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 Ca(2+) 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.     

The ingi and RIME non-LTR retrotransposons are not randomly distributed in the genome of Trypanosoma brucei

The ingi (long and autonomous) and RIME (short and nonautonomous) non--long-terminal repeat retrotransposons are the most abundant mobile elements characterized to date in the genome of the African trypanosome Trypanosoma brucei. These retrotransposons were thought to be randomly distributed, but a detailed and comprehensive analysis of their genomic distribution had not been performed until now. To address this question, we analyzed the ingi/RIME sequences and flanking sequences from the ongoing T. brucei genome sequencing project (TREU927/4 strain). Among the 81 ingi/RIME elements analyzed, 60% are complete, and 7% of the ingi elements (approximately 15 copies per haploid genome) appear to encode for their own transposition. The size of the direct repeat flanking the ingi/RIME retrotransposons is conserved (i.e., 12-bp), and a strong 11-bp consensus pattern precedes the 5'-direct repeat. The presence of a consensus pattern upstream of the retroelements was confirmed by the analysis of the base occurrence in 294 GSS containing 5'-adjacent ingi/RIME sequences. The conserved sequence is present upstream of ingis and RIMEs, suggesting that ingi-encoded enzymatic activities are used for retrotransposition of RIMEs, which are short nonautonomous retroelements. In conclusion, the ingi and RIME retroelements are not randomly distributed in the genome of T. brucei and are preceded by a conserved sequence, which may be the recognition site of the ingi-encoded endonuclease.     

Mitochondrial implications in human pregnancies with intrauterine growth restriction and associated cardiac remodelling

Intrauterine growth restriction (IUGR) is an obstetric complication characterised by placental insufficiency and secondary cardiovascular remodelling that can lead to cardiomyopathy in adulthood. Despite its aetiology and potential therapeutics are poorly understood, bioenergetic deficits have been demonstrated in adverse foetal and cardiac development. We aimed to evaluate the role of mitochondria in human pregnancies with IUGR. In a single‐site, cross‐sectional and observational study, we included placenta and maternal peripheral and neonatal cord blood mononuclear cells (PBMC and CBMC) from 14 IUGR and 22 control pregnancies. The following mitochondrial measurements were assessed: enzymatic activities of mitochondrial respiratory chain (MRC) complexes I, II, IV, I + III and II + III, oxygen consumption (cell and complex I‐stimulated respiration), mitochondrial content (citrate synthase [CS] activity and mitochondrial DNA copy number), total ATP levels and lipid peroxidation. Sirtuin3 expression was evaluated as a potential regulator of bioenergetic imbalance. Intrauterine growth restriction placental tissue showed a significant decrease of MRC CI enzymatic activity (P < 0.05) and CI‐stimulated oxygen consumption (P < 0.05) accompanied by a significant increase of Sirtuin3/β‐actin protein levels (P < 0.05). Maternal PBMC and neonatal CBMC from IUGR patients presented a not significant decrease in oxygen consumption (cell and CI‐stimulated respiration) and MRC enzymatic activities (CII and CIV). Moreover, CS activity was significantly reduced in IUGR new‐borns (P < 0.05). Total ATP levels and lipid peroxidation were preserved in all the studied tissues. Altered mitochondrial function of IUGR is especially present at placental and neonatal level, conveying potential targets to modulate obstetric outcome through dietary interventions aimed to regulate Sirtuin3 function.     

Gibberellic Acid (GA3) Inhibits ROS Increase in Chloroplasts During Dark-Induced Senescence of Pelargonium Cuttings

The temporal and spatial changes in reactive oxygen species (ROS) during dark treatment of Pelargonium cuttings and the effect of gibberellic acid (GA₃) on ROS levels were studied. ROS-related fluorescence was detected in mitochondria and cytoplasm of epidermal cells and in chloroplasts. By monitoring dichlorofluorescein (DCF) fluorescence, an initial decrease in ROS was observed under darkness in the epidermal cell cytoplasm and the chloroplasts, which was followed by an increase on the third day. Following 3 days under darkness, the size and the structure of the chloroplasts also changed, and they became more sensitive to illumination as judged by a higher accumulation of ROS. Pretreatment of leaves with GA₃ did not prevent the structural changes in the chloroplasts, but it inhibited the increase in ROS levels in all cell compartments, including the chloroplasts. It is suggested that the inhibition of ROS increase by GA₃ prevented complete disintegration of chloroplasts during dark-induced senescence and thereby enabled the maintenance of chlorophyll levels in the tissue.     

Expression variation of the porcine ADRB2 has a complex genetic background

Porcine adrenergic receptor beta 2 (ADRB2) gene exhibits differential allelic expression in skeletal muscle, and its genetic variation has been associated with muscle pH. Exploring the molecular–genetic background of expression variation for porcine ADRB2 will provide insight into the mechanisms driving its regulatory divergence and may also contribute to unraveling the genetic basis of muscle-related traits in pigs. In the present study, we therefore examined haplotype effects on the expression of porcine ADRB2 in four tissues: longissimus dorsi muscle, liver, subcutaneous fat, and spleen. The diversity and structure of haplotypes of the proximal gene region segregating in German commercial breeds were characterized. Seven haplotypes falling into three clades were identified. Two clades including five haplotypes most likely originated from introgression of Asian genetics during formation of modern breeds. Expression analyses revealed that the Asian-derived haplotypes increase expression of the porcine ADRB2 compared to the major, wild-type haplotype independently of tissue type. In addition, several tissue-specific differences in the expression of the Asian-derived haplotypes were found. Inspection of haplotype sequences showed that differentially expressed haplotypes exhibit polymorphisms in a polyguanine tract located in the core promoter region. These findings demonstrate that expression variation of the porcine ADRB2 has a complex genetic basis and suggest that the promoter polyguanine tract is causally involved. This study highlights the challenges of finding causal genetic variants underlying complex traits.     

Assessing the Semelparity Hypothesis: Egg-guarding and Fecundity in the Malaysian Treehopper Pyrgauchenia tristaniopsis

According to the semelparity hypothesis, iteroparous insects should provide either no maternal care or less care than related semelparous species. We present field data on reproductive output and maternal care in the Southeast Asian treehopper Pyrgauchenia tristaniopsis (Mt. Kinabalu, Borneo) relevant to a preliminary assessment of the hypothesis. In a mark‐recapture experiment, more females than expected under semelparity were found to have oviposited a second clutch (37%). Female longevity was a of 75 d. Both these estimates were highly conservative. Oviposition was successive resulting in a of 46 eggs per clutch. Females provided care for eggs only, occasionally scraping their legs along the sides of the clutch apparently attempting to deter Brachygrammatella sp. egg parasitoids (Trichogrammatidae). Females straddled their clutch for a of 27 d, i.e. until 8 d after the beginning of first instar hatching. First instars hatched successively over a period of 11 d. When a female deserted her clutch, it contained about 37% yet unhatched eggs. Egg‐guarding effectively reduced egg mortality: the earlier a female was experimentally removed from her clutch the higher the egg mortality. Displacement experiments demonstrated that egg‐guarding is a behaviour actively maintained despite disturbances and specifically directed towards the egg clutch but not to the feeding site. We interpret our findings as being in accordance with the weaker claim of the semelparity hypothesis, i.e. the iteroparous P. tristaniopsis provided less maternal care than semelparous membracid species. Continued female feeding is discussed as a mechanism to display some level of care despite iteroparity.