Autophagy: The missing link between non-enzymatically glycated proteins inducing apoptosis and premature senescence of endothelial cells?

In a series of studies into the fate of endothelial cells exposed to non-enzymatically glycated collagen I, a model of cytotoxic molecules relevant to diabetic vasculopathy, we demonstrate that cells either undergo apoptosis or become prematurely senescent despite relatively spared telomeres and telomerase activity. Our most recent work shows that long-lived advanced glycation end product (AGE)-modified proteins induce (1) lysosomal permeabilization leading to the inefficiency of autophagy due to the reduced digestion (early) and non-fusion (later) of lysosomes with phagosomes--a frustrated autophagy; and (2) accumulation of lipid mediators, such as ceramide and sphingosine-1-phosphate, known to be involved in autophagic cell death. Under the experimental conditions described here, the seesaw relations between premature senescence and apoptosis are integrated by autophagy, which plays a novel function of a cellular switch between states of premature senescence and apoptosis.     

Correlation of hemocyte counts with different developmental parameters during the last larval instar of the tobacco hornworm, Manduca sexta

We determined the changes in hemocyte titer and in the abundance of hemocyte types of the tobacco hornworm Manduca sexta during the fourth and fifth larval stadium and the beginning of the pupal stadium. As we analyzed the samples of individual insects at daily intervals, we were able to correlate phenotypical features, body weight, as well as total protein content and lysozyme activity in the hemolymph with the observations on hemocytes. In the course of the fifth larval stadium, the hemocyte titer decreased slightly and declined further after pupation. Using calculated values for total hemocyte numbers, females had about five times and males three times more hemocytes in the circulating population at the beginning of the wandering stage (in the middle of the fifth larval stadium) than immediately after the last larval--larval molt (from the fourth to the fifth larval stadium). This sexual difference was mainly due to an increase in the number of plasmatocytes, which was more prominent in females than in males. Granular cells were dominant in early fifth larval stadium while plasmatocytes were the most abundant cells in pupae. Oenocytoids and spherule cells disappeared during the wandering stage. Lysozyme activity in the hemolymph rose to a maximum during the wandering stage, with females having lysozyme values twice as high as those for males. These changes in lysozyme activity, however, did not correlate with the increase of total hemolymph protein titer which occurred already at the beginning of the wandering stage. We postulate that changes in hemocyte titers are under direct hormonal control, which has to be proven in future experiments.     

Community dynamics within a bacterial consortium during growth on toluene under sulfate-reducing conditions

A sulfate-reducing bacterial consortium was enriched from an anoxic aquifer contaminated with BTEX compounds, using toluene as a growth substrate. Total cell counts, protein contents and sulfide production were determined to follow growth at the in situ temperature (14 °C) and at 25 °C, respectively. Community members were identified by 16S rRNA gene cloning and sequencing. Phylogenetic analysis revealed 12 sequence types belonging to Deltaproteobacteria (several groups) , Epsilonproteobacteria, Bacteroidetes, Spirochaetaceae and an unclassified bacterial clade. The most prominent phylotype comprising 34% of all clones was affiliated to the Desulfobulbaceae and closely related to environmental clones retrieved from hydrocarbon-contaminated aquifers. Flow-cytometric methods were applied to analyze the community dynamics and to identify key organisms involved in toluene assimilation. Flow-cytometric measurement of DNA contents and scatter behavior served to detect and quantify dominant and newly emerging clusters of subcommunities. Up to seven subcommunities, two of them dominant, were distinguished. Cell sorting was used to facilitate the analysis of conspicuous clusters for phylogenetic identity by terminal restriction fragment length polymorphism profiling of the 16S rRNA genes. The Desulfobulbaceae phylotype accounted for up to 87% in proliferating subcommunities, indicating that it represents the key organism of toluene degradation within this complex anaerobic consortium.     

Aurora1 phosphorylation activity on histone H3 and its cross-talk with other post-translational histone modifications in Arabidopsis

The enzymological properties of AtAurora1, a kinase responsible for the cell cycle-dependent phosphorylation of histone H3 at S10, and its cross-talk with other post-translational histone modifications, were determined. In vitro phosphorylation of H3S10 by AtAurora1 is strongly increased by K9 acetylation, and decreased by K14 acetylation and T11 phosphorylation. However, S10 phosphorylation activity is unaltered by mono-, di- or trimethylation of K9. An interference of H3K9 dimethylation by SUVR4 occurs by a pre-existing phosphorylation at S10. Hence, cross-talk in plants exists between phosphorylation of H3S10 and methylation, acetylation or phosphorylation of neighbouring amino acid residues. AtAurora1 undergoes autophosphorylation in vivo regardless of the presence of substrate, and forms dimers in planta . Of the three ATP-competitive Aurora inhibitors tested, Hesperadin was most effective in reducing the in vivo kinase activity of AtAurora1. Hesperadin consistently inhibited histone H3S10 phosphorylation during mitosis in Arabidopsis cells, but did not affect other H3 post-translational modifications, suggesting a specific inhibition of AtAurora in vivo . Inactivation of AtAurora also caused lagging chromosomes in a number of anaphase cells, but, unlike the situation in mammalian cells, Hesperadin did not influence the microtubule dynamics in dividing cells.     

