Real‐time imaging of photodynamic action in bacteria

Fluorescence imaging studies of the processes leading to photodynamic inactivation of bacteria have been limited due to the small size of microorganisms as well as by the faint fluorescence of most photosensitizers. A versatile method based on highly‐sensitive fluorescence microscopy is presented which allows to study, in real time, the incorporation of photosensitizers inside S. aureus upon photodynamic action. The method takes advantage of the fluorescence enhancement of phenothiazine and porphyrin photosensitizers upon entering the bacterial cytosol after the cell wall has been compromised. In combination with typical assays, such as the addition of specific enhancers of reactive oxygen species, it is possible to extract mechanistic information about the pathway of photodynamic damage at the single‐cell level. Imaging experiments in deuterated buffer strongly support a Type‐I mechanism for methylene blue and a very minor role of singlet oxygen.

     

1,25(OH)2 vitamin D3 signalling on immature rat Sertoli cells: gamma-glutamyl transpeptidase and glucose metabolism

1α,25-Dihydroxyvitamin D₃ (1,25-D₃) is critical for the maintenance of normal male reproduction since reduced fertility is observed in vitamin D-deficient rats. Gamma-glutamyl transpeptidase (GGT) is a membrane-bound enzyme that is localized on Sertoli cells and catalyses the transfer of the gamma-glutamyl residues to an amino acid or peptide acceptor. Sertoli cells are also responsible for providing nutrients, as lactate, to the development of germ cells. The aim of this study was to investigate the effect and the mechanism of action of 1,25-D₃ on GGT on Sertoli cell functions from 30-day-old immature rat testis. Results demonstrated that 1,25-D₃ stimulates GGT activity at Sertoli cells plasma membrane through a PKA-dependent mechanism of action, which was not dependent of active de novo protein synthesis. The hormone increases glucose uptake, as well as lactate production and release by Sertoli cells without altering the reactive oxygen species (ROS) generation. In addition, 1,25-D₃ did not change reduced glutathione (GSH) amount or oxygen consumption, and diminished Sertoli cell death. These findings demonstrate that 1,25-D₃ stimulatory effect on GGT activity, glucose uptake, LDH activity and lactate production seem to be an important contribution of Sertoli cells for germ cells nutrition and for a full and active ongoing spermatogenesis.     

A comparison between Ca and Sr cycling in forest ecosystems

In favourable conditions, the ⁸⁷Sr/⁸⁶Sr isotope ratios of the Sr delivered by rain and soil mineral weathering differ. Assuming that Ca and Sr behave similarly in forest ecosystems, several authors have used the ⁸⁷Sr/⁸⁶Sr variation in forest compartments to calculate the contribution of rain and mineral weathering to Ca fluxes and pools. However, there are a number of experimental reports showing that Ca and Sr may behave differently in the soil and in the plant. We have tested this Ca–Sr analogy in the field by measuring the variation of Sr and Ca concentrations, fluxes and pools in spruce, beech and maple stands on granite, sandstone and limestone. Results show that (1) variations of Ca and Sr concentrations are generally correlated at each level of the ecosystems. (2) In spruce on acid soils, a preferential uptake of Ca over Sr occurs (Aubure spruce Sr/Ca = 0.8×10⁻³; soil exchangeable Sr/Ca between 2 and 6×10⁻³). On calcareous soils, a preferential uptake of Sr over Ca by spruce may occur. (3) In spruce and beech on acid and calcareous soils, a preferential translocation of Ca over Sr from roots to leaves occurs ((Sr/Ca) in leaves was between 10 and 90% of that in roots). (4) The biological cycling of Ca and Sr leads to an enrichment of the upper soil layers in Ca and Sr. Compared to Sr, Ca accumulates in the upper layer of acid soils because Ca cycling through litterfall is favoured over Sr cycling, and possibly because of the selectivity of acid organic exchangers for Ca. This revised version was published online in June 2006 with corrections to the Cover Date.     

Down‐regulation of striatin,a neuronal calmodulin‐binding protein,impairs rat locomotor activity

Striatin, an intraneuronal, calmodulin‐binding protein addressed to dendrites and spines, is expressed in the motor system, particularly the striatum and motoneurons. Striatin contains a high number of domains mediating protein–protein interactions, suggesting a role within a dendritic Ca²⁺‐signaling pathway. Here, we explored the hypothesis of a direct role of striatin in the motor control of behaving rats, by using an antisense strategy based on oligodeoxynucleotides (ODN). Rats were treated by intracerebroventricular infusion of a striatin antisense ODN (A‐ODN) or mismatch ODN (M‐ODN) delivered by osmotic pumps over 6 days. A significant decrease in the nocturnal locomotor activity of A‐ODN–treated rats was observed after 5 days of treatment. Hypomotricity was correlated with a 60% decrease in striatin content of the striata of A‐ODN–treated rats sacrificed on day 6. Striatin thus plays a role in the control of motor function. To approach the cellular mechanisms in which striatin is involved, striatin down‐regulation was studied in a comparatively simpler model: purified rat spinal motoneurons which retain their polarity in culture. Treatment of cells by the striatin A‐ODN resulted in the impairement of the growth of dendrites but not axon. The decrease in dendritic growth paralleled the loss of striatin. This model allows analysis of the molecular basis of striatin function in the dynamic changes occurring in growing dendrites, and offers clues to unravel its function within spines. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 234–243, 1999     

