Marcus G. Bell

Inhibition of apoptosis in neuronal cells infected with Chlamydophila (Chlamydia) pneumoniae

BackgroundChlamydophila (Chlamydia) pneumoniae is an intracellular bacterium that has been identified within cells in areas of neuropathology found in Alzheimer disease (AD), including endothelia, glia, and neurons. Depending on the cell type of the host, infection by C. pneumoniae has been shown to influence apoptotic pathways in both pro- and anti-apoptotic fashions. We have hypothesized that persistent chlamydial infection of neurons may be an important mediator of the characteristic neuropathology observed in AD brains. Chronic and/or persistent infection of neuronal cells with C. pneumoniae in the AD brain may affect apoptosis in cells containing chlamydial inclusions.ResultsSK-N-MC neuroblastoma cells were infected with the respiratory strain of C. pneumoniae, AR39 at an MOI of 1. Following infection, the cells were either untreated or treated with staurosporine and then examined for apoptosis by labeling for nuclear fragmentation, caspase activity, and membrane inversion as indicated by annexin V staining. C. pneumoniae infection was maintained through 10 days post-infection. At 3 and 10 days post-infection, the infected cell cultures appeared to inhibit or were resistant to the apoptotic process when induced by staurosporine. This inhibition was demonstrated quantitatively by nuclear profile counts and caspase 3/7 activity measurements.ConclusionThese data suggest that C. pneumoniae can sustain a chronic infection in neuronal cells by interfering with apoptosis, which may contribute to chronic inflammation in the AD brain.

Activation Kinetics of Skinned Cardiac Muscle by Laser Photolysis of Nitrophenyl-EGTA

The kinetics of Ca(2+)-induced contractions of chemically skinned guinea pig trabeculae was studied using laser photolysis of NP-EGTA. The amount of free Ca(2+) released was altered by varying the output from a frequency-doubled ruby laser focused on the trabeculae, while maintaining constant total [NP-EGTA] and [Ca(2+)]. The time courses of the rise in stiffness and tension were biexponential at 23 degrees C, pH 7.1, and 200 mM ionic strength. At full activation (pCa < 5.0), the rates of the rapid phase of the stiffness and tension rise were 56 +/- 7 s(-1) (n = 7) and 48 +/- 6 s(-1) (n = 11) while the amplitudes were 21 +/- 2 and 23 +/- 3%, respectively. These rates had similar dependencies on final [Ca(2+)] achieved by photolysis: 43 and 50 s(-1) per pCa unit, respectively, over a range of [Ca(2+)] producing from 15% to 90% of maximal isometric tension. At all [Ca(2+)], the rise in stiffness initially was faster than that of tension. The maximal rates for the slower components of the rise in stiffness and tension were 4.1 +/- 0.8 and 6.2 +/- 1.0 s(-1). The rate of this slower phase exhibited significantly less Ca(2+) sensitivity, 1 and 4 s(-1) per pCa unit for stiffness and tension, respectively. These data, along with previous studies indicating that the force-generating step in the cross-bridge cycle of cardiac muscle is marginally sensitive to [Ca(2+)], suggest a mechanism of regulation in which Ca(2+) controls the attachment step in the cross-bridge cycle via a rapid equilibrium with the thin filament activation state. Myosin kinetics sets the time course for the rise in stiffness and force generation with the biexponential nature of the mechanical responses to steps in [Ca(2+)] arising from a shift to slower cross-bridge kinetics as the number of strongly bound cross-bridges increases.

