Mitochondrial permeability transition involves dissociation of F1FO ATP synthase dimers and C‐ring conformation

The impact of the mitochondrial permeability transition (MPT) on cellular physiology is well characterized. In contrast, the composition and mode of action of the permeability transition pore complex (PTPC), the supramolecular entity that initiates MPT, remain to be elucidated. Specifically, the precise contribution of the mitochondrial F₁F_O ATP synthase (or subunits thereof) to MPT is a matter of debate. We demonstrate that F₁F_O ATP synthase dimers dissociate as the PTPC opens upon MPT induction. Stabilizing F₁F_O ATP synthase dimers by genetic approaches inhibits PTPC opening and MPT. Specific mutations in the F₁F_O ATP synthase c subunit that alter C‐ring conformation sensitize cells to MPT induction, which can be reverted by stabilizing F₁F_O ATP synthase dimers. Destabilizing F₁F_O ATP synthase dimers fails to trigger PTPC opening in the presence of mutants of the c subunit that inhibit MPT. The current study does not provide direct evidence that the C‐ring is the long‐sought pore‐forming subunit of the PTPC, but reveals that PTPC opening requires the dissociation of F₁F_O ATP synthase dimers and involves the C‐ring.     

Effect of lacidipine on ischaemic and reperfused isolated rabbit hearts

Lacidipine is a new developed dihydropyridine calcium-antagonist, showing a slow onset and long lasting-selective activity.To assess whether the administration of lacidipine protects the myocardium in a dose-dependent manner against ischaemia and reperfusion, isolated rabbit heart were infused with three different concentrations of lacidipine: 10^–10; 10^–9; 10^–8 M. Diastolic and developed pressures were monitored; coronary effluent was collected and assayed for CPK activity and for noradrenaline concentration; mitochondria were harvested and assayed for respiratory activity, ATP production and calcium content and tissue concentration of ATP, creatine phosphate (CP) and calcium were determined. Occurrence of oxidative stress during ischaemia and reperfusion was also monitored in terms of tissue content and release of reduced (GSH) and oxidized (GSSG) glutatione. Treatment with lacidipine at 10^–10 and 10^–9 M had no effects on the hearts when perfused under aerobic condition, whilst the higher dose reduced developed pressure of 36%. The ischaemic-induced deterioration of mitochondrial function was attenuated. On reperfusion treated hearts recovered better than the untreated hearts with respect to left ventricular performance, replenishment of ATP and CP stores and mitochondrial function. The reperfusion-induced tissue and mitochondrial calcium overload, release of CPK and of noradrenaline and oxidative stress were also significantly reduced. The effects of lacidipine were dose-dependent. The lower concentration (10^–10 M) failed to modify ischaemic and reperfusion damage. The dose of 10^–9 M was cardioprotective, but the best effect was found at 10^–8 M.It is concluded that lacidipine infusion provides a dose dependent protection of the heart against ischaemia and reperfusion. Because this protection occurred also at 10^–9 M, in the absence of negative inotropic effect during normoxia and of a coronary dilatory effect during ischaemia, it cannot be attributed to an energy sparing effect or to improvement of oxygen delivery. From our data we can envisage two other major mechanism:-1) membrane protection-2) reduction of oxygen toxicity. The ATP sparing effect occurring at 10^–8 M is likely to be responsable for the further protection.     

Sulphide release from anoxic sediments in relation to iron availability and organic matter recalcitrance and its effects on inorganic phosphorus recycling

