Respiratory complex III dysfunction in humans and the use of yeast as a model organism to study mitochondrial myopathy and associated diseases.

The bc1 complex or complex III is a central component of the aerobic respiratory chain in prokaryotic and eukaryotic organisms. It catalyzes the oxidation of quinols and the reduction of cytochrome c, establishing a proton motive force used to synthesize adenosine triphosphate (ATP) by the F1Fo ATP synthase. In eukaryotes, the complex III is located in the inner mitochondrial membrane. The genes coding for the complex III have a dual origin. While cytochrome b is encoded by the mitochondrial genome, all the other subunits are encoded by the nuclear genome. In this review, we compile an exhaustive list of the known human mutations and associated pathologies found in the mitochondrially-encoded cytochrome b gene as well as the fewer mutations in the nuclear genes coding for the complex III structural subunits and accessory proteins such as BCS1L involved in the assembly of the complex III. Due to the inherent difficulties of studying human biopsy material associated with complex III dysfunction, we also review the work that has been conducted to study the pathologies with the easy to handle eukaryotic microorganism, the yeast Saccharomyces cerevisiae. Phenotypes, biochemical data and possible effects due to the mutations are also discussed in the context of the known three-dimensional structure of the eukaryotic complex III. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

Membrane-bound oxygen reductases of the anaerobic sulfate-reducing Desulfovibrio vulgaris Hildenborough: roles in oxygen defence and electron link with periplasmic hydrogen oxidation.

Cytoplasmic membranes of the strictly anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough contain two terminal oxygen reductases, a bd quinol oxidase and a cc(b/o)o3 cytochrome oxidase (Cox). Viability assays pointed out that single Δbd, Δcox and double ΔbdΔcox deletion mutant strains were more sensitive to oxygen exposure than the WT strain, showing the involvement of these oxygen reductases in the detoxification of oxygen. The Δcox strain was slightly more sensitive than the Δbd strain, pointing to the importance of the cc(b/o)o3 cytochrome oxidase in oxygen protection. Decreased O2 reduction rates were measured in mutant cells and membranes using lactate, NADH, ubiquinol and menadiol as substrates. The affinity for oxygen measured with the bd quinol oxidase (Km, 300 nM) was higher than that of the cc(b/o)o3 cytochrome oxidase (Km, 620 nM). The total membrane activity of the bd quinol oxidase was higher than that of the cytochrome oxidase activity in line with the higher expression of the bd oxidase genes. In addition, analysis of the ΔbdΔcox mutant strain indicated the presence of at least one O2-scavenging membrane-bound system able to reduce O2 with menaquinol as electron donor with an O2 affinity that was two orders of magnitude lower than that of the bd quinol oxidase. The lower O2 reductase activity in mutant cells with hydrogen as electron donor and the use of specific inhibitors indicated an electron transfer link between periplasmic H2 oxidation and membrane-bound oxygen reduction via the menaquinol pool. This linkage is crucial in defence of the strictly anaerobic bacterium Desulfovibrio against oxygen stress.

Oxygen reduction in the strict anaerobe Desulfovibrio vulgaris Hildenborough: characterization of two membrane-bound oxygen reductases.

Although Desulfovibrio vulgaris Hildenborough (DvH) is a strictly anaerobic bacterium, it is able to consume oxygen in different cellular compartments, including extensive periplasmic O2 reduction with hydrogen as electron donor. The genome of DvH revealed the presence of cydAB and cox genes, encoding a quinol oxidase bd and a cytochrome c oxidase, respectively. In the membranes of DvH, we detected both quinol oxygen reductase [inhibited by heptyl-hydroxyquinoline-N-oxide (HQNO)] and cytochrome c oxidase activities. Spectral and HPLC data for the membrane fraction revealed the presence of o-, b- and d-type haems, in addition to a majority of c-type haems, but no a-type haem, in agreement with carbon monoxide-binding analysis. The cytochrome c oxidase is thus of the cc(o/b)o3 type, a type not previously described. The monohaem cytochrome c553 is an electron donor to the cytochrome c oxidase; its encoding gene is located upstream of the cox operon and is 50-fold more transcribed than coxI encoding the cytochrome c oxidase subunit I. Even when DvH is grown under anaerobic conditions in lactate/sulfate medium, the two terminal oxidase-encoding genes are expressed. Furthermore, the quinol oxidase bd-encoding genes are more highly expressed than the cox genes. The cox operon exhibits an atypical genomic organization, with the gene coxII located downstream of coxIV. The occurrence of these membrane-bound oxygen reductases in other strictly anaerobic Deltaproteobacteria is discussed.

