The supramolecular assemblies of voltage-dependent anion channels in the native membrane

Voltage-dependent anion channels (VDACs) are major constituents of the outer mitochondrial membrane (OMM). These primary transporters of nucleotides, ions and metabolites mediate a substantial portion of the OMM molecular traffic. To study the native supramolecular organization of the VDAC, we have isolated, characterized and imaged OMMs from potato tubers. SDS-PAGE and mass spectrometry of OMMs revealed the presence of the VDAC isoforms POM34 and POM36, as well as the translocase of the OMM complex. Tubular two-dimensional crystals of the VDAC spontaneously formed after incubation of OMMs for two to three months at 4 degrees C. Transmission electron microscopy revealed an oblique lattice and unit cells housing six circular depressions arranged in a hexagon. Atomic force microscopy of freshly isolated OMMs demonstrated (i) the existence of monomers to tetramers, hexamers and higher oligomers of the VDAC and (ii) its spatial arrangement within the oligomers in the native membrane. We discuss the importance of the observed oligomerization for modulation of the VDAC function, for the binding of hexokinase and creatine kinase to the OMM and for mitochondria-mediated apoptosis.     

Primary cell model for activation-inducible human immunodeficiency virus

Quiescent T lymphocytes containing latent human immunodeficiency virus (HIV) provide a long-lived viral reservoir. This reservoir may be the source of active infection that is reinitiated following the cessation of antiretroviral therapy. Therefore, it is important to understand the mechanisms involved in latent infection to develop new strategies to eliminate the latent HIV reservoir. We have previously demonstrated that latently infected quiescent lymphocytes can be generated during thymopoiesis in vivo in the SCID-hu mouse system. However, there is still a pressing need for an in vitro model of HIV latency in primary human cells. Here, we present a novel in vitro model that recapitulates key aspects of dormant HIV infection. Using an enhanced green fluorescent protein-luciferase fusion protein-containing reporter virus, we have generated a stable infection in primary human CD4(+) CD8(+) thymocytes in the absence of viral gene expression. T-cell activation induces a >200-fold induction of reporter activity. The induced reporter activity originates from a fully reverse-transcribed and integrated genome. We further demonstrate that this model can be useful to study long terminal repeat regulation, as previously characterized NF-kappaB response element mutations decrease the activation of viral gene expression. This model can therefore be used to study intricate molecular aspects of activation-inducible HIV infection in primary cells.     

Does the calcein-AM method assay the total cellular 'labile iron pool' or only a fraction of it?

The calcein-AM (calcein-acetoxymethyl ester) method is a widely used technique that is supposed to assay the intracellular 'labile iron pool' (LIP). When cells in culture are exposed to this ester, it passes the plasma membrane and reacts with cytosolic unspecific esterases. One of the reaction products, calcein, is a fluorochrome and a hydrophilic alcohol to which membranes are non-permeable and which, consequently, is retained within the cytosol of cells. Calcein fluorescence is quenched following chelation of low-mass labile iron, and the degree of quenching gives an estimate of the amounts of chelatable iron. However, a requirement for the assay to be able to demonstrate cellular LIP in total is that such iron be localized in the cytosol and not in a membrane-limited compartment. For some time it has been known that a major part of cellular, redox-active, labile, low-mass iron is temporarily localized in the lysosomal compartment as a result of the autophagic degradation of ferruginous materials, such as mitochondrial complexes and ferritin. Even if some calcein-AM may escape cytosolic esterases and enter lysosomes to be cleaved by lysosomal acidic esterases, the resulting calcein does not significantly chelate iron at 相似文献   

Mechanisms of programmed cell death during oogenesis in <Emphasis Type="Italic">Drosophila virilis</Emphasis>

We describe the features of programmed cell death occurring in the egg chambers of Drosophila virilis during mid-oogenesis and late oogenesis. During mid-oogenesis, the spontaneously degenerating egg chambers exhibit typical characteristics of apoptotic cell death. As revealed by propidium iodide, rhodamine-conjugated phalloidin staining, and the TUNEL assay, respectively, the nurse cells contain condensed chromatin, altered actin cytoskeleton, and fragmented DNA. In vitro caspase activity assays and immunostaining procedures demonstrate that the atretic egg chambers possess high levels of caspase activity. Features of autophagic cell death are also observed during D. virilis mid-oogenesis, as shown by monodansylcadaverine staining, together with an ultrastructural examination by transmission electron microscopy. During the late stages of oogenesis in D. virilis, once again, the two mechanisms, viz., nurse cell cluster apoptosis and autophagy, operate together, manifesting features of cell death similar to those detailed above. Moreover, an altered form of cytochrome c seems to be released from the mitochondria in the nurse cells proximal to the oocyte. We propose that apoptosis and autophagy function synergistically during oogenesis in D. virilis in order to achieve a more efficient elimination of the degenerated nurse cells and abnormal egg chambers. The present study was co-financed within Op. Education by the European Social Fund and by National Resources via a grant (HRAKLEITOS 70/3/7164) to Professor L.H. Margaritis.     

