Acute and Chronic Altitude-Induced Cognitive Dysfunction in Children and Adolescents

OBJECTIVE: To assess whether exposure to high altitude induces cognitive dysfunction in young healthy European children and adolescents during acute, short-term exposure to an altitude of 3450 m and in an age-matched European population permanently living at this altitude. STUDY DESIGN: We tested executive function (inhibition, shifting, and working memory), memory (verbal, short-term visuospatial, and verbal episodic memory), and speed processing ability in: (1) 48 healthy nonacclimatized European children and adolescents, 24 hours after arrival at high altitude and 3 months after return to low altitude; (2) 21 matched European subjects permanently living at high altitude; and (3) a matched control group tested twice at low altitude. RESULTS: Short-term hypoxia significantly impaired all but 2 (visuospatial memory and processing speed) of the neuropsychological abilities that were tested. These impairments were even more severe in the children permanently living at high altitude. Three months after return to low altitude, the neuropsychological performances significantly improved and were comparable with those observed in the control group tested only at low altitude. CONCLUSIONS: Acute short-term exposure to an altitude at which major tourist destinations are located induces marked executive and memory deficits in healthy children. These deficits are equally marked or more severe in children permanently living at high altitude and are expected to impair their learning abilities

The Targeting of Plasmalemmal Ceramide to Mitochondria during Apoptosis

Ceramide is a key lipid mediator of cellular processes such as differentiation, proliferation, growth arrest and apoptosis. During apoptosis, ceramide is produced within the plasma membrane. Although recent data suggest that the generation of intracellular ceramide increases mitochondrial permeability, the source of mitochondrial ceramide remains unknown. Here, we determine whether a stress-mediated plasmalemmal pool of ceramide might become available to the mitochondria of apoptotic cells. We have previously established annexin A1—a member of a family of Ca2+ and membrane-binding proteins—to be a marker of ceramide platforms. Using fluorescently tagged annexin A1, we show that, upon its generation within the plasma membrane, ceramide self-associates into platforms that subsequently invaginate and fuse with mitochondria. An accumulation of ceramide within the mitochondria of apoptotic cells was also confirmed using a ceramide-specific antibody. Electron microscopic tomography confirmed that upon the formation of ceramide platforms, the invaginated regions of the plasma membrane extend deep into the cytoplasm forming direct physical contacts with mitochondrial outer membranes. Ceramide might thus be directly transferred from the plasma membrane to the mitochondrial outer membrane. It is conceivable that this “kiss-of-death” increases the permeability of the mitochondrial outer membrane thereby triggering apoptosis

Click chemistry for the generation of cell permeable apoptotic peptides

he use of proteins and peptides as drug molecules has been held back by their proteolytic instability and inability to cross-cellular membranes. Proteins and long peptides are often produced by expression in E. coli rather than by solid phase peptide synthesis. A drawback of in vivo protein and peptide synthesis is the difficulty to selectively modify the product peptide by the attachment of fluorescent dyes or ligation to other macromolecules like polysaccharides, lipids or peptides. Here we present a facile method to modify an expressed protein or peptide to create a C-terminal alkyne group. This functionality is then used inter alia for conjugation to the cell-penetrating peptide octa-arginine. This will provide a vector for delivery across the plasma membrane of cells. To demonstrate our method, we have produced in E. coli a peptide derived from the Bak protein; one of the key regulators of apoptosis in eukaryotic cells. In the cell it is usually found bound to Bcl-xL at the outer mitochondrial membrane. If this interaction is disrupted, Bak oligomerizes and forms pores which trigger mitochondria dependent apoptosis through cytochrome c release. Small peptides derived from the BH3 helix of Bak have been shown to induce apoptosis. We have expressed such a peptide in E. coli as a fusion protein. The ketosteroid isomerase fusion protein is insoluble and readily purified from cell extracts. The peptide is then cleaved from the fusion protein by reaction with cyanogen bromide at a strategically inserted methionine residue to generate a homoserine lactone at the C-terminus of the Bak peptide. This lactone is then used for direct amide formation with inexpensive propargylamine. The resulting alkynyl peptide serves as a reagent for highly efficient ‘click’ reactions to couple to a wide range of azides. Since the Bak peptide is not able to cross the cell membrane, the well-known octa-arginine cell penetrating peptide sequence was added as a delivery vector. Here we discuss the synthesis of this semi-synthetic peptide and its interaction with, and uptake into, cancer cell lines

Chemical synthesis of cell-permeable apoptotic peptides from in vivo produced proteins

In vivo synthesis of peptides by bacterial expression has developed into a reliable alternative to solid-phase peptide synthesis. A significant drawback of in vivo methods is the difficulty with which gene products can be modified post-translationally. Here, we present a method for the facile modification of peptides generated in bacterial hosts after cyanogen bromide cleavage at C-terminal methionines. Reaction of the resulting homoserine lactones with propargylamine allows efficient and selective modification with a wide variety of chemicals such as fluorescent dyes, biotin derivatives, polyprenyls, lipids, polysaccharides, or peptides. Attachment of the cell penetrating peptide octa-arginine (R8) to peptides derived from the proapoptotic tumor suppressor Bak BH3 led to efficient cellular uptake and subsequent cytochrome c release from mitochondria, culminating in induction of apoptosis similar to that observed with peptides linked to R8 via the peptide backbone. These results highlight the significant potential for use of such tools in live cells

BH3 helix-derived biophotonic nanoswitches regulate cytochrome c release in permeabilised cells

Dynamic physical interactions between proteins underpin all key cellular processes and are a highly attractive area for the development of research tools and medicines. Protein–protein interactions frequently involve α-helical structures, but peptides matching the sequences of these structures usually do not fold correctly in isolation. Therefore, much research has focused on the creation of small peptides that adopt stable α-helical structures even in the absence of their intended protein targets. We show that short peptides alkylated with azobenzene crosslinkers can be used to photo-stimulate mitochondrial membrane depolarization and cytochrome c release in permeabilised cells, the initial events of the intrinsic apoptosis pathway

Adherence to long-term therapies in cystic fibrosis: a French cross-sectional study linking prescribing, dispensing, and hospitalization data

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