Stratospheric response to trace gas perturbations: changes in ozone and temperature distributions.

The stratospheric concentration of trace gases released in the atmosphere as a result of human activities is increasing at a rate of 5 to 8 percent per year in the case of the chlorofluorocarbons (CFCs), 1 percent per year in the case of methane (CH(4)), and 0.25 percent per year in the case of nitrous oxide (N(2)O). The amount of carbon dioxide (CO(2)) is expected to double before the end of the 21st century. Even if the production of the CFCs remains limited according to the protocol for the protection of the ozone layer signed in September 1987 in Montreal, the abundance of active chlorine (2 parts per billion by volume in the early 1980s) is expected to reach 6 to7 parts per billion by volume by 2050. The impact of these increases on stratospheric temperature and ozone was investigated with a two-dimensional numerical model. The model includes interactive radiation, wave and mean flow dynamics, and 40 trace species. An increase in CFCs caused ozone depletion in the model, with the largest losses near the stratopause and, in the vertical mean, at high latitudes. Increased CO(2) caused ozone amounts to increase through cooling, with the largest increases again near 45 kilometers and at high latitudes. This CO(2)-induced poleward increase reduced the CFC-induced poleward decrease. Poleward and downward ozone transport played a major role in determining the latitudinal variation in column ozone changes.

Mount pinatubo aerosols, chlorofluorocarbons, and ozone depletion.

The injection into the stratosphere of large quantities of sulfur during the June 1991 eruption of Mount Pinatubo (Philippines) and the subsequent formation of sulfate aerosol particles have generated a number of perturbations in the atmosphere with potential effects on the Earth's climate. Changes in the solar and infrared radiation budget caused by the eruption should produce a cooling of the troposphere and a warming of the lower stratosphere. These changes could affect atmospheric circulation. In addition, heterogeneous chemical reactions on the surface of sulfate aerosol particles render the ozone molecules more vulnerable to atmospheric chlorine and hence to man-made chlorofluorocarbons.

Paclitaxel induces release of cytochrome c from mitochondria isolated from human neuroblastoma cells'.

Paclitaxel is an antimicrotubule agent that induces mitotic block and apoptosis. We show for the first time that paclitaxel acts directly or mitochondria isolated from human cancer cells. In isolated yeast mito chondria, paclitaxel (15 microM) induced an 18% increase in the respiration rate, with no concomitant release of cytochrome c. In isolated neuroblas toma mitochondria, paclitaxel (10-100 microM) induced a 27-72% release o cytochrome c. Release was prevented by cyclosporin A, suggesting the involvement of the permeability transition pore. Doxorubicin did no induce cytochrome c release, whereas vinorelbine, another antimicrotu bule agent, did. Thus, antimicrotubule agents can directly affect mito chondria to induce apoptosis.

A compilation of mutations located in the cytochrome b subunit of the bacterial and mitochondrial bc1 complex.

In anticipation of the structure of the bc1 complex which is now imminent, we present here a preliminary compilation of all available cytochrome b mutants that have been isolated or constructed to date both in prokaryotic and eukaryotic species. We have briefly summarized their salient properties with respect to the structure and function of cytochrome b and to the Qo and Qi sites of the bc1 complex. In conjunction with the high resolution structure of the bc1 complex, this database is expected to serve as a useful reference point for the available data and help to focus and stimulate future experimental work in this field.

Analysis of suppressor mutation reveals long distance interactions in the bc(1) complex of Saccharomyces cerevisiae.

Four totally conserved glycines are involved in the packing of the two cytochrome b hemes, b(L) and b(H), of the bc(1) complex. The conserved glycine 131 is involved in the packing of heme b(L) and is separated by only 3 A from this heme in the bc(1) complex structure. The cytochrome b respiratory deficient mutant G131S is affected in the assembly of the bc(1) complex. An intragenic suppressor mutation was obtained at position 260, in the ef loop, where a glycine was replaced by an alanine. This respiratory competent revertant exhibited a low bc(1) complex activity and was affected in the electron transfer at the Q(P) site. The k(min) for the substrate DBH(2) was diminished by an order of magnitude and EPR spectra showed a partially empty Q(P) site. However, the binding of the Q(P) site inhibitors stigmatellin and myxothiazol remained unchanged in the suppressor strain. Optical spectroscopy revealed that heme b(L) is red shifted by 0.8 nm and that the E(m) of heme b(L) was slightly increased (+20 mV) in the revertant strain as compared to wild type strain values. Addition of a methyl group at position 260 is thus sufficient to allow the assembly of the bc(1) complex and the insertion of heme b(L) despite the presence of the serine at position 131. Surprisingly, reversion at position 260 was located 13 A away from the original mutation and revealed a long distance interaction in the yeast bc(1) complex.

