Role of the carboxyl-terminal region of the PVP protein (F0I subunit) in the H+ conduction of F0F1 H+-ATP synthase of bovine heart mitochondria

By means of protein sequencing, labelling with thiol reagents and reconstitution studies it is shown that the carboxyl-terminal region of the PVP protein (F0I subunit, nuclear-encoded protein of Mr 25,000) of mitochondrial F0 promotes transmembrane proton conduction by F0 and the sensitivity of this process to oligomycin.     

The same nuclear proteins bind the proximal CACCC box of the human beta-globin promoter and a similar sequence in the enhancer

Using in vitro assays, we show that nuclear proteins related to the Sp1 and GT-1 factors bind to a CACCC box sequence in the human beta-globin enhancer, adjacent to binding sites for the erythroid-specific factor NFE1 and the ubiquitous factor CP1. The same proteins are known to bind to the proximal, but not to the distal, CACCC, box in the human beta-globin promoter. A C G mutation in the promoter CACCC box, known to cause beta-thalassemia, greatly decreases protein binding to the CACCC box; the same effect is obtained when this mutation is introduced into the enhancer CACCC box.     

Interplay between intrinsic plasma rotation and magnetic island evolution in disruptive discharges

The behavior of the intrinsic toroidal rotation of the plasma column during the growth and eventual saturation of m/n = 2/1 magnetic islands, triggered by programmed density rise, has been carefully investigated in disruptive discharges in TCABR. The results show that, as the island starts to grow and rotate at a speed larger than that of the plasma column, the angular frequency of the intrinsic toroidal rotation increases and that of the island decreases, following the expectation of synchronization. As the island saturates at a large size, just before a major disruption, the angular speed of the intrinsic rotation decreases quite rapidly, even though the island keeps still rotating at a reduced speed. This decrease of the toroidal rotation is quite reproducible and can be considered as an indicative of disruption.     

γ-Conglutin,the Lupinus albus XEGIP-like protein,whose expression is elicited by chitosan,lacks of the typical inhibitory activity against GH12 endo-glucanases

γ-Conglutin, a glycoprotein from Lupinus albus seed, has been characterized at molecular level but its physiological function is still unknown. γ-Conglutin shares a high structural similarity with xyloglucan-specific endo-β-1,4-glucanase inhibitor proteins (XEGIPs) and Triticum aestivum xylanase inhibitor (TAXI-I), which act specifically against fungal glycosyl hydrolase belonging to families 12 and 11, respectively. To assess the possible involvement of γ-conglutin in plant defense, germinating lupin seeds were incubated with chitosan. The relative quantification of γ-conglutin mRNA extracted from cotyledons was then carried out by RT-qPCR and indicated that chitosan strongly elicited the expression of γ-conglutin. Moreover, biochemical trials aimed to test the inhibitory capacity of the protein have been also carried out. γ-Conglutin failed to inhibit representative fungal endo-glucanases and other cell wall-degrading enzymes. To explain the lack of inhibitory capacity we investigated the possible structural differences between γ-conglutin and XEGIPs and TAXI-I, including the construction of a predictive 3D model of the protein. Bioinformatic analysis suggests that the lack of inhibitory activity of γ-conglutin can be attributed to sequence differences in the inhibitor interaction domains, and in particular to a sequence deletion in one of the functional loops.     

Chronic in vivo hypoxia in various organs: hypoxia-inducible factor-1alpha and apoptosis

We studied the in vivo persistence of hypoxia-inducible factor-1alpha (HIF-1alpha), main transducer of hypoxia, the differential response in organs exposed to the same degree of hypoxemia and the relationship with apoptosis. We measured HIF-1alpha (immunohistochemistry peroxidase and Western blot) and apoptosis (TUNEL) in heart, liver, kidney, gastrocnemius, and brain of rats exposed to chronic normobaric hypoxia (10% O2) or normoxia (21% O2) for 2 weeks. Despite same arterial O2 pressure and increased hemoglobin concentration (219 +/- 5 vs. 124 +/- 4 g/L), the organs responded differently. While marked in brain, muscle, and kidney cortex, HIF-1alpha was undetectable in heart and liver. In kidney medulla, HIF-1alpha was high in both normoxia and hypoxia. By contrast, apoptosis was marked in heart, slight in kidney medulla, and undetectable in other organs. We conclude that the HIF-1alpha response to chronic hypoxia can be a sustained phenomenon, but not in all organs, and that apoptosis responds differently from HIF-1alpha.     

High-precision targeting workflow for volume electron microscopy

Cells are 3D objects. Therefore, volume EM (vEM) is often crucial for correct interpretation of ultrastructural data. Today, scanning EM (SEM) methods such as focused ion beam (FIB)–SEM are frequently used for vEM analyses. While they allow automated data acquisition, precise targeting of volumes of interest within a large sample remains challenging. Here, we provide a workflow to target FIB-SEM acquisition of fluorescently labeled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeted trimming guided by confocal maps of the fluorescence signal in the resin block. Laser branding is used to create landmarks on the block surface to position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as 3D cultures of mouse mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae, and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before.