Structural analysis of the specific capsular polysaccharide of Streptococcus pneumoniae type 45 (American type 72)

The specific capsular polysaccharide of Streptococcus pneumoniae type 45 (American type 72) was found to be a high molecular weight polymer composed of D-galactose, 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-L-fucose, L-rhamnose, glycerol, and phosphate (2:1:1:1:1:1:1). Partial hydrolysis, dephosphorylation, methylation analysis, periodate oxidation studies, and one- and two-dimensional 1H and 13C high-field nuclear magnetic resonance experiments showed the polysaccharide to be a branched polymer of a 1-phosphoglycerol-substituted hexasaccharide repeating unit having the structure: (formula; see text).     

Dietary n-3 PUFAs affect the blood pressure rise and cardiac impairments in a hyperinsulinemia rat model in vivo

The cardiovascular consequences of eicosapentaenoic acid (EPA)- and docosahexaenoic acid (DHA)-specific intake were evaluated in vivo in a hyperinsulinemia (HI) model induced by dietary fructose intake. Wistar rats were fed a diet containing (or not for control) either EPA or DHA. The rise in blood pressure (BP), heart rate, and ECG were continuously monitored using an intra-abdominal telemetry system. The myocardial phospholipid fatty acid profile was significantly affected by DHA intake but less by EPA intake. The data indicated a reduced rise in BP in both DHA and EPA HI groups compared with controls. This result was confirmed by tail-cuff measurement after 5 wk [133.3 +/- 1.67 and 142.5 +/- 1.12 mmHg in n-3 polyunsaturated fatty acid (PUFA) and control groups, respectively], whereas n-3 PUFA did not affect BP in non-HI rats (116.3 +/- 3.33 mmHg). The heart rate was lower in the HI DHA group than in the other two dietary HI groups. Moreover, DHA induced a significantly shorter QT interval. It is concluded that the cardioactive component of fish oils is DHA through a mechanism that may involve the cardiac adrenergic system.     

Osteopromotion of Biphasic Calcium Phosphate granules in critical size defects after osteonecrosis induced by focal heating insults

Heat-induced osteonecrosis represents a simple, rapid, and inexpensive method for reproducing the effects of bone disease. In the present study, we employed this technique to induce osteonecrosis in femoral defects in rabbits and assessed the efficacy of treatment using Biphasic Calcium Phosphate (BCP) granules (MBCP+?, Biomatlante SA). After 3 weeks, the osteopromotion effects of BCP granules could be statistically proven (P < 0.05) through image analysis of newly formed bone in osteonecrosed sites containing BCP granules when compared to empty control sites. Increasing mature and woven bone presence was observed after 6 and 12 weeks, forming new trabeculae in necrosed site. Significant statistical differences were evidenced at each time between empty necrosed and filled necrosed defects in terms of new bone volume.     

Colonisation and Diversification of the Zenaida Dove (Zenaida aurita) in the Antilles: Phylogeography,Contemporary Gene Flow and Morphological Divergence

Caribbean avifaunal biogeography has been mainly studied based on mitochondrial DNA. Here, we investigated both past and recent island differentiation and micro-evolutionary changes in the Zenaida Dove (Zenaida aurita) based on combined information from one mitochondrial (Cytochrome c Oxydase subunit I, COI) and 13 microsatellite markers and four morphological characters. This Caribbean endemic and abundant species has a large distribution, and two subspecies are supposed to occur: Z. a. zenaida in the Greater Antilles (GA) and Z. a. aurita in the Lesser Antilles (LA). Doves were sampled on two GA islands (Puerto Rico and the British Virgin Islands) and six LA islands (Saint Barthélemy, Guadeloupe, Les Saintes, Martinique, Saint Lucia and Barbados). Eleven COI haplotypes were observed that could be assembled in two distinct lineages, with six specific to GA, four to LA, the remaining one occurring in all islands. However, the level of divergence between those two lineages was too moderate to fully corroborate the existence of two subspecies. Colonisation of the studied islands appeared to be a recent process. However, both phenotypic and microsatellite data suggest that differentiation is already under way between all of them, partly associated with the existence of limited gene flow. No isolation by distance was observed. Differentiation for morphological traits was more pronounced than for neutral markers. These results suggest that despite recent colonisation, genetic drift and/or restricted gene flow are promoting differentiation for neutral markers. Variation in selective pressures between islands may explain the observed phenotypic differentiation.     