This research aims to analyse the sediment capacity to buffer free sulphide release in three coastal lagoons which differ in terms of eutrophication level, tide influence and primary producer communities. A preliminary estimate of soluble reactive phosphorus (SRP) regeneration coupled with sulphide fluxes is also made. Sediment profiles of ferrous and ferric iron and reduced sulphur pools were determined in three stations in the Bassin d'Arcachon (South West France), in one site in the Etang du Prévost lagoon (Southern France), and in three stations in the Sacca di Goro lagoon (Northern Italy). Laboratory experiments were also conducted by incubating sediment slurries. Slurries from the French lagoons were also enriched with about 2% d.w. of organic detritus obtained from the dominant macrophytes of each site, namely Zostera noltii and Ruppia cirrhosa (Bassin d'Arcachon), and Ulva rigida (Etang du Prévost). In the Sacca di Goro, slurry experiments were conducted at two sites with different salinity range, sediment composition and hydrodynamics.Field data showed that concentrations of available iron (Fe(II)+Fe(III)) ranged from a minimum of 28.5 µmol cm^–3 (Etang du Prévost) to a maximum of 275.7 µmol cm^–3 (Sacca di Goro). Moreover, in the French lagoons, acid volatile sulphide (AVS) accumulation in the superficial sediment was related to ferrous iron concentrations. Laboratory experiments showed that in spite of strong reducing conditions, sulphide and SRP release was weaker in iron-rich sediments and in those enriched with the most refractory organic matter. The highest fluxes were detected in sediment slurries from the Etang du Prévost, which had the lowest iron content, supplied by 2% of the labile detritus from Ulva rigida. In this case, SRP release was directly related to sulphide production.Two factors seem significant to evaluate the buffer capacity against free sulphide and SRP release from anoxic sediment: organic matter biodegradability, which forces sediment toward reducing conditions, and iron availability, which can affect sulphide mobility as well as the iron hydroxide-phosphate-sulphide system.     

The role of host sex in parasite dynamics: field experiments on the yellow-necked mouse Apodemus flavicollis

We investigated the role of host sex in parasite transmission and questioned: ‘Is host sex important in influencing the dynamics of infection in free living animal populations?’ We experimentally reduced the helminth community of either males or females in a yellow‐necked mice (Apodemus flavicollis) population using an anthelmintic, in replicated trapping areas, and subsequently monitored the prevalence and intensity of macroparasites in the untreated sex. We focussed on the dominant parasite Heligmosomoides polygyrus and found that reducing parasites in males caused a consistent reduction of parasitic intensity in females, estimated through faecal egg counts, but the removal of parasites in females had no significant influence on the parasites in males. This finding suggests that males are responsible for driving the parasite infection in the host population and females may play a relatively trivial role. The possible mechanisms promoting such patterns are discussed.     

Detection of two TaqI polymorphisms in the VTR region of the human HRAS1 oncogene

Restriction enzyme analysis of the variable tandem repetition (VTR) region of the human HRAS1 oncogene revealed two TaqI polymorphisms. The first polymorphism overlaps that detected with MspI/HpaII restriction enzymes and is due to a variable number of repeat units which form the VTR region at the 3' end of the oncogene. The second polymorphism appears to be due to two new TaqI restriction sites created within the VTR region itself. This novel polymorphism was detected in 26 of 145 human DNA samples and was found to segregate in a Mendelian fashion.     

The C2 fragment from Neisseria meningitidis antigen NHBA increases endothelial permeability by destabilizing adherens junctions

Neisseria meningitidis is a human pathogen that can cause fatal sepsis and meningitis once it reaches the blood stream and the nervous system. Here we demonstrate that a fragment, released upon proteolysis of the surface‐exposed protein Neisserial Heparin Binding Antigen (NHBA), by the bacterial protease NalP, alters the endothelial permeability by inducing the internalization of the adherens junction protein VE‐cadherin. We found that C2 rapidly accumulates in mitochondria where it induces the production of reactive oxygen species: the latter are required for the phosphorylation of the junctional protein and for its internalization that, in turn, is responsible for the endothelial leakage. Our data support the notion that the NHBA‐derived fragment C2 might contribute to the extensive vascular leakage typically associated with meningococcal sepsis.     

The effect of residual water on antacid properties of sucralfate gel dried by microwaves

The aim of this work was to study the acid neutralization characteristics of microwave-dried sucralfate gel in relation to the water content and physical structure of the substance. Several dried sucralfate gels were compared with humid sucralfate gel and sucralfate nongel powder in terms of neutralization rate and buffering capacity. Humid sucralfate gel and microwave-dried gel exhibited antacid effectiveness. In particular, the neutralization rate of dried gel powders was inversely related to the water content: as the water content of dried powders decreased, the acid reaction rate linearly increased. The relationship was due to the different morphology of dried sucralfate gels. In fact, the porosity of the dried samples increased with the water reduction. However, the acid neutralization equivalent revealed that the dried sucralfate gel became more resistant to acid attack in the case of water content below 42%. Then, the microwave drying procedure had the opposite effect on the reactivity of the aluminum hydroxide component of dried sucralfate gel powders, since the rate of the reaction increased whereas the buffering capacity decreased as the amount of water was reduced. Published: January 20, 2006     