Polarized fluorescence depletion reports orientation distribution and rotational dynamics of muscle cross-bridges

The method of polarized fluorescence depletion (PFD) has been applied to enhance the resolution of orientational distributions and dynamics obtained from fluorescence polarization (FP) experiments on ordered systems, particularly in muscle fibers. Previous FP data from single fluorescent probes were limited to the 2(nd)- and 4(th)-rank order parameters, and , of the probe angular distribution (beta) relative to the fiber axis and , a coefficient describing the extent of rapid probe motions. We applied intense 12-micros polarized photoselection pulses to transiently populate the triplet state of rhodamine probes and measured the polarization of the ground-state depletion using a weak interrogation beam. PFD provides dynamic information describing the extent of motions on the time scale between the fluorescence lifetime (e.g., 4 ns) and the duration of the photoselection pulse and it potentially supplies information about the probe angular distribution corresponding to order parameters above rank 4. Gizzard myosin regulatory light chain (RLC) was labeled with the 6-isomer of iodoacetamidotetramethylrhodamine and exchanged into rabbit psoas muscle fibers. In active contraction, dynamic motions of the RLC on the PFD time scale were intermediate between those observed in relaxation and rigor. The results indicate that previously observed disorder of the light chain region in contraction can be ascribed principally to dynamic motions on the microsecond time scale.

P3-223: Caspase activity is inhibited in neuronal cells infected with Chlamydia pneumoniae: Implications for apoptosis in Alzheimer’s disease

of A uptake 2) the requirement of A internalization for A -induced toxicity. Methods: Primary neurons cultured in three-compartmented dishes were treated with oligomeric fluorescein-conjugated A 1-42 exclusively in the distal axonor the cell body-containing compartments.A uptake and intracellular transport were analyzed by fluorescent microscopy and/or ELISA.The role of clathrinand caveolamediated endocytosis was analyzed in neurons infected with recombinant adenoviruses expressing HA-tagged wild-type dynamin-1, HA-tagged dynK44A mutant, or T7tagged clathrin hub. Neuronal apoptosis was determined by nuclear fragmentation and caspase activation. Results: A 1-42 was internalized mainly through the distal axons and retrogradely transported to cell bodies. Conversely, neurons treated with A 1-42 exclusively in the cell bodies showed very little A 1-42 internalization and anterograde transport . This result suggests that A 1-42 enters the cell by a regulated mechanism. Two main internalization pathways exist: namely clathrinand caveolae-mediated endocytosis. Both mechanisms require dynamin, but only the former is mediated by clathrin. Neurons expressing the dominat negative mutant of dynamin 1, showed a dramatic decrease in A 1-42 transport from distal axons to cell bodies. Conclusions: Our data suggest the oligomeric A 1-42 internalization is independent of clathrin. Our model is relevant to the death of basal forebrain cholinergic neurons in AD.

778.1-Pos Board # B363.1 – Effects of Ca2+ and Temperature on the Force-generating Transition in Cardiac Muscle Studied by Photolysis of Caged-phosphate ☆

We have shown that below 20°C the step determining kPi in skinned guinea pig trabeculae may shift from the force generating transition to a different step, perhaps cross-bridge formation (Biophys J. 80:586a, 2001) implying kPi should be calcium sensitive at lower temperatures. To test this hypothesis, we have measured kPi at a fixed final [Pi] (initial + photoreleased @ 1.4 mM) at different [Ca2+] and at either 24° or 14°C. At 14°C and full activation (pCa 4.5) kPi was 5.87 ± 0.54 sec−1 (mean ± sem, n = 9) decreasing ∼40% to 3.42 ± 0.92 sec−1 at pCa 5.34 (P/Po∼0.4). At 24°C pCa 5.93 (P/Po∼0.4) kPi was not significantly different than kPi at full activation (26.30 ± 3.9 sec−1 at pCa 5.93 vs. 28.4 ± 2.87 sec−1 at pCa 4.5). This suggests that Ca2+ control of cross-bridge kinetics differs at the two temperatures: at higher temperatures the force-generating transition is Ca2+-insensitive, while both cross-bridge formation and the force-generating transition are Ca2+-sensitive at lower temperatures. Alternatively, Ca2+ control is exerted at the cross-bridge formation step at both temperatures, but at 14°C, kPi is governed by a force generating transition that is slow compared to a Ca2+-sensitive equilibrium at the cross-bridge formation step and therefore sensitive to it.