Characteristics of the rat cardiac sphingolipid pool in two mitochondrial subpopulations

Mitochondrial sphingolipids play a diverse role in normal cardiac function and diseases, yet a precise quantification of cardiac mitochondrial sphingolipids has never been performed. Therefore, rat heart interfibrillary mitochondria (IFM) and subsarcolemmal mitochondria (SSM) were isolated, lipids extracted, and sphingolipids quantified by LC-tandem mass spectrometry. Results showed that sphingomyelin (∼10,000 pmol/mg protein) was the predominant sphingolipid regardless of mitochondrial subpopulation, and measurable amounts of ceramide (∼70 pmol/mg protein) sphingosine, and sphinganine were also found in IFM and SSM. Both mitochondrial populations contained similar quantities of sphingolipids except for ceramide which was much higher in SSM. Analysis of sphingolipid isoforms revealed ten different sphingomyelins and six ceramides that differed from 16- to 24-carbon units in their acyl side chains. Sub-fractionation experiments further showed that sphingolipids are a constituent part of the inner mitochondrial membrane. Furthermore, inner membrane ceramide levels were 32% lower versus whole mitochondria (45 pmol/mg protein). Three ceramide isotypes (C₂₀-, C₂₂-, and C₂₄-ceramide) accounted for the lower amounts. The concentrations of the ceramides present in the inner membranes of SSM and IFM differed greatly. Overall, mitochondrial sphingolipid content reflected levels seen in cardiac tissue, but the specific ceramide distribution distinguished IFM and SSM from each other.     

Plasma membrane helps autophagosomes grow

The membrane origin of autophagosomes has long been a mystery and it may involve multiple sources. In this punctum, we discuss our recent finding that the plasma membrane contributes to the formation of pre-autophagic structures via clathrin-mediated endocytosis. Our study suggests that Atg16L1 interacts with clathrin heavy-chain/AP2 and is also localized on vesicles (positive for clathrin or cholera toxin B) close to the plasma membrane. Live-cell imaging studies revealed that the plasma membrane contributes to Atg16L1-positive structures and that this process and autophagosome formation are impaired by knockdowns of genes regulating clathrin-mediated endocytosis.Key words: autophagy, plasma membrane, endocytosis, phagophore, originWhere do autophagosomes get their membrane from? Although the field of autophagy has grown tremendously since its discovery a few decades ago, the origin(s) of the membranes that contribute to autophagosome biogenesis has been a mystery among autophagy researchers until recently. Mammalian autophagosomes are formed randomly throughout the cytoplasm via a process that involves elongation and fusion of phagophores to form double-membraned autophagosomes. This process involves two ubiquitin-like conjugation systems: conjugation of Atg12 to Atg5 that later forms a macromolecular complex with Atg16L1, and conjugation of phosphatidylethanolamine (PE) with Atg8/LC3-I. The Atg12-Atg5-Atg16L1 complex is targeted to the preautophagic structures, which then acquire Atg8. Atg12-Atg5-Atg16L1 dissociates from completed autophagosomes, while LC3-PE (LC3-II) is associated both with pre-autophagic structures and completed autophagosomes.Some recent studies have explored the contribution of membranes from different organelles supporting the general idea that autophagosomes derive membranes from pre-existing organelles. It is quite possible that there may be multiple membrane sources involved. A few groups have revisited the hypothesis that the endoplasmic reticulum (ER) may be one of the membrane donors. High-resolution 2D electron microscopy (EM) and 3D EM-tomography studies have revealed connections between the ER and the growing autophagosomes. Whether the ER contributes to general autophagy or a specific form of autophagy, reticulophagy, remains to be determined. In addition, it has not been shown if ER membrane is required for autophagosome formation. Recently another study has reported that autophagosomes receive lipids from the outer mitochondrial membrane, but only under starvation conditions, again fueling the multiple-membrane source hypothesis.We have now found evidence for plasma membrane contribution to pre-autophagic structures via endocytosis. Unlike the previous studies, which have focused on LC3- positive structures, we looked specifically at the Atg5-, Atg12- and Atg16-positive pre-autophagic structures, an idea that stemmed from our finding that clathrin heavy-chain immunoprecipitates with Atg16L1. We think that this interaction is partly mediated by the adaptor protein AP2, since knockdown of AP2 decreases the clathrin heavy-chain-Atg16L1 interaction. Immunogold EM also shows clathrin localization on Atg16L1-labeled vesicles close to the plasma membrane.These findings led us to test whether knockdown of proteins involved in clathrin-mediated endocytosis affected Atg16L1-positive pre-autophagic structures. Indeed, knockdown of key proteins in the clathrin-mediated endocytic pathway results in a decrease in the formation of Atg16L1-positive structures both under basal or autophagy-induced conditions (starvation or trehalose treatment). This correlates with a decrease in the number of LC3-labeled autophagosomes. When we directly analyzed vesicle fusion by livecell microscopy, we observed that vesicles endocytosed from the plasma membrane fuse to the Atg16L1-positive vesicles close to the plasma membrane. This was confirmed by immuno-EM when we found cholera toxin B-labeling (used to label plasma membrane that is subsequently internalized by endocytosis) on Atg16L1-vesicles. We noticed that overexpression of an Atg16L1 mutant that does not bind clathrin heavy-chain does not form Atg16L1-vesicular structures in the way we see with wild-type Atg16L1, suggesting that the binding of Atg16L1 to AP2/clathrin is required for the subsequent formation of the Atg16L1 vesicles.When we blocked endocytic vesicle scission (using both genetic and chemical inhibitors) we found that Atg16L1 strongly immunoprecipitates with clathrin-heavy chain probably due to the accumulation of clathrin-Atg16L1 structures at the plasma membrane that failed to pinch off. This was strongly supported by our fluorescence microscopy and immuno-EM studies that showed what we predicted—accumulation of Atg16L1 at the plasma membrane. This suggests that Atg16L1 in a complex with AP2/clathrin is targeted to the plasma membrane and subsequently internalized as Atg16L1-positive structures. Thus, our data strongly suggest that plasma membrane contributes to early autophagic precursors that subsequently mature to form phagophores (Fig. 1).Open in a separate windowFigure 1Plasma membrane contributes to the formation of early autophagic precursors. Previous studies show that delivery of fully formed autophagosomes to lysosomes requires fusion of such autophagosomes with early or late endosomes to form amphisomes, which are Atg16L1-negative, LC3-positive and are also positive for endosomal markers. We show that blocking clathrin-mediated endocytosis inhibits formation of Atg16L1-positive structures that mature to form phagophores and later autophagosomes. These Atg16L1-vesicles are positive for other early autophagosomal markers like Atg5 and Atg12, but are negative for early endosomal markers like EEA1, suggesting that they are high up in the autophagosome biogenesis cascade. Inhibition of dynamin with Dynsasore or the use of a dominant negative K44A mutant blocks scission and results in Atg16L1 accumulation on the plasma membrane, suggesting that endosomal scission is critical for this process.Although previous studies suggest that completely formed autophagosomes need to fuse with early or late endosomes in order for subsequent autophagosomelysosome fusion to occur, they did not look at the formation of pre-autophagic structures. Our study shows that active endocytosis is required both for the formation of autophagosomes, when very early endocytic intermediates immediately pinching off the plasma membrane (not early endosomes) fuse with Atg16L1-positive structures to form phagophores, and also for maturation of autophagosomes when early or late endosomes fuse with Atg16L1-negative but LC3-positive autophagosomes to form amphisomes. Since blocking clathrin-mediated endocytosis does not completely abrogate autophagosome formation, we believe that other endocytic pathways may have a similar role. Depending on the cell type or the physiological conditions, the contributions from the different endocytic pathways may vary accordingly. It will be interesting to know if the endocytic pathway continuously delivers membrane for early steps in autophagy as the preautophagic structures grow and mature to form autophagosomes, deriving membrane from other sources.     

