Ultrastructure of the membrane attack complex of complement: detection of the tetramolecular C9-polymerizing complex C5b-8.

The ultrastructure of the membrane attack complex (MAC) of complement had been described as representing a hollow cylinder of defined dimensions that is composed of the proteins C5b, C6, C7, C8, and C9. After the characteristic cylindrical structure was identified as polymerized C9 [poly(C9)], the question arose as to the ultrastructural identity and topology of the C9-polymerizing complex C5b-8. An electron microscopic analysis of isolated MAC revealed an asymmetry of individual complexes with respect to their length. Whereas the length of one boundary (+/- SEM) was always 16 +/- 1 nm, the length of the other varied between 16 and 32 nm. In contrast, poly(C9), formed spontaneously from isolated C9, had a uniform tubule length (+/- SEM) of 16 +/- 1 nm. On examination of MAC-phospholipid vesicle complexes, an elongated structure was detected that was closely associated with the poly(C9) tubule and that extended 16-18 nm beyond the torus of the tubule and 28-30 nm above the membrane surface. The width of this structure varied depending on its two-dimensional projection in the electron microscope. By using biotinyl C5b-6 in the formation of the MAC and avidin-coated colloidal gold particles for the ultrastructural analysis, this heretofore unrecognized subunit of the MAC could be identified as the tetramolecular C5b-8 complex. Identification also was achieved by using anti-C5 Fab-coated colloidal gold particles. A similar elongated structure of 25 nm length (above the surface of the membrane) was observed on single C5b-8-vesicle complexes. It is concluded that the C5b-8 complex, which catalyzes poly(C9) formation, constitutes a structure of discrete morphology that remains as such identifiable in the fully assembled MAC, in which it is closely associated with the poly(C9) tubule.

Protein-binding site at the immunoglobulin mu membrane polyadenylylation signal: possible role in transcription termination.

mRNAs specifying immunoglobulin mu and delta heavy chains are encoded by a single large, complex transcription unit (mu + delta gene). The transcriptional activity of delta gene segments in terminally differentiated, IgM-secreting B lymphocytes is 10-20 times lower than in earlier B-lineage cells expressing delta mRNA. We find that transcription of the mu + delta gene in IgM-secreting murine myeloma cells terminates within a region of 500-1000 nucleotides immediately following the mu membrane (mu m) polyadenylylation site. Transcription decreases only minimally through this region in murine cell lines representative of earlier stages in B-cell development. A DNA fragment containing the mu m polyadenylylation signal gives protein-DNA complexes with different mobilities in gel retardation assays with nuclear extracts from myeloma cells than with nuclear extracts from earlier B-lineage cells. However, using a recently developed "footprinting" procedure in which protein-DNA complexes resolved in gel retardation assays are subjected to nucleolytic cleavage while still in the polyacrylamide gel, we find that the DNA sequences protected by factors from the two cell types are indistinguishable. The factor-binding site on the DNA is located 5' of the mu m polyadenylylation signal AATAAA and includes the 15-nucleotide-long A + T-rich palindrome CTGTAAACAAATGTC. This type of palindromic binding site exhibits orientation-dependent activity consistent with the reported properties of polymerase II termination signals. This binding site is followed by two sets of directly repeated DNA sequences with different helical conformation as revealed by their reactivity with the chemical nuclease 1,10-phenanthroline-copper. The close proximity of these features to the signals for mu m mRNA processing may reflect a linkage of the processes of developmentally regulated mu m polyadenylylation and transcription termination.

Catalytic core of a membrane-associated eukaryotic polyphosphate polymerase.

Polyphosphate (polyP) occurs ubiquitously in cells, but its functions are poorly understood and its synthesis has only been characterized in bacteria. Using x-ray crystallography, we identified a eukaryotic polyphosphate polymerase within the membrane-integral vacuolar transporter chaperone (VTC) complex. A 2.6 angstrom crystal structure of the catalytic domain grown in the presence of adenosine triphosphate (ATP) reveals polyP winding through a tunnel-shaped pocket. Nucleotide- and phosphate-bound structures suggest that the enzyme functions by metal-assisted cleavage of the ATP gamma-phosphate, which is then in-line transferred to an acceptor phosphate to form polyP chains. Mutational analysis of the transmembrane domain indicates that VTC may integrate cytoplasmic polymer synthesis with polyP membrane translocation. Identification of the polyP-synthesizing enzyme opens the way to determine the functions of polyP in lower eukaryotes.

Extracorporeal membrane oxygenation support in acardia.

