Effects of ceramide on liquid-ordered domains investigated by simultaneous AFM and FCS

The sphingolipid ceramides are known to influence lipid lateral organization in biological membranes. In particular, ceramide-induced alterations of microdomains can be involved in several cell functions, ranging from apoptosis to immune response. We used a combined approach of atomic force microscopy, fluorescence correlation spectroscopy, and confocal fluorescence imaging to investigate the effects of ceramides in model membranes of biological relevance. Our results show that physiological quantities of ceramide in sphingomyelin/dioleoylphosphatidylcholine/cholesterol supported bilayers lead to a significant rearrangement of lipid lateral organization. Our experimental setup allowed a simultaneous characterization of both structural and dynamic modification of membrane microdomains, induced by the presence of ceramide. Formation of similar ceramide-enriched domains and, more general, alterations of lipid-lipid interactions can be of crucial importance for the biological function of cell membranes.     

High-density genetic linkage maps with over 2,400 sequence-anchored DArT markers for genetic dissection in an F2 pseudo-backcross of Eucalyptus grandis × E. urophylla

Traits that differentiate cross-fertile plant species can be dissected by genetic linkage analysis in interspecific hybrids. Such studies have been greatly facilitated in Eucalyptus tree species by the recent development of Diversity Arrays Technology (DArT) markers. DArT is an affordable, high-throughput marker technology for the construction of high-density genetic linkage maps. Eucalyptus grandis and Eucalyptus urophylla are commonly used to produce fast-growing, disease tolerant hybrids for clonal eucalypt plantations in tropical and subtropical regions. We analysed 7,680 DArT markers in an F2 pseudo-backcross mapping pedigree based on an F1 hybrid clone of E. grandis and E. urophylla. A total of 2,440 markers (31.7%) were polymorphic and could be placed in linkage maps of the F1 hybrid and two pure-species backcross parents. An integrated genetic linkage map was constructed for the pedigree resulting in 11 linkage groups (n = 11) with 2,290 high-confidence (LOD ≥ 3.0) markers and a total map length of 1,107.6 cM. DNA sequence analysis of the mapped DArT marker fragments revealed that 43% were located in protein coding regions and 90% could be placed in the recently completed draft genome assembly of E. grandis. Together with the anchored genomic sequence information, this linkage map will allow detailed genetic dissection of quantitative traits and hybrid fitness characters segregating in the F2 progeny and will facilitate the development of markers for molecular breeding in Eucalyptus.     

CD14 major role during lipopolysaccharide-induced inflammation in chick embryo cardiomyocytes

CD14 is a surface differentiation antigen that functions as a receptor for bacterial lipopolysaccharide. The cellular signaling events that lead to lipopolysaccharide-induced production of inflammatory mediators are the primary cause of myocardial dysfunction observed in sepsis. Here, we evaluated the role of CD14 in chick embryo cardiomyocytes stimulated with lipopolysaccharide. CD14 expression was detected by confocal laser microscopy observation and by immunoblotting analysis. Moreover, we provided evidence for CD14-dependent functional responses of lipopolysaccharide-stimulated cardiomyocytes in terms of tumor necrosis factor (TNF)-alpha and nitric oxide (NO) production. Attenuated TNF-alpha and NO secretion, following anti-CD14 treatment of cardiomyocytes, suggested a role for this receptor in lipopolysaccharide-mediated cell responses. We also evidenced that labeled lipopolysaccharide was internalized and localized next to the Golgi complex, at the level of lysosomes, and in the perinuclear zone. The intracytoplasmatic transport seems to depend on the contractile apparatus, because cell pretreatment with cytochalasin D prevented lipopolysaccharide internalization and reduced both TNF-alpha and NO release. Lipopolysaccharide internalization was dependent on CD14 receptor, since anti-CD14 pre-treatment prevented endotoxin uptake by cardiomyocytes. Results demonstrated: (1) CD14 is expressed on the surface membrane of cardiomyocytes; (2) CD14 is involved in cytoskeletal dependent lipopolysaccharide internalization at specific cytoplasmatic locations; (3) CD14 plays a role in lipopolysaccharide-mediated responses by cardiomyocytes after lipopolysaccharide internalization.     

