Effects of amphipathic peptides,including presequences,on the functional integrity of rat liver mitochondrial membranes

A number of amphipathic peptides were tested for their effects on structural and functional properties of isolated rat liver mitochondria. The peptides included the matrix targeting sequence of subunit IV of (yeast) cytochromec oxidase. Titration experiments in which the mitochondria were incubated with increasing concentrations of the peptides revealed two major stages in the interaction. First, at low peptide/mitochondria ratios, peptide binding to the outer membrane occurred which was accompanied by gradual lysis of the outer membrane at higher ratios. The latter was deduced from the release of adenylate kinase, the classical marker enzyme of the intermembrane space. Secondly, at still higher peptide/mitochondria ratios, the permeability of the inner membrane progressively increased, as evidenced by measurements of respiratory control and of the membrane potential. Complete uncoupling of respiration seemed to precede dissipation of the membrane potential.     

Ex situ transportation of coral larvae for research,conservation, and aquaculture

Due to the lack of appropriate methods to transport high amounts of larvae ex situ over large distances, the availability of coral larvae was so far mainly limited to their place of origin. For a research project at Rotterdam Zoo, The Netherlands, we transported several thousand larvae of three broadcast spawners (Acropora tenuis, A. digitifera, Diploria strigosa) from the Indo Pacific and the Caribbean to Europe. Beside logistics and packing techniques, post-transport survival rates were mainly influenced by larvae density and transport duration. Our results indicate optimum survival rates of >90% at densities of 4 larvae ml⁻¹ when not exceeding a transportation time of 4 days. The ex situ transport of coral larvae over large distances might offer new possibilities for research, conservation, and aquaculture.     

Properties of microtubule sliding disintegration in isolated tetrahymena cilia

Properties of the sliding disintegration response of demembranated tetrahymena cilia have been studied by measuring the spectrophotomeric response or turbidity of cilia suspensions at a wavelength of 350 nm relative to changes in the dynein substrate (MgATP(2-)) concentration. The maximum decrease in turbidity occurs in 20 muM ATP, and 90 percent of the decrease occurs in approximately 5.9 s. At lower ATP concentrations (1-20 muM), both the velocity and magnitude of the turbidity decreases are proportional to ATP concentration. The velocity data for 20 muM ATP permit construction of a reaction velocity curve suggesting that changes in turbidity are directly proportional to the extent and velocity of disintegration. At ATP concentrations more than 20 muM (50muM to 5mM), both velocity and magnitude of the turbidimetric response are reduced by approximately 50 percent. This apparent inhibition results in a biphasic response curve that may be related to activation of residual shear resistance or regulatory components at the higher ATP concentrations. The inhibitory effects of elevated ATP can be eliminated by mild trypsin proteolysis, whereupon the reaction goes to completion at any ATP concentration. The turbidimetric responses of the axoneme-substrate suspensions are consistent with the extent and type of axoneme disintegration revealed by electron microscope examination of the various suspensions, suggesting that the turbidimetric assay may prove to be a reliable means for assessing the state of axoneme integrity.     

Sexual recruitment of the corals Favia fragum and Agaricia humilis in a 30‐m3 exhibit aquarium: species‐specific limitations and implications on reproductive ecology

We studied the recruitment of the Caribbean reef building corals Favia fragum (F. fragum) and Agaricia humilis(A. humilis) in captivity. Thirty colonies of each species collected in Curaçao, Netherlands Antilles, reproduced sexually during a temporary stay in a 30‐m³ closed system from November 2001–January 2002. Twelve months later, the F1 generation of F. fragum started reproducing and formed an F2 generation. No reproduction of the F1 recruits of A. humilis was observed. Two years after the introduction of the field colonies, sexual recruits of both species were assessed using two different methods: recruits were estimated using quadrats (quadrat sampling method) and the total number of recruits was counted by creating a map of the artificial rock work divided in distinct areas (area census method). Recruitment rates of F. fragum were highest for the F2 generation on horizontal surfaces (area census method: 137.9±191.7 recruits m^?2; quadrat sampling method: 272.0±254.8 recruits m^?2; mean±SD) and were overall lowest for the F1 generation on overhanging areas of the tank (area census method: 3.8±4.0 recruits m^?2; quadrat sampling method: 4.0±6.1 recruits m^?2; mean±SD recruits m^?2). The populations of both species showed similar patterns independent of the applied assessment method with highest densities on horizontal surfaces followed by vertical surfaces and lowest densities on overhanging surfaces; however, both methods showed conflicting results when the influence of the surface orientation on the population densities was statistically analyzed. The maximum density of A. humilis (1.4±2.7 recruits m^?2 on horizontal surfaces) was much lower than the maximum density of the F1 generation of F. fragum (24.7±18.3 recruits m^?2). Colony sizes of recruits within each population (F. fragum F1: 3.12±0.98 cm, F2: 0.83±0.41 cm; A. humilis F1: 3.79±1.35 cm; maximum diameter±SD) did not differ between different orientations (horizontal vs. vertical vs. overhanging). Calculated growth rates in the aquarium reflected those observed for F. fragum in the field, whereas A. humilis showed slower growth in the aquarium than in the field. Factors such as competition, sedimentation, and predation, that generally reduce in situ recruitment may be excluded in captivity resulting in far higher recruitment rates. This study confirms that aquarium exhibits can serve as a comparative model to study the ecology of corals under semi‐controlled conditions. Zoo Biol 0:1–17, 2007. © 2007 Wiley‐Liss, Inc.     