In extreme situations, such as hyperacute rejection of heart transplant or major heart trauma, heart preservation may not be possible. Our experimental team works on a project of peripheral extracorporeal membrane oxygenation (ECMO) support in acardia as a bridge to heart transplantation or artificial heart implantation. An ECMO support was established in five calves (58.6 ± 6.9 kg) by the transjugular insertion to the caval axis of a self-expanded cannula, with carotid artery return. After baseline measurements, ventricular fibrillation was induced, great arteries were clamped, heart was excised, and right and left atria remnants, containing pulmonary veins, were sutured together leaving an atrial septal defect over the caval axis cannula. Measurements of pump flow and arterial pressure were taken with the pulmonary artery clamped and anastomosed with the caval axis for a total of 6 hours. Pulmonary artery anastomosis to the caval axis provided an acceptable 6 hour hemodynamic stability, permitting a peripheral access ECMO support in extreme scenarios indicating a heart explantation.

Hybrid Multiscale Finite Volume method for two-phase flow in porous media

We present a novel hybrid (or multiphysics) algorithm, which couples pore-scale and Darcy descriptions of two-phase flow in porous media. The flow at the pore-scale is described by the Navier?Stokes equations, and the Volume of Fluid (VOF) method is used to model the evolution of the fluid?fluid interface. An extension of the Multiscale Finite Volume (MsFV) method is employed to construct the Darcy-scale problem. First, a set of local interpolators for pressure and velocity is constructed by solving the Navier?Stokes equations; then, a coarse mass-conservation problem is constructed by averaging the pore-scale velocity over the cells of a coarse grid, which act as control volumes; finally, a conservative pore-scale velocity field is reconstructed and used to advect the fluid?fluid interface. The method relies on the localization assumptions used to compute the interpolators (which are quite straightforward extensions of the standard MsFV) and on the postulate that the coarse-scale fluxes are proportional to the coarse-pressure differences. By numerical simulations of two-phase problems, we demonstrate that these assumptions provide hybrid solutions that are in good agreement with reference pore-scale solutions and are able to model the transition from stable to unstable flow regimes. Our hybrid method can naturally take advantage of several adaptive strategies and allows considering pore-scale fluxes only in some regions, while Darcy fluxes are used in the rest of the domain. Moreover, since the method relies on the assumption that the relationship between coarse-scale fluxes and pressure differences is local, it can be used as a numerical tool to investigate the limits of validity of Darcy's law and to understand the link between pore-scale quantities and their corresponding Darcy-scale variables.

Hydrophobic labeling of (Na+,K+)-ATPase: further evidence that the beta subunit is embedded in the membrane bilayer.

O-Hexanoyl-3,5-diiodo-N-(4-azido-2-nitro-phenyl)tyramine has been used after photochemical conversion into the reactive nitrene to label (Na+,K+)-ATPase from Bufo marinus toad kidney. Immunochemical evidence indicates that the reagent labels both subunits of the enzyme in partially purified form as well as in microsomal membranes. These results support the view that the glycoprotein subunit, like the catalytic subunit, possesses hydrophobic domains by which it is integrated into the plasma membrane.

Merging the hypothetical extraction method and the classical multiplier approach: a hybrid possibility for identifying key distributive sectors

The two main alternative methods used to identify key sectors within the input-output approach, the Classical Multiplier method (CMM) and the Hypothetical Extraction method (HEM), are formally and empirically compared in this paper. Our findings indicate that the main distinction between the two approaches stems from the role of the internal effects. These internal effects are quantified under the CMM while under the HEM only external impacts are considered. In our comparison, we find, however that CMM backward measures are more influenced by within-block effects than the proposed forward indices under this approach. The conclusions of this comparison allow us to develop a hybrid proposal that combines these two existing approaches. This hybrid model has the advantage of making it possible to distinguish and disaggregate external effects from those that a purely internal. This proposal has also an additional interest in terms of policy implications. Indeed, the hybrid approach may provide useful information for the design of ''second best'' stimulus policies that aim at a more balanced perspective between overall economy-wide impacts and their sectoral distribution.

Regulation of membrane trafficking and organ separation by the NEVERSHED ARF-GAP protein.

Cell separation, or abscission, is a highly specialized process in plants that facilitates remodeling of their architecture and reproductive success. Because few genes are known to be essential for organ abscission, we conducted a screen for mutations that alter floral organ shedding in Arabidopsis. Nine recessive mutations that block shedding were found to disrupt the function of an ADP-ribosylation factor-GTPase-activating protein (ARF-GAP) we have named NEVERSHED (NEV). As predicted by its homology to the yeast Age2 ARF-GAP and transcriptional profile, NEV influences other aspects of plant development, including fruit growth. Co-localization experiments carried out with NEV-specific antiserum and a set of plant endomembrane markers revealed that NEV localizes to the trans-Golgi network and endosomes in Arabidopsis root epidermal cells. Interestingly, transmission electron micrographs of abscission zone regions from wild-type and nev flowers reveal defects in the structure of the Golgi apparatus and extensive accumulation of vesicles adjacent to the cell walls. Our results suggest that NEV ARF-GAP activity at the trans-Golgi network and distinct endosomal compartments is required for the proper trafficking of cargo molecules required for cell separation.