Apparent redundancy of electron transfer pathways via bc(1) complexes and terminal oxidases in the extremophilic chemolithoautotrophic Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotrophic bacterium that can grow in the presence of either the weak reductant Fe(2+), or reducing sulfur compounds that provide more energy for growth than Fe(2+). We have previously shown that the uphill electron transfer pathway between Fe(2+) and NAD(+) involved a bc(1) complex that functions only in the reverse direction [J. Bacteriol. 182, (2000) 3602]. In the present work, we demonstrate both the existence of a bc(1) complex functioning in the forward direction, expressed when the cells are grown on sulfur, and the presence of two terminal oxidases, a bd and a ba(3) type oxidase expressed more in sulfur than in iron-grown cells, besides the cytochrome aa(3) that was found to be expressed only in iron-grown cells. Sulfur-grown cells exhibit a branching point for electron flow at the level of the quinol pool leading on the one hand to a bd type oxidase, and on the other hand to a bc(1)-->ba(3) pathway. We have also demonstrated the presence in the genome of transcriptionally active genes potentially encoding the subunits of a bo(3) type oxidase. A scheme for the electron transfer chains has been established that shows the existence of multiple respiratory routes to a single electron acceptor O(2). Possible reasons for these apparently redundant pathways are discussed.

Advanced glycation end products can induce glial reaction and neuronal degeneration in retinal explants.

BACKGROUND/AIMS: Neuronal degeneration has been reported to occur in diabetic retinopathy before the onset of detectable microvascular abnormalities. To investigate whether advanced glycation end products (AGE) could be directly responsible for retinal neurodegeneration, retinal explants were incubated with glycated bovine serum albumin (BSA). METHODS: Retinal explants obtained from non-diabetic adult rats were incubated 4 days with or without 200 mug/ml glycated BSA. Neural apoptosis was quantified by terminal dUTP nick end labelling (TUNEL) binding and immunostaining with anti-cleaved caspase-3 antibody. Expression of glial fibrillary acidic protein (GFAP) was localised by immunofluorescence. RESULTS: TUNEL and cleaved caspase-3 positive cells increased significantly by 2.2-fold and 2.5-fold in retinal explants incubated in glycated BSA (p<0.05), respectively. The ganglion cell layer was the most sensitive retinal layer to the glycated BSA. Neuronal degeneration was confirmed by the increased GFAP labelling in Müller glial cells from retinal explants treated with glycated BSA. CONCLUSION: These results suggest that AGE could induce retinal neurodegeneration in the absence of blood perfusion. Cells in the ganglion cell layer appeared to be the most sensitive as in diabetic retinopathy and its animal models. AGE toxicity could therefore contribute to the early pathological mechanisms of diabetic retinopathy.

[Optic neuropathy associated with primary antiphospholipid syndrome]. / Neuropathie optique associée à un syndrome primaire des anti-phospholipides.

We describe a 45-year-old male patient with an atypical unilateral optic neuropathy who was diagnosed with primary antiphospholipid syndrome. The initially poor vision lasting several months completely recovered and long-term oral anticoagulation therapy prevented potential further systemic thrombotic complications.

Specificities of the two center N inhibitors of mitochondial bc1 complex, antimycin and funiculosin: strong involvement of cytochrome b-asparagine-208 in funiculosin binding.

Funiculosin, a center N inhibitor of the bc1 complex, induces a blue-shift in the cytochrome b spectrum. A thermosensitive revertant [Coppee, J.Y. et al., J. Biol. Chem. 269 (1994) 4221-4226] isolated from a cytochrome b respiratory-deficient mutant, exhibits a red-shift instead of the blue-shift retained in the original mutant and shows resistance to this inhibitor. Replacing cytochrome b-Asparagine-208 by Lysine in this revertant, keeping the original mutation S206L, leads, when mitochondria are incubated at non-permissive temperature, to complete loss of bc1 complex activity and funiculosin-binding, while the antimycin-binding is conserved. These data suggest some inhibitor site specificity and close proximity between the funiculosin-binding site and the catalytic center N domain (QN).

Subretinal fluid in primary rhegmatogenous retinal detachment: physiopathology and composition.

