies were embedded in extracellular polymeric material. By day 120, these microcolonies had begun to coalesce, and by day 180 the biofilm had developed into a multilayered, heterogeneous mixture of microcolonies. The mean aerobic plate counts of colony-forming units of planktonic and biofilm populations were, in log10 values, 5.9 +/- 0.54/mL and 4.2 +/- 0.82/cm2, respectively. Early colonizers were predominantly Pseudomonas spp., but included Pasteurella, Moraxella, Ochrobactrum, and Aeromonas spp. Flavobacterium and Acinetobacter spp. were observed later. Many of these organisms are opportunistic pathogens. These results demonstrate the longitudinal dynamics of biofilm formation.

Molecular computing for edge-enhanced laser imaging.

In order to illustrate the self-assembly capability, we consider a laser imaging experiment on a wet film that is made of bacteriorhodopsin (BR) molecules suspended in a diffusion-limited viscous medium. BR wet film is similar to a wet photograph film but having a finer resolution and adaptive pixel locations due to laser-induced thermal diffusion. The synergism between thermal diffusion of BR molecules (induced externally by a write-laser) and molecular photochromism (generated internally by a read-laser) is exploited naturally for edge-enhanced image applications.

Ammonia Induces Settlement Behavior in Oyster Larvae.

Oyster larvae exposed to solutions of NH4Cl exhibit stereotypical settlement behavior similar to that which normally precedes cementation and metamorphosis. Un-ionized ammonia is the active chemical species. At pH = 8.0, the threshold concentration of NH4Cl (pH = 8.0) for newly competent larvae is 2.5 mM; maximum activity is at 7.9 mM, corresponding to calculated NH3 concentrations of 100 {mu}M and 310 {mu}M, respectively. Induction of settlement behavior is rapid, with >90% of larvae exposed to 310 {mu}M NH3 responding within less than 5 min. After 15 to 30 min, larvae become habituated to NH3 and resume swimming so that the percent exhibiting settlement behavior after 30 min is <10%. Other weak bases, such as methylamine and trimethylamine, induce similar behavior suggesting that NH3 acts by increasing intracellular pH. Evidence that NH3 and L-3,4-dihyrodxyphenylalanine (L-DOPA) induce settlement behavior through different mechanisms is presented. Ammonia may be a natural environmental cue that promotes oyster settlement behavior and, ultimately, recruitment.

Factors influencing bacterial production of inducers of settlement behavior of larvae of the oysterCrassostrea gigas.

Dissolved chemical inducers of settlement behavior of veliger larvae of the oysterCrassostrea gigas are found in supernatants of both pigmented species of bacteria (Alteromonas colwelliana, Vibrio cholerae strain HTX) as well as nonpigmented bacteria (Excherichia coli, Vibrio cholerae strain 596-B). Usually less than 10% of veligers exhibited settlement behavior in response to supernatants from the early bacterial growth phases, whereas 30-90% of larvae responded when exposed to supernatant from late-log and stationary phase cultures. Percentages of larvae exhibiting settlement behavior were inversely correlated with oxygen levels in the culture. Furthermore, the behavioral response decreased with pigment formation, suggesting that quantities of noxious compounds such as quinones may build up in the supernatants of cultures of pigmented bacteria. Tyrosinase, an enzyme that converts L-tyrosine to L-DOPA in the first step of melanogenesis, was detected both in the bacterial pellet and the supernatant during growth of the pigmented species. The enzyme is not required for the production of settlement inducer as the nonpigmented speciesE. coli andV. cholerae (596-B) also released inducer into the supernatant and had no detectable tyrosinase. The data suggest either that there is more than one inducer of settlement behavior found in bacterial supernatants or that the inducer is not L-DOPA or an L-DOPA-mimetic associated with the melanin biochemical pathway.