Exogenous selection shapes germination behaviour and seedling traits of populations at different altitudes in a Senecio hybrid zone.

BACKGROUND AND AIMS: The Senecio hybrid zone on Mt Etna, Sicily, is characterized by steep altitudinal clines in quantitative traits and genetic variation. Such clines are thought to be maintained by a combination of 'endogenous' selection arising from genetic incompatibilities and environment-dependent 'exogenous' selection leading to local adaptation. Here, the hypothesis was tested that local adaptation to the altitudinal temperature gradient contributes to maintaining divergence between the parental species, S. chrysanthemifolius and S. aethnensis. METHODS: Intra- and inter-population crosses were performed between five populations from across the hybrid zone and the germination and early seedling growth of the progeny were assessed. KEY RESULTS: Seedlings from higher-altitude populations germinated better under low temperatures (9-13 °C) than those from lower altitude populations. Seedlings from higher-altitude populations had lower survival rates under warm conditions (25/15 °C) than those from lower altitude populations, but also attained greater biomass. There was no altitudinal variation in growth or survival under cold conditions (15/5 °C). Population-level plasticity increased with altitude. Germination, growth and survival of natural hybrids and experimentally generated F(1)s generally exceeded the worse-performing parent. CONCLUSIONS: Limited evidence was found for endogenous selection against hybrids but relatively clear evidence was found for divergence in seed and seedling traits, which is probably adaptive. The combination of low-temperature germination and faster growth in warm conditions might enable high-altitude S. aethnensis to maximize its growth during a shorter growing season, while the slower growth of S. chrysanthemifolius may be an adaptation to drought stress at low altitudes. This study indicates that temperature gradients are likely to be an important environmental factor generating and maintaining adaptive divergence across the Senecio hybrid zone on Mt Etna.

Cardiopulmonary support and extracorporeal membrane oxygenation for cardiac assist.

BACKGROUND: Use of cardiopulmonary bypass for emergency resuscitation is not new. In fact, John Gibbon proposed this concept for the treatment of severe pulmonary embolism in 1937. Significant progress has been made since, and two main concepts for cardiac assist based on cardiopulmonary bypass have emerged: cardiopulmonary support (CPS) and extracorporeal membrane oxygenation (ECMO). The objective of this review is to summarize the state of the art in these two technologies. METHODS: Configuration of CPS is now fairly standard. A mobile cart with relatively large wheels allowing for easy transportation carries a centrifugal pump, a back-up battery with a charger, an oxygen cylinder, and a small heating system. Percutaneous cannulation, pump-driven venous return, rapid availability, and transportability are the main characteristics of a CPS system. Cardiocirculatory arrest is a major predictor of mortality despite the use of CPS. In contrast, CPS appears to be a powerful tool for patients in cardiogenic shock before cardiocirculatory arrest, requiring some type of therapeutic procedures, especially repair of anatomically correctable problems or bridging to other mechanical circulatory support systems such as ventricular assist devices. CPS is in general not suitable for long-term applications because of the small-bore cannulas, resulting in significant pressure gradients and eventually hemolysis. RESULTS: In contrast, ECMO can be designed for longer-term circulatory support. This requires large-bore cannulas and specifically designed oxygenators. The latter are either plasma leakage resistent (true membranes) or relatively thrombo-resistant (heparin coated). Both technologies require oxygenator changeovers although the main reason for this is different (clotting for the former, plasma leakage for the latter). Likewise, the tubing within a roller pump has to be displaced and centrifugal pump heads have to be replaced over time. ECMO is certainly the first choice for a circulatory support system in the neonatal and pediatric age groups, where the other assist systems are too bulky. ECMO is also indicated for patients improving on CPS. Septic conditions are, in general, considered as contraindications for ECMO. CONCLUSIONS: Ease of availability and moderate cost of cardiopulmonary bypass-based cardiac support technologies have to be balanced against the significant immobilization of human resources, which is required to make them successful.

La chirurgie combinée phacoémulsification/pelage de membrane épirétinienne et oedème maculaire [Combined phacoemulsification/membrane resection and macular edema].