During retinal detachment, subretinal fluid is present, whose composition and physiopathology are still little known. Under normal conditions, osmotic and oncotic pressures help keep the retina in place, but the main retinal attachment force is provided by active transport in the pigment epithelium. Subretinal fluid composition varies according to detachment duration; total protein concentration in subretinal fluid increases with time. In addition, all proteins are qualitatively modified. The detached retina loses its oxygen supply, and it then uses the anaerobic pathway to degrade glucose. Thus, long-duration retinal detachments feature increased lactic acid and dextrose concentrations. Phospholipids are also increased in subretinal fluid, reflecting retinal degradation. This review presents data on the physiopathology and composition of the subretinal fluid in retinal detachments.

Use of denaturing HPLC and automated sequencing to screen the VMD2 gene for mutations associated with Best's vitelliform macular dystrophy.

We identified three novel VMD2 mutations in patients with Best's macular dystrophy. DHPLC analysis of the 11 VMD2 exons revealed abnormal profiles in exon 8. Direct sequencing showed that these abnormal profiles were due to monoallelic transitions and transversions. We also found three polymorphic sequence changes that have been reported previously and annotated to an online database (http://www.uni-wuerzburg.de/humangenetics/vmd2.html).

Analysing the various obstacles to cornea postmortem procurement.

AIMS: In many countries the number of corneal donations is far too low to graft all patients on waiting lists within reasonable time. The aim of this study was to define specifically what practical changes are to be implemented to fully meet corneal graft demand. METHODS: The list of potential donors drawn by the coordination team from 1 January to 31 December 1999 was compared with that of all patients who had died during the same period. In each identified record, the parameters which permitted or precluded effective collection of cornea specimens were analysed, and the reasons why other records were not identified were investigated. RESULTS: Among the 1112 patients who died in 1999, coordinating nurses were able to identify 451 records (40.5 %) including 329 patients aged between 18 and 85 years (29.5%). After excluding 184 patients (55.9 %) who presented with medical contraindications, the coordinating nurses were able to meet the relatives of only 55 out of 145 patients (38%) and obtained their agreement in 39 cases (71% approval rate). Therefore, relatives' refusal was the cause for the absence of collection in only 5.5% of cases (16/290). The number of corneas procured amounted to 11.8% of identified records and 3.5% of all deceased patients. CONCLUSION: French law and regulations regarding tissue collection are based on consent presumption but it requires that verifications be made with the relatives to ensure that potential donors were not, before their death, opposed to such tissue procurement. That provision implies a high degree of organisation on the part of coordinating teams. It was demonstrated that donation shortage is no longer the result of relatives' refusal but rather because of logistical difficulties (potential donors not identified and problems in reaching relatives). It appears necessary therefore to strengthen coordinating teams with sufficient staff levels for wider donor identification. Those teams should also find ways to keep closer contact with relatives, so as to meet the maximum transparency targets required by public opinion and regulations and to graft all patients awaiting corneal transplantation.

Peribulbar anaesthesia during keratoplasty: a prospective study of 100 cases.

AIMS: A prospective study was carried out in order to evaluate the efficacy and safety of peribulbar anaesthesia during keratoplasty and to describe surgical conditions. METHODS: Of 137 consecutive keratoplasties, 100 (73%) were performed under peribulbar anaesthesia. Patients received a mean volume of 16.5 (SD 4) ml (range 9-22 ml) of a mixture of etidocaine, bupivacaine, and hyaluronidase. Ocular compression duration was at least 20 minutes and intraocular pressure (IOP) was measured with a Tonopen after injection, compression, and before trephination. Degree of akinesia, pain scoring, complications, and surgical conditions were studied. RESULTS: Before trephination, IOP was 5.73 mm Hg below the preinjection value and was never above 21 mm Hg. Akinesia was complete in 80% of cases and 94% of patients found that surgery was painless. Two patients (2%) were very agitated during surgery. The last patient presented with an acute intraoperative suprachoroidal haemorrhage that did not result in a true expulsive haemorrhage despite an "open sky" situation. Surgical conditions were judged to be optimal by the patients in 92% of cases and by the surgeon in 98% of cases. CONCLUSION: These results demonstrate that peribulbar anaesthesia offers excellent anaesthesia and akinesia during keratoplasty and may be recommended for this type of surgery.

Deep lamellar keratoplasty as surgical management of anterior and posterior segment injuries to the eye.