Climate disruption and parasite-host dynamics: patterns and processes associated with warming and the frequency of extreme climatic events

Levels of parasitism and the dynamics of helminth systems is subject to the impact of environmental conditions such that we may expect long term increases in temperature will increase the force of infection and the parasite's basic reproduction number, R0. We postulate that an increase in the force of infection will only lead to an increase in mean intensity of adults when adult parasite mortality is not determined by acquired immunity. Preliminary examination of long term trends of parasites of rabbits and grouse confirm these predictions. Parasite development rate increases with temperature and while laboratory studies indicate this is linear some recent studies indicate that this may be non-linear and would have an important impact on R0. Warming would also reduce the selective pressure for the development of arrestment and this would increase R0 so that in systems like the grouse and Trichostrongylus tenuis this would increase the instability and lead to larger disease outbreaks. Extreme climatic events that act across populations appear important in synchronizing transmission and disease outbreaks, so it is speculated that climate disruption will lead to increased frequency and intensity of disease outbreaks in parasite populations not regulated by acquired immunity.     

Effect of different cryopreservation protocols on cytoskeleton and gap junction mediated communication integrity in feline germinal vesicle stage oocytes

Oocyte cryopreservation in carnivores can significantly improve assisted reproductive technologies in animal breeding and preservation programs for endangered species. However, the cooling process severely affects the integrity and the survival of the oocyte after thawing and may irreversibly compromise its subsequent developmental capability.In the present study, two different methods of oocyte cryopreservation, slow freezing and vitrification, were evaluated in order to assess which of them proved more suitable for preserving the functional coupling with cumulus cells as well as nuclear and cytoplasmic competence after warming of immature feline oocytes.From a total of 422 cumulus enclosed oocytes (COCs) obtained from queens after ovariectomy, 137 were stored by vitrification in open pulled straws, 147 by slow freezing and 138 untreated oocytes were used as controls. Immediately after collection and then after warming, functional coupling was assessed by lucifer yellow injection and groups of fresh and cryopreserved oocytes were fixed to analyze tubulin and actin distribution, and chromatin organization. Finally, COCs cryopreserved with both treatments were matured in vitro after warming. In most cases, oocytes cryopreserved by slow freezing showed a cytoskeletal distribution similar to control oocytes, while the process of vitrification induced a loss of organization of cytoskeletal elements. The slow freezing protocol ensured a significantly higher percentage of COCs with functionally open and partially open communications (37.2 vs. 19.0) and higher maturational capability (32.5 vs. 14.1) compared to vitrified oocytes. We conclude that although both protocols impaired intercellular junctions, slow freezing represents a suitable method of GV stage cat oocytes banking since it more efficiently preserves the functional coupling with cumulus cells after thawing as well as nuclear and cytoplasmic competence. Further studies are needed to technically overcome the damage induced by the cryopreservation procedures on immature mammalian oocytes.     

Lmx1b is essential for survival of periocular mesenchymal cells and influences Fgf-mediated retinal patterning in zebrafish

To gain insight into the mechanisms of Lmx1b function during ocular morphogenesis, we have studied the roles of lmx1b.1 and lmx1b.2 during zebrafish eye development. In situ hybridization and characterization of transgenic lines in which GFP is expressed under lmx1b.1 regulatory sequence show that these genes are expressed in periocular tissues and in a pattern conserved with other vertebrates. Anti-sense morpholinos against lmx1b.1 and lmx1b.2 result in defective migration of periocular mesenchymal cells around the eye and lead to apoptosis of these cells. These defects in the periocular mesenchyme are correlated with a failure in fusion of the choroid fissure or in some instances, more severe ventral optic cup morphogenesis phenotypes. Indeed, by blocking the death of the periocular mesenchyme in Lmx1b morphants, optic vesicle morphogenesis is largely restored. Within the retina of lmx1b morphants, Fgf activity is transiently up-regulated and these morphants show defective naso-temporal patterning. Epistasis experiments indicate that the increase in Fgf activity is partially responsible for the ocular anomalies caused by loss of Lmx1b function. Overall, we propose zebrafish lmx1b.1 and lmx1b.2 promote the survival of periocular mesenchymal cells that influence multiple signaling events required for proper ocular development.