Analysis of chromosome conservation in <Emphasis Type="Italic">Lemur catta</Emphasis> studied by chromosome paints and BAC/PAC probes

A panel of human chromosome painting probes and bacterial and P1 artificial chromosome (BAC/PAC) clones were used in fluorescence in situ hybridization (FISH) experiments to investigate the chromosome conservation of the ring-tailed lemur (Lemur catta, LCA) with respect to human. Whole chromosome paints specific for human chromosomes 7, 9, 11, 13, 14, 17, 18, 20, 21, and X were found to identify a single chromosome or an uninterrupted chromosomal region in LCA. A large set of partial chromosome paints and BAC/PAC probes were then used to refine the characterization of the rearrangements differentiating the two karyotypes. The results were also used to reconstruct the ancestral Lemuridae karyotype. Lemur catta, indeed, can be used as an outgroup, allowing symplesiomorphic (ancestral) rearrangements to be distinguished from apomorphic (derived) rearrangements in lemurs. Some LCA chromosomes are difficult to distinguish morphologically. The 'anchorage' of most LCA chromosomes to specific probes will contribute to the standardization of the karyotype of this species.     

Relaxation kinetics following sudden Ca(2+) reduction in single myofibrils from skeletal muscle

To investigate the roles of cross-bridge dissociation and cross-bridge-induced thin filament activation in the time course of muscle relaxation, we initiated force relaxation in single myofibrils from skeletal muscles by rapidly (approximately 10 ms) switching from high to low [Ca(2+)] solutions. Full force decay from maximal activation occurs in two phases: a slow one followed by a rapid one. The latter is initiated by sarcomere "give" and dominated by inter-sarcomere dynamics (see the companion paper, Stehle, R., M. Krueger, and G. Pfitzer. 2002. Biophys. J. 83:2152-2161), while the former occurs under nearly isometric conditions and is sensitive to mechanical perturbations. Decreasing the Ca(2+)-activated force preceding the start of relaxation does not increase the rate of the slow isometric phase, suggesting that cycling force-generating cross-bridges do not significantly sustain activation during relaxation. This conclusion is strengthened by the finding that the rate of isometric relaxation from maximum force to any given Ca(2+)-activated force level is similar to that of Ca(2+)-activation from rest to that given force. It is likely, therefore, that the slow rate of force decay in full relaxation simply reflects the rate at which cross-bridges leave force-generating states. Because increasing [P(i)] accelerates relaxation while increasing [MgADP] slows relaxation, both forward and backward transitions of cross-bridges from force-generating to non-force-generating states contribute to muscle relaxation.     

Protein synthesis in the visual cortex is needed for ocular dominance plasticity

Experience-dependent remodelling of neural connections progresses through stages, and early phases eventually give way to later long-lasting ones. The transition from early to late stages, often associated with structural changes, depends on protein synthesis. Suppression of cortical but not geniculate protein synthesis blocks ocular dominance plasticity at its earliest stage, suggesting that structural changes occur rapidly in the visual cortex following monocular deprivation.     

Effect of a topical treatment in organotypic culture of human breast skin after exposure to gamma-rays

The early radiation of epidermal reactions can lead to healing of the lesion or radiation necrosis. There is no general agreement for either the prevention and/or treatment of radiation skin response, also as little is known about the immediate phases of this phenomenon. We investigated the early effects exerted by Healing and Wound Emulsion (HWE) on human skin response after ionizing radiation. Epidermal morphology, Heat Shock Protein (HSP) 70, and Transforming Growth Factor-beta1 (TGF-beta1) gene expression were investigated in organotypic human skin cultures undergoing a double dose of gamma-rays (2 Gy). HSP70 gene expression tended to be induced in the HWE group 6 hours after cream administration and was significantly up-regulated after 48 hours, when epidermal morphological alterations were evident. TGF-beta1 seems not affected in cream treated samples. HWE may stimulate skin to mount an early defensive response against damage induced by gamma rays.