PURPOSE: To report on the feasibility of combined deep lamellar keratoplasty and vitreoretinal surgery in one patient with corneal opacity associated with retinal detachment. METHODS: A 35-year-old man presented with a major hematocornea and retinal detachment after experiencing a right ocular trauma with corneoscleral wound 1 month earlier. We elected to perform deep lamellar keratoplasty to perform vitreoretinal surgery through the bared Descemet's membrane within the same surgical procedure. RESULTS: Deep lamellar keratoplasty offered perfect visibility of the anterior and posterior segments of the eye through the bared Descemet's membrane during the 4-hour operation. Descemet's membrane was resilient enough to maintain remarkable tightness of the anterior chamber throughout vitreoretinal surgery procedures (vitrectomy, peeling of epiretinal membranes, encircling scleral buckling). Unfortunately, despite our efforts and extended operative time, the retina could not be restored to its position because of the high baseline level of ocular impairment. CONCLUSION: The combined procedure (deep lamellar keratoplasty and pars plana vitrectomy) appeared to be a good and feasible alternative to the temporary keratoprosthesis usually applied in that situation.

Successful treatment of Acanthamoeba keratitis by hexamidine.

Acanthamoeba keratitis remains very difficult to treat because of the lack of antiamoebic agents completely effective against cysts. Currently, the recommended treatment includes the use of topical neomycin sulfate, various imidazoles, and propamidine isethionate (Brolene) 0.1% eye drops, a compound of the diamidine family. In the present article, we describe the successful management of two patients with an Acanthamoeba keratitis, treated with hexamidine diisethionate (Desomedine) 0.1% eye drops, another diamidine derivative, which was found amoebicidal in vitro on the isolated Acanthamoeba strains. This is to our knowledge the first report on the amoebicidal effectiveness of hexamidine, simultaneously in vitro and in vivo.

The interview with the donor's family before postmortem cornea procurement.

PURPOSE: Asking the family of a deceased patient to consider eye donation is one of the most difficult aspects of the donation process. The aim of this prospective study was to describe the content of interviews with the families of potential donors and to analyze their reactions to improve the process of eye donation. METHODS: We consecutively met with 151 families of suitable corneal donors at the Rouen University Hospital. All interviews with donor families were analyzed using a preestablished questionnaire. RESULTS: In only 17.9% of cases was the family aware of the potential donor's last will. In 77.7% of these cases, the patient wished to donate. Procurement rate was 71.5%. This acceptance was mostly facilitated by the awareness and motivation of the hospital staff, the experience of the physician, and the 13.3-h period of time allowed after the donor's death. The commitment on the part of the ophthalmologist to carry out optimal anatomical restoration was a very important point for 32% of families who accepted donation. Twenty-one percent of families asked for a delay for reflection. This delay helped to obtain a positive response in 72.7% of cases and even sometimes helped families to reconsider a previously negative position (14% of initial refusals). CONCLUSION: We demonstrate that a high positive response (71.5%) can be obtained from the donor's family when a trained and motivated group manages the post-mortem cornea donation request.

Sensitivity of the terrestrial biosphere to climatic changes: impact on the carbon cycle.

The biosphere is a major pool in the global carbon cycle; its response to climatic change is therefore of great importance. We developed a 5 degrees x 5 degrees longitude-latitude resolution model of the biosphere in which the global distributions of the major biospheric variables, i.e. the vegetation types and the main carbon pools and fluxes, are determined from climatic variables. We defined nine major broad vegetation types: perennial ice, desert and semi-desert, tundra, coniferous forest, temperate deciduous forest, grassland and shrubland, savannah, seasonal tropical forest and evergreen tropical forest. Their geographical repartition is parameterized using correlations between observed vegetation type, precipitation and biotemperature distributions. The model computes as a function of climate and vegetation type, the variables related to the continental biospheric carbon cycle, i.e. the carbon pools such as the phytomass, the litter and the soil organic carbon; and carbon fluxes such as net primary production, litter production and heterotrophic respiration. The modeled present-day biosphere is in good agreement with observation. The model is used to investigate the response of the terrestrial biosphere to climatic changes as predicted by different General Circulation Models (GCM). In particular, the impact on the biosphere of climatic conditions corresponding to the last glacial climate (LGM), 18 000 years ago, is investigated. Comparison with results from present-day climate simulations shows the high sensitivity of the geographical distribution of vegetation types and carbon content as well as biospheric trace gases emissions to climatic changes. The general trend for LGM compared to the present is an increase in low density vegetation types (tundra, desert, grassland) to the detriment of forested areas, in tropical as well as in other regions. Consequently, the biospheric activity (carbon fluxes and trace gases emissions) was reduced.