Mutations within the programmed cell death 10 gene cause cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are hamartomatous vascular malformations characterized by abnormally enlarged capillary cavities without intervening brain parenchyma. They cause seizures and cerebral hemorrhages, which can result in focal neurological deficits. Three CCM loci have been mapped, and loss-of-function mutations were identified in the KRIT1 (CCM1) and MGC4607 (CCM2) genes. We report herein the identification of PDCD10 (programmed cell death 10) as the CCM3 gene. The CCM3 locus has been previously mapped to 3q26-27 within a 22-cM interval that is bracketed by D3S1763 and D3S1262. We hypothesized that genomic deletions might occur at the CCM3 locus, as reported previously to occur at the CCM2 locus. Through high-density microsatellite genotyping of 20 families, we identified, in one family, null alleles that resulted from a deletion within a 4-Mb interval flanked by markers D3S3668 and D3S1614. This de novo deletion encompassed D3S1763, which strongly suggests that the CCM3 gene lies within a 970-kb region bracketed by D3S1763 and D3S1614. Six additional distinct deleterious mutations within PDCD10, one of the five known genes mapped within this interval, were identified in seven families. Three of these mutations were nonsense mutations, and two led to an aberrant splicing of exon 9, with a frameshift and a longer open reading frame within exon 10. The last of the six mutations led to an aberrant splicing of exon 5, without frameshift. Three of these mutations occurred de novo. All of them cosegregated with the disease in the families and were not observed in 200 control chromosomes. PDCD10, also called "TFAR15," had been initially identified through a screening for genes differentially expressed during the induction of apoptosis in the TF-1 premyeloid cell line. It is highly conserved in both vertebrates and invertebrates. Its implication in cerebral cavernous malformations strongly suggests that it is a new player in vascular morphogenesis and/or remodeling.     

Evidence for specific RNA/protein interactions in the differential segment of the W sex chromosome in the amphibian Pleurodeles waltl

Pleurodeles exhibits a ZZ/ZW system of GSD (genotype sex determination). However, the Z and W sex chromosomes appear to be morphologically identical. A short RNA sequence is described that was specifically bound to lampbrush loops in the differential segment of the sexual bivalent IV. The distribution of these labeled loops in experimentally produced ZZ and WW females enabled us to demonstrate that such labeled loops were perfectly correlated with the W chromosome. Therefore, this RNA sequence constitutes an excellent marker for the W differential segment. Furthermore, analysis of the labeled loops under various experimental conditions suggested that their labeling is caused by specific interactions between this RNA sequence and lampbrush loop-associated proteins (RNA/protein interactions). North-western assays revealed that nuclear polypeptide(s) of 65 kDa could be responsible for such binding.