Preserving and using germplasm and dissociated embryonic cells for conserving Caribbean and Pacific coral

Coral reefs are experiencing unprecedented degradation due to human activities, and protecting specific reef habitats may not stop this decline, because the most serious threats are global (i.e., climate change), not local. However, ex situ preservation practices can provide safeguards for coral reef conservation. Specifically, modern advances in cryobiology and genome banking could secure existing species and genetic diversity until genotypes can be introduced into rehabilitated habitats. We assessed the feasibility of recovering viable sperm and embryonic cells post-thaw from two coral species, Acropora palmata and Fungia scutaria that have diffferent evolutionary histories, ecological niches and reproductive strategies. In vitro fertilization (IVF) of conspecific eggs using fresh (control) spermatozoa revealed high levels of fertilization (>90% in A. palmata; >84% in F. scutaria; P>0.05) that were unaffected by tested sperm concentrations. A solution of 10% dimethyl sulfoxide (DMSO) at cooling rates of 20 to 30°C/min most successfully cryopreserved both A. palmata and F. scutaria spermatozoa and allowed producing developing larvae in vitro. IVF success under these conditions was 65% in A. palmata and 53% in F. scutaria on particular nights; however, on subsequent nights, the same process resulted in little or no IVF success. Thus, the window for optimal freezing of high quality spermatozoa was short (～5 h for one night each spawning cycle). Additionally, cryopreserved F. scutaria embryonic cells had～50% post-thaw viability as measured by intact membranes. Thus, despite some differences between species, coral spermatozoa and embryonic cells are viable after low temperature (-196°C) storage, preservation and thawing. Based on these results, we have begun systematically banking coral spermatozoa and embryonic cells on a large-scale as a support approach for preserving existing bio- and genetic diversity found in reef systems.     

The quantitation of ADP diffusion gradients across the outer membrane of heart mitochondria in the presence of macromolecules

We have previously provided evidence that diffusion of metabolites across the porin pores of mitochondrial outer membrane is hindered. A functional consequence of this diffusion limitation is the dynamic compartmentation of ADP in the intermembrane space. These earlier studies were done on isolated mitochondria suspended in isotonic media without macromolecules, in which intermembrane space of mitochondria is enlarged. The present study was undertaken to assess the diffusion limitation of outer membrane in the presence of 10% (w/v) dextran M20, in order to mimic the action of cytosolic macromolecules on mitochondria. Under these conditions, mitochondria have a more native, condensed configuration.Flux-dependent concentration gradients of ADP were estimated by measuring the ADP diffusion fluxes across the porin pores of isolated rat heart mitochondria incubated together with pyruvate kinase (PK), both of which compete for ADP regenerated by mitochondrial creatine kinase (mtCK) within the intermembrane space or by yeast hexokinase (HK) extramitochondrially. From diffusion fluxes and bulk phase concentrations of ADP, its concentrations in the intermembrane space were calculated using Fick's law of diffusion. Flux-dependent gradients up to 23 microM ADP (for a diffusion rate of J(Dif)=1.9 micromol ADP/min/mg mitochondrial protein) were observed. These gradients are about twice those estimated in the absence of dextran and in the same order of magnitude as the cytosolic ADP concentration (30 microM), but they are negligibly low for cytosolic ATP (5 mM). Therefore, it is concluded that the dynamic ADP compartmentation is of biological importance for intact heart cells.If mtCK generates ADP within the intermembrane space, the local ADP concentration can be clearly higher than in the cytosol resulting in higher extramitochondrial phosphorylation potentials. In this way, mtCK contributes to ensure optimal kinetic conditions for ATP-splitting reactions in the extramitochondrial compartment.     

Protocol for Birmingham Atrial Fibrillation Treatment of the Aged study (BAFTA): a randomised controlled trial of warfarin versus aspirin for stroke prevention in the management of atrial fibrillation in an elderly primary care population [ISRCTN89345269]

Background

Statins effectively lower blood cholesterol and the risk of cardiovascular death. Immunomodulatory actions, independent of their lipid-lowering effect, have also been ascribed to these compounds. Since macrophages participate in several vascular pathologies, we examined the effect of statin treatment on the survival and differentiation of primary human monocytes.

Methods

Peripheral blood mononuclear cells (PBMCs) from healthy individuals were cultured in the presence or absence of mevastatin. Apoptosis was monitored by annexin V / PI staining and flow cytometry. In parallel experiments, cultures were stimulated with LPS in the presence or absence of mevastatin and the release of IL-1β and IL-1Ra was measured by ELISA.

Results

Among PBMCs, mevastatin-treated monocytes were particularly susceptible to apoptosis, which occurred at doses >1 microM and was already maximal at 5 microM. However, even at the highest mevastatin dose used (10 microM), apoptosis occurred only after 24 h of culture, possibly reflecting a requirement for cell commitment to differentiation. After 72 h of treatment the vast majority (>50%) of monocytes were undergoing apoptosis. Stimulation with LPS revealed that mevastatin-treated monocytes retained the high IL-1β output characteristic of undifferentiated cells; conversely, IL-1Ra release was inhibited. Concurrent treatment with mevalonolactone prevented the induction of apoptosis and suppressed both IL-1β and IL-1Ra release in response to LPS, suggesting a rate-limiting role for HMG-CoA reductase in monocyte differentiation.

Conclusions

Our findings indicate that statins arrest the functional differentiation of monocytes into macrophages and steer these cells into apoptosis, suggesting a novel mechanism for the vasculoprotective properties of HMG-CoA reductase inhibitors.