Inhibition of histone deacetylation protects wild-type but not gelsolin-deficient neurons from oxygen/glucose deprivation

Histone acetylation and deacetylation participate in the epigenetic regulation of gene expression. In this paper, we demonstrate that pre-treatment with the histone deacetylation inhibitor trichostatin A (TSA) enhances histone acetylation in primary cortical neurons and protects against oxygen/glucose deprivation, a model for ischaemic cell death in vitro. The actin-binding protein gelsolin was identified as a mediator of neuroprotection by TSA. TSA enhanced histone acetylation of the gelsolin promoter region, and up-regulated gelsolin messenger RNA and protein expression in a dose- and time-dependent manner. Double-label confocal immunocytochemistry visualized the up-regulation of gelsolin and histone acetylation within the same neuron. Together with gelsolin up-regulation, TSA pre-treatment decreased levels of filamentous actin. The neuroprotective effect of TSA was completely abolished in neurons lacking gelsolin gene expression. In conclusion, we demonstrate that the enhancement of gelsolin gene expression correlates with neuroprotection induced by the inhibition of histone deacetylation.     

Regulation of LHCII aggregation by different thylakoid membrane lipids

In the present study the influence of the lipid environment on the organization of the main light-harvesting complex of photosystem II (LHCII) was investigated by 77K fluorescence spectroscopy. Measurements were carried out with a lipid-depleted and highly aggregated LHCII which was supplemented with the different thylakoid membrane lipids. The results show that the thylakoid lipids are able to modulate the spectroscopic properties of the LHCII aggregates and that the extent of the lipid effect depends on both the lipid species and the lipid concentration. Addition of the neutral galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) seems to induce a modification of the disorganized structures of the lipid-depleted LHCII and to support the aggregated state of the complex. In contrast, we found that the anionic lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) exert a strong disaggregating effect on the isolated LHCII. LHCII disaggregation was partly suppressed under a high proton concentration and in the presence of cations. The strongest suppression was visible at the lowest pH value (pH 5) and the highest Mg(2+) concentration (40 mM) used in the present study. This suggests that the negative charge of the anionic lipids in conjunction with negatively charged domains of the LHCII proteins is responsible for the disaggregation. Additional measurements by photon correlation spectroscopy and sucrose gradient centrifugation, which were used to gain information about the size and molecular mass of the LHCII aggregates, confirmed the results of the fluorescence spectroscopy. LHCII treated with MGDG and DGDG formed an increased number of aggregates with large particle sizes in the micromm-range, whereas the incubation with anionic lipids led to much smaller LHCII particles (around 40 nm in the case of PG) with a homogeneous distribution.     

Distribution and diversity of Gallionella-like neutrophilic iron oxidizers in a tidal freshwater marsh

Microbial iron oxidation is an integral part of the iron redox cycle in wetlands. Nonetheless, relatively little is known about the composition and ecology of iron-oxidizing communities in the soils and sediments of wetlands. In this study, sediment cores were collected across a freshwater tidal marsh in order to characterize the iron-oxidizing bacteria (FeOB) and to link their distributions to the geochemical properties of the sediments. We applied recently designed 16S rRNA primers targeting Gallionella-related FeOB by using a nested PCR-denaturing gradient gel electrophoresis (DGGE) approach combined with a novel quantitative PCR (qPCR) assay. Gallionella-related FeOB were detected in most of the samples. The diversity and abundance of the putative FeOB were generally higher in the upper 5 to 12 cm of sediment than in deeper sediment and higher in samples collected in April than in those collected in July and October. Oxygen supply by macrofauna appears to be a major force in controlling the spatial and temporal variations in FeOB communities. The higher abundance of Gallionella-related FeOB in April coincided with elevated concentrations of extractable Fe(III) in the sediments. Despite this coincidence, the distributions of FeOB did not exhibit a simple relationship to the redox zonation inferred from the geochemical depth profiles.     

Crystal structures exploring the origins of the broader specificity of escherichia coli heat-labile enterotoxin compared to cholera toxin

Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are structurally and functionally related and share the same primary receptor, the GM1 ganglioside. Despite their extensive similarities, these two toxins exhibit distinct ligand specificities, with LT being more promiscuous than CT. Here, we have attempted to rationalize the broader binding specificity of LT and the subtle differences between the binding characteristics of LTs from human and porcine origins (mediated by their B subunit pentamers, hLTB and pLTB, respectively). The analysis is based on two crystal structures of pLTB in complexes with the pentasaccharide of its primary ligand, GM1, and with neolactotetraose, the carbohydrate determinant of a typical secondary ligand of LTs, respectively. Important molecular determinants underlying the different binding specificities of LTB and CTB are found to be contributed by Ser95, Tyr18 and Thr4 (or Ser4 of hLTB), which together prestabilize the binding site by positioning Lys91, Glu51 and the adjacent loop region (50-61) containing Ile58 for ligand binding. Glu7 and Ala1 may also play an important role. Many of these residues are closely connected with a recently identified second binding site, and there appears to be cross-talk between the two sites. Binding to N-acetyllactosamine-terminated receptors is further augmented by Arg13 (present in pLT and some hLT variants), as previously predicted.