Visualizing mitochondrial FoF1-ATP synthase as the target of the immunomodulatory drug Bz-423

Targeting the mitochondrial enzyme FoF1-ATP synthase and modulating its catalytic activities with small molecules is a promising new approach for treatment of autoimmune diseases. The immuno-modulatory compound Bz-423 is such a drug that binds to subunit OSCP of the mitochondrial FoF1-ATP synthase and induces apoptosis via increased reactive oxygen production in coupled, actively respiring mitochondria. Here we review the experimental progress to reveal the binding of Bz-423 to the mitochondrial target and discuss how subunit rotation of FoF1-ATP synthase is affected by Bz-423. Briefly, we report how F\"orster resonance energy transfer (FRET) can be employed to colocalize the enzyme and the fluorescently tagged Bz-423 within the mitochondria of living cells with nanometer resolution.Comment: 10 pages, 2 figure

Stage progression and neurological symptoms in Trypanosoma brucei rhodesiense sleeping sickness: role of the CNS inflammatory response

Background: Human African trypanosomiasis progresses from an early (hemolymphatic) stage, through CNS invasion to the late (meningoencephalitic) stage. In experimental infections disease progression is associated with neuroinflammatory responses and neurological symptoms, but this concept requires evaluation in African trypanosomiasis patients, where correct diagnosis of the disease stage is of critical therapeutic importance. Methodology/Principal Findings: This was a retrospective study on a cohort of 115 T.b.rhodesiense HAT patients recruited in Eastern Uganda. Paired plasma and CSF samples allowed the measurement of peripheral and CNS immunoglobulin and of CSF cytokine synthesis. Cytokine and immunoglobulin expression were evaluated in relation to disease duration, stage progression and neurological symptoms. Neurological symptoms were not related to stage progression (with the exception of moderate coma). Increases in CNS immunoglobulin, IL-10 and TNF-α synthesis were associated with stage progression and were mirrored by a reduction in TGF-β levels in the CSF. There were no significant associations between CNS immunoglobulin and cytokine production and neurological signs of disease with the exception of moderate coma cases. Within the study group we identified diagnostically early stage cases with no CSF pleocytosis but intrathecal immunoglobulin synthesis and diagnostically late stage cases with marginal CSF pleocytosis and no detectable trypanosomes in the CSF. Conclusions: Our results demonstrate that there is not a direct linkage between stage progression, neurological signs of infection and neuroinflammatory responses in rhodesiense HAT. Neurological signs are observed in both early and late stages, and while intrathecal immunoglobulin synthesis is associated with neurological signs, these are also observed in cases lacking a CNS inflammatory response. While there is an increase in inflammatory cytokine production with stage progression, this is paralleled by increases in CSF IL-10. As stage diagnostics, the CSF immunoglobulins and cytokines studied do not have sufficient sensitivity to be of clinical value

Layering Assume-Guarantee Contracts for Hierarchical System Design

Specifications for complex engineering systems are typically decomposed into specifications for individual subsystems in a manner that ensures they are implementable and simpler to develop further. We describe a method to algorithmically construct component specifications that implement a given specification when assembled. By eliminating variables that are irrelevant to realizability of each component, we simplify the specifications and reduce the amount of information necessary for operation. We parametrize the information flow between components by introducing parameters that select whether each variable is visible to a component. The decomposition algorithm identifies which variables can be hidden while preserving realizability and ensuring correct composition, and these are eliminated from component specifications by quantification and conversion of binary decision diagrams to formulas. The resulting specifications describe component viewpoints with full information with respect to the remaining variables, which is essential for tractable algorithmic synthesis of implementations. The specifications are written in TLA + , with liveness properties restricted to an implication of conjoined recurrence properties, known as GR(1). We define an operator for forming open systems from closed systems, based on a variant of the “while-plus” operator. This operator simplifies the writing of specifications that are realizable without being vacuous. To convert the generated specifications from binary decision diagrams to readable formulas over integer variables, we symbolically solve a minimal covering problem. We show with examples how the method can be applied to obtain contracts that formalize the hierarchical structure of system design

04491 Abstracts Collection -- Synchronous Programming - SYNCHRON\u2704

From 28.11.04 to 03.12.04, the Dagstuhl Seminar Perspectives Workshop 04491 ``Synchronous Programming - SYNCHRON\u2704\u27\u27 was held in the International Conference and Research Center (IBFI), Schloss Dagstuhl. During the seminar, several participants presented their current research, and ongoing work and open problems were discussed. Abstracts of the presentations given during the seminar as well as abstracts of seminar results and ideas are put together in this paper. The first section describes the seminar topics and goals in general. Links to extended abstracts or full papers are provided, if available

Enhanced charge-independent Mitochondrial Free Ca\u3csup\u3e2+\u3c/sup\u3e and Attenuated ADP-induced NADH Oxidation by Isoflurane: Implications for Cardioprotection

Modulation of mitochondrial free Ca2 + ([Ca2 +]m) is implicated as one of the possible upstream factors that initiates anesthetic-mediated cardioprotection against ischemia–reperfusion (IR) injury. To unravel possible mechanisms by which volatile anesthetics modulate [Ca2 +]m and mitochondrial bioenergetics, with implications for cardioprotection, experiments were conducted to spectrofluorometrically measure concentration-dependent effects of isoflurane (0.5, 1, 1.5, 2 mM) on the magnitudes and time-courses of [Ca2 +]m and mitochondrial redox state (NADH), membrane potential (ΔΨm), respiration, and matrix volume. Isolated mitochondria from rat hearts were energized with 10 mM Na+- or K+-pyruvate/malate (NaPM or KPM) or Na+-succinate (NaSuc) followed by additions of isoflurane, 0.5 mM CaCl2 (≈ 200 nM free Ca2 + with 1 mM EGTA buffer), and 250 μM ADP. Isoflurane stepwise: (a) increased [Ca2 +]m in state 2 with NaPM, but not with KPM substrate, despite an isoflurane-induced slight fall in ΔΨm and a mild matrix expansion, and (b) decreased NADH oxidation, respiration, ΔΨm, and matrix volume in state 3, while prolonging the duration of state 3 NADH oxidation, respiration, ΔΨm, and matrix contraction with PM substrates. These findings suggest that isoflurane\u27s effects are mediated in part at the mitochondrial level: (1) to enhance the net rate of state 2 Ca2 + uptake by inhibiting the Na+/Ca2 + exchanger (NCE), independent of changes in ΔΨm and matrix volume, and (2) to decrease the rates of state 3 electron transfer and ADP phosphorylation by inhibiting complex I. These direct effects of isoflurane to increase [Ca2 +]m, while depressing NCE activity and oxidative phosphorylation, could underlie the mechanisms by which isoflurane provides cardioprotection against IR injury at the mitochondrial level

Specifying the Caltech asynchronous microprocessor

The action systems framework for modelling parallel programs is used to formally specify a microprocessor. First the microprocessor is specified as a sequential program. The sequential specification is then decomposed and refined into a concurrent program using correctness-preserving program transformations. Previously this microprocessor has been specified at Caltech, where an asynchronous circuit for the microprocessor was derived from the specification. We propose a specification strategy that is based on the idea of spatial decomposition of the program variable space