Bioenergetics of lung tumors: alteration of mitochondrial biogenesis and respiratory capacity.

Little is known on the metabolic profile of lung tumors and the reminiscence of embryonic features. Herein, we determined the bioenergetic profiles of human fibroblasts taken from lung epidermoid carcinoma (HLF-a) and fetal lung (MRC5). We also analysed human lung tumors and their surrounding healthy tissue from four patients with adenocarcinoma. On these different models, we measured functional parameters (cell growth rates in oxidative and glycolytic media, respiration, ATP synthesis and PDH activity) as well as compositional features (expression level of various energy proteins and upstream transcription factors). The results demonstrate that both the lung fetal and cancer cell lines produced their ATP predominantly by glycolysis, while oxidative phosphorylation was only capable of poor ATP delivery. This was explained by a decreased mitochondrial biogenesis caused by a lowered expression of PGC1alpha (as shown by RT-PCR and Western blot) and mtTFA. Consequently, the relative expression of glycolytic versus OXPHOS markers was high in these cells. Moreover, the re-activation of mitochondrial biogenesis with resveratrol induced cell death specifically in cancer cells. A consistent reduction of mitochondrial biogenesis and the subsequent alteration of respiratory capacity was also observed in lung tumors, associated with a lower expression level of bcl2. Our data give a better characterization of lung cancer cells' metabolic alterations which are essential for growth and survival. They designate mitochondrial biogenesis as a possible target for anti-cancer therapy.

Physical continuity of the perimysium from myofibers to tendons: involvement in lateral force transmission in skeletal muscle.

Advances in muscle physiology suggest that the perimysium plays a role in the transmission of lateral contractile forces. This hypothesis is strongly supported by our recent demonstration of the existence of "Perimysial Junctional Plates" in bovine Flexor carpi radialis muscle [Passerieux, E., Rossignol, R., Chopard, A., Carnino, A., Marini, J.F., Letellier, T., Delage, J.P. 2006. Structural organization of the perimysium in bovine skeletal muscle: junctional plates and associated intracellular subdomains. J. Struct. Biol. 154 (2), 206-216] However, the overall organization of the perimysium collagen network, as well as its continuity and heterogeneity, have still not been described in detail throughout the entire muscle. We used an extension of the standard NaOH digestion technique and scanning electron microscopy to analyze perimysium architecture in bovine Flexor carpi radialis muscle. First, we observed that the perimysium is made of a highly ordered network of collagen fibers, binding the myofibers from tendon to tendon. We identified basic collagen cable structures, characterized by a straight portion (3 cm long) in the direction of the myofibers and a curved terminal portion at 60 degrees. These cables reach the myofiber surface at the level of the previously described "Perimysial Junctional Plates". At a higher level of organization, these cables stick together to form the walls of numerous tubes arranged in a overlapping honeycomb pattern around the myofibers. At the ends of these tubes, the straight portions of the collagen cables ramify in large bundles that merge with the tendons. Taken together, these observations identify four levels of organization in the perimysium: (i) Perimysial Junctional Plates that constitute the focal attachment between the perimysium and the myofibers, (ii) collagen plexi attaching adjacent myofibers, (iii) a loose lattice of large interwoven fibers, and (iv) honeycomb tubes connecting two tendons. This spatial arrangement of the perimysium supports the view of a complex pattern of lateral force transmission from myofibers to tendons and adjacent muscles.

Physiological diversity of mitochondrial oxidative phosphorylation.

To investigate the physiological diversity in the regulation and control of mitochondrial oxidative phosphorylation, we determined the composition and functional features of the respiratory chain in muscle, heart, liver, kidney, and brain. First, we observed important variations in mitochondrial content and infrastructure via electron micrographs of the different tissue sections. Analyses of respiratory chain enzyme content by Western blot also showed large differences between tissues, in good correlation with the expression level of mitochondrial transcription factor A and the activity of citrate synthase. On the isolated mitochondria, we observed a conserved molar ratio between the respiratory chain complexes and a variable stoichiometry for coenzyme Q and cytochrome c, with typical values of [1-1.5]:[30-135]:[3]:[9-35]:[6.5-7.5] for complex II:coenzyme Q:complex III:cytochrome c:complex IV in the different tissues. The functional analysis revealed important differences in maximal velocities of respiratory chain complexes, with higher values in heart. However, calculation of the catalytic constants showed that brain contained the more active enzyme complexes. Hence, our study demonstrates that, in tissues, oxidative phosphorylation capacity is highly variable and diverse, as determined by different combinations of 1) the mitochondrial content, 2) the amount of respiratory chain complexes, and 3) their intrinsic activity. In all tissues, there was a large excess of enzyme capacity and intermediate substrate concentration, compared with what is required for state 3 respiration. To conclude, we submitted our data to a principal component analysis that revealed three groups of tissues: muscle and heart, brain, and liver and kidney.

Structural organization of the perimysium in bovine skeletal muscle: Junctional plates and associated intracellular subdomains.

We analyzed the structural features of the perimysium collagen network in bovine Flexor carpi radialis muscle using various sample preparation methods and microscopy techniques. We first observed by scanning electron microscopy that perimysium formed a regular network of collagen fibers with three hierarchical levels including (i) a loose lattice of large interwoven fibers ramified in (ii) numerous collagen plexi attaching together adjacent myofibers at the level of (iii) specific structures that we call perimysial junctional plates. Second, we looked more closely at the intracellular organization underneath each plate using transmission electron microscopy, immunohistochemistry, and a three-dimensional reconstruction from serial sections. We observed the accumulation of myonuclei arranged in clusters surrounded by a high density of subsarcolemmal mitochondria and the proximity of capillary branches. Third, we analyzed the distribution of these perimysial junctional plates, subsarcolemmal mitochondria, and myonuclei clusters along the myofibers using a statistical analysis of the distances between these structures. This revealed a global colocalization and the existence of adhesion domains between endomysium and perimysium. Taken together, our observations give a better description of the perimysium organization in skeletal muscle, and provide evidence that perimysial junctional plates with associated intracellular subdomains may participate in the lateral transmission of contractile forces as well as mechanosensing.

rDNA units are highly polymorphic in Scutellospora castanea (glomales, zygomycetes).

The ribosomal DNA (rDNA) units in the glomalean zygomycete fungus Scutellospora castanea were analyzed. Dot-blot assays allowed an estimation of 75 copies per genome. After constructing a genomic library in a phage lambdaEMBL3 vector, 13 rDNA clones were screened and explored. PCR experiments confirmed their nature and allowed homologous probes to be obtained. Restriction-fragment length polymorphism (RFLP) analysis and hybridizations with 18 s and 25 s probes allowed their grouping into nine families. The 18 s gene from these 13 clones was partially sequenced. The resulting 550 bases sequences were analyzed, and a phylogenetic tree was inferred. This revealed that two clones contain one highly divergent rDNA family (rUSc1) by comparison with other known 18 s sequences from the database. A phylogenetic tree was constructed with the entire 18 s sequences of rUSc1, rUSc3 and those of seven species representative of the glomalean fungi, Glomus, Entrophospora, Acaulospora, Scutellospora and Gigaspora. This tree confirmed that the rUSc1 sequence is the neighbor of 18 s sequences from Glomus (Glomineae), while rUSc3 remained in the group of the Gigaspora and Scutellospora (Gigasporineae). A specific primer, rUSc1-1, was generated from the ITS region of rUSc1, and used for PCR amplification from single spores, depicting the presence of rUSc1 in the genome of S. castanea at a lower frequency than other units.