Colloidal stability of carbon nanotubes in an aqueous dispersion of phospholipid

Within the family of nanomaterials, carbon nanotubes (CNTs) have emerged as a new efficient scaffold for studying molecular interactions at interfaces. Poor dispersability of CNTs in any solvent presents a considerable drawback for the development of novel functional composite structures. Previous studies have demonstrated that the solubility of CNTs can be greatly enhanced by employing appropriate surfactants, some of them being biological molecules. In this work, we study the noncovalent wrapping of lipid chains onto the graphitic surface of single-walled material (SWCNTs) by electron microscopy and Raman spectroscopy. Stable and homogenous aqueous suspensions of SWCNTs in the presence of lipids have been prepared, whereas their electrophoretic mobility was confirmed by zeta-potential measurements. Raman measurements revealed that smaller diameter SWCNTs are preferentially dispersed by lipid molecules in the aqueous supernatant part of the prepared suspension.     

Multidrug-resistant endosymbiotic bacteria account for the emergence of zygomycosis: A hypothesis

Although the extensive use of Aspergillus-active antifungals has been recently associated with an increase in zygomycosis in several cancer centers, the frequency of this opportunistic mycosis began to rise earlier, since the mid 1990s. The reasons for that emergence are unclear. Recent evidence suggests that endosymbiotic bacteria of Rhizopus species produce toxins that enhance fungal pathogenicity. We postulate that, although Zygomycetes appear equally ubiquitous and virulent to Aspergillus, zygomycosis was rare in the past in immunosuppressed patients specifically because of the widespread use of antibacterials in this patient population. Such use may have resulted in inhibition of endosymbiotic, toxin-producing bacteria and led indirectly in attenuation of Zygomycetes virulence. Thus, the growing rates of antimicrobial resistance over the past decade selected for multidrug-resistant endosymbiotic bacteria of Zygomycetes, which could facilitate the emergence of zygomycosis. This hypothesis, if true, will be the first paradigm of modulation of virulence of opportunistic fungi by antibacterials.     

Inhibition of ozone-induced SP-A oxidation by plant polyphenols

Surfactant protein-A (SP-A) is the best studied and most abundant of the protein components of lung surfactant and plays an important role in host defense of the lung. It has been shown that ozone-induced oxidation of SP-A protein changes its functional and biochemical properties. In the present study, eight plant polyphenols (three flavonoids, three hydroxycinnamic acids, and two hydroxybenzoic acids) known as strong antioxidants, were tested for their ability to inhibit ozone-induced SP-A oxidation as a mechanism for chemoprevention against lung damage. SP-A isolated from alveolar proteinosis patients was exposed to ozone (1 ppm) for 4 h. The flavonoids protected SP-A from oxidation in a dose dependent manner. ( - )-Epicatechin was the most potent flavonoid and exhibited inhibition of ozone-induced formation of carbonyls by 35% at a concentration as low as 5 μM. Hydroxybenzoic acids inhibited SP-A oxidation in a dose-dependent manner although they were less potent than flavonoids. On the other hand, hydroxycinnamic acids exhibited a different inhibitory pattern. Inhibition was observed only at medium concentrations. The results indicate that inhibition of SP-A oxidation by plant polyphenols may be a mechanism accounting for the protective activity of natural antioxidants against the effects of ozone exposure on lungs.     

Morphologic and morphometrical study of the muscle spindle in muscular dystrophy

OBJECTIVE: To determine the morphologic and the morphometrical features of spindles in biopsies of patients with different types of muscular dystrophy and investigate the possible involvement of the spindle in the pathologic process of these diseases. STUDY DESIGN: The following variables were studied in biopsy specimens from 10 patients with Duchenne or Becker dystrophy, 9 with limb-girdle dystrophy, 3 with congenital dystrophy and 3 with facioscapulohumeral dystrophy: diameter and area of spindle; thickness of the capsule; number, diameter and area of intrafusal fibers; and number and area of nuclei. RESULTS: The statistical evaluation of the data showed significant differences regarding the thickness of the capsule, which was greater in patients than in controls, while the diameter and the area of the fibers were all smaller in patients than in controls. The area of nuclei of fibers was increased; this was a common feature for all types of muscular dystrophy. CONCLUSION: These findings indicate that the spindle possibly participates in the pathologic process of different types of muscular dystrophies.     

Oxidative stress biomarkers responses to physical overtraining: implications for diagnosis

Overtraining syndrome is characterized by declining performance and transient inflammation following periods of severe training with major health implications for the athletes. Currently, there is no single diagnostic marker for overtraining. The present investigation examined the responses of oxidative stress biomarkers to a resistance training protocol of progressively increased and decreased volume/intensity. Twelve males (21.3+/-2.3 years) participated in a 12-week resistance training consisting of five 3-week periods (T1, 2 tones/week; T2, 8 tones/week; T3, 14 tones/week; T4, 2 tones/week), followed by a 3-week period of complete rest. Blood/urine samples were collected at baseline and 96 h following the last training session of each period. Performance (strength, power, jumping ability) increased after T2 and declined thereafter, indicating an overtraining response. Overtraining (T3) induced sustained leukocytosis, an increase of urinary isoprostanes (7-fold), TBARS (56%), protein carbonyls (73%), catalase (96%), glutathione peroxidase, and oxidized glutathione (GSSG) (25%) and a decline of reduced glutathione (GSH) (31%), GSH/GSSG (56%), and total antioxidant capacity. Isoprostanes and GSH/GSSG were highly (r=0.764-0.911) correlated with performance drop and training volume increase. In conclusion, overtraining induces a marked response of oxidative stress biomarkers which, in some cases, was proportional to training load, suggesting that they may serve as a tool for overtraining diagnosis.