PURPOSE: To evaluate the occurrence of macular edema (ME) after epiretinal membrane resection, managed either with simple vitrectomy or with combined vitrectomy and phacoemulsification. MATERIAL AND METHODS: Two groups of 12 patients had a vitrectomy for epiretinal membrane associated or not to a phacoemulsification. A fundus fluorescein angiography was performed pre and postoperatively and at least 3 months after the surgery. RESULTS: In the group of patients who had a simple vitrectomy, a ME was observed in 50% of the cases preoperatively and in 25% of the cases at the end of follow-up. In 3 cases, preoperative ME was worsened after the surgery. In the group of patients who were treated by a combined vitrectomy and phacoemulsification, a ME was observed in 25% of the cases preoperatively and in 50% of the cases at the end of follow-up. A de novo ME was observed in 3 cases. CONCLUSION: Combined vitrectomy and cataract surgery could allow a rapid recovery of visual acuity but might increase the occurrence of ME.

Evaluation of organ-specific peripheral doses after 2-dimensional, 3-dimensional and hybrid intensity modulated radiation therapy for breast cancer based on Monte Carlo and convolution/superposition algorithms: implications for secondary cancer risk assessment.

To make a comprehensive evaluation of organ-specific out-of-field doses using Monte Carlo (MC) simulations for different breast cancer irradiation techniques and to compare results with a commercial treatment planning system (TPS). Three breast radiotherapy techniques using 6MV tangential photon beams were compared: (a) 2DRT (open rectangular fields), (b) 3DCRT (conformal wedged fields), and (c) hybrid IMRT (open conformal+modulated fields). Over 35 organs were contoured in a whole-body CT scan and organ-specific dose distributions were determined with MC and the TPS. Large differences in out-of-field doses were observed between MC and TPS calculations, even for organs close to the target volume such as the heart, the lungs and the contralateral breast (up to 70% difference). MC simulations showed that a large fraction of the out-of-field dose comes from the out-of-field head scatter fluence (>40%) which is not adequately modeled by the TPS. Based on MC simulations, the 3DCRT technique using external wedges yielded significantly higher doses (up to a factor 4-5 in the pelvis) than the 2DRT and the hybrid IMRT techniques which yielded similar out-of-field doses. In sharp contrast to popular belief, the IMRT technique investigated here does not increase the out-of-field dose compared to conventional techniques and may offer the most optimal plan. The 3DCRT technique with external wedges yields the largest out-of-field doses. For accurate out-of-field dose assessment, a commercial TPS should not be used, even for organs near the target volume (contralateral breast, lungs, heart).

Studies of membrane fluidity and heart contractile force in Trypanosoma cruzi infected mice

In Chagas disease serious cardiac dysfunction can appear. We specifically studied the cardiac function by evaluating: ventricle contractile force and norepinephrine response, affinity and density of beta-adrenergic receptors, dynamic properties of myocardial membranes, and electrocardiography. Albino swiss mice (n = 250) were infected with 55 trypomastigotes, Tulahuen strain and studied at 35, 75, and 180 days post-infection, that correspond to the acute, indeterminate, and chronic phase respectively. Cardiac beta-adrenergic receptors' affinity, myocardial contractility, and norepinephrine response progressively decreased from the acute to the chronic phase of the disease (p < 0.01). The density (expressed as fmol/mg.prot) of the receptors was similar to non-infected mice (71.96 ± 0.36) in both the acute (78.24 ± 1.67) and indeterminate phases (77.28 ± 0.91), but lower in the chronic disease (53.32 ± 0.71). Electrocardiographic abnormalities began in the acute phase and were found in 65% of the infected-mice during the indeterminate and chronic phases. Membrane contents of triglycerides, cholesterol, and anisotropy were similar in all groups. A quadratic correlation between the affinity to beta-adrenergic receptors and cardiac contractile force was obtained. In conclusion the changes in cardiac beta-adrenergic receptors suggests a correlation between the modified beta-adrenergic receptors affinity and the cardiac contractile force.

SCAP is required for timely and proper myelin membrane synthesis.

Myelination requires a massive increase in glial cell membrane synthesis. Here, we demonstrate that the acute phase of myelin lipid synthesis is regulated by sterol regulatory element-binding protein (SREBP) cleavage activation protein (SCAP), an activator of SREBPs. Deletion of SCAP in Schwann cells led to a loss of SREBP-mediated gene expression involving cholesterol and fatty acid synthesis. Schwann cell SCAP mutant mice show congenital hypomyelination and abnormal gait. Interestingly, aging SCAP mutant mice showed partial regain of function; they exhibited improved gait and produced small amounts of myelin indicating a slow SCAP-independent uptake of external lipids. Accordingly, extracellular lipoproteins partially rescued myelination by SCAP mutant Schwann cells. However, SCAP mutant myelin never reached normal thickness and had biophysical abnormalities concordant with abnormal lipid composition. These data demonstrate that SCAP-mediated regulation of glial lipogenesis is key to the proper synthesis of myelin membrane, and provide insight into abnormal Schwann cell function under conditions affecting lipid metabolism.