The splicing‐regulatory lncRNA NTRAS sustains vascular integrity

Vascular integrity is essential for organ homeostasis to prevent edema formation and infiltration of inflammatory cells. Long non‐coding RNAs (lncRNAs) are important regulators of gene expression and often expressed in a cell type‐specific manner. By screening for endothelial‐enriched lncRNAs, we identified the undescribed lncRNA NTRAS to control endothelial cell functions. Silencing of NTRAS induces endothelial cell dysfunction in vitro and increases vascular permeability and lethality in mice. Biochemical analysis revealed that NTRAS, through its CA‐dinucleotide repeat motif, sequesters the splicing regulator hnRNPL to control alternative splicing of tight junction protein 1 (TJP1; also named zona occludens 1, ZO‐1) pre‐mRNA. Deletion of the hnRNPL binding motif in mice (Ntras ^∆CA/∆CA) significantly repressed TJP1 exon 20 usage, favoring expression of the TJP1α‐ isoform, which augments permeability of the endothelial monolayer. Ntras ^∆CA/∆CA mice further showed reduced retinal vessel growth and increased vascular permeability and myocarditis. In summary, this study demonstrates that NTRAS is an essential gatekeeper of vascular integrity.     

The tandem PDZ protein Syntenin interacts with the aminoacyl tRNA synthetase complex in a lysyl-tRNA synthetase-dependent manner

Syntenin-1 is a tandem PDZ protein that binds a diverse array of signaling molecules that are often associated with cell adhesion and intracellular trafficking. With the use of a MS-based functional proteomics approach, we identified several members of the aminoacyl-tRNA synthetase macromolecular (ARS) complex in a syntenin-1 pull down assay. Interaction of these proteins with syntenin-1 was confirmed by co-immunoprecipitation from cultured cells. We demonstrate a direct interaction of syntenin-1 with lysyl-tRNA synthetase (KRS), which contains a PDZ binding motif at its C-terminus. This motif is important for the interaction of the entire complex with syntenin-1. A point mutation in the PDZ2 domain of syntenin-1 abrogates interaction with KRS. As a result, other components of the ARS complex no longer co-immunoprecipitate with syntenin-1. We further show that syntenin-1 regulates KRS activity. These findings suggest that syntenin-1 is an adaptor modulating the activity of KRS.     

Mirror-induced behavior in the magpie (Pica pica): evidence of self-recognition

Comparative studies suggest that at least some bird species have evolved mental skills similar to those found in humans and apes. This is indicated by feats such as tool use, episodic-like memory, and the ability to use one's own experience in predicting the behavior of conspecifics. It is, however, not yet clear whether these skills are accompanied by an understanding of the self. In apes, self-directed behavior in response to a mirror has been taken as evidence of self-recognition. We investigated mirror-induced behavior in the magpie, a songbird species from the crow family. As in apes, some individuals behaved in front of the mirror as if they were testing behavioral contingencies. When provided with a mark, magpies showed spontaneous mark-directed behavior. Our findings provide the first evidence of mirror self-recognition in a non-mammalian species. They suggest that essential components of human self-recognition have evolved independently in different vertebrate classes with a separate evolutionary history.     

Occurrence of Tn4371-Related Mobile Elements and Sequences in (Chloro)biphenyl-Degrading Bacteria

Tn4371, a 55-kb transposable element involved in the degradation and biphenyl or 4-chlorobiphenyl identified in Ralstonia eutropha A5, displays a modular structure including a phage-like integrase gene (int), a Pseudomonas-like (chloro)biphenyl catabolic gene cluster (bph), and RP4- and Ti-plasmid-like transfer genes (trb) (C. Merlin, D. Springael, and A. Toussaint, Plasmid 41:40–54, 1999). Southern blot hybridization was used to examine the presence of different regions of Tn4371 in a collection of (chloro)biphenyl-degrading bacteria originating from different habitats and belonging to different bacterial genera. Tn4371-related sequences were never detected on endogenous plasmids. Although the gene probes containing only bph sequences hybridized to genomic DNA from most strains tested, a limited selection of strains, all β-proteobacteria, displayed hybridization patterns similar to the Tn4371 bph cluster. Homology between Tn4371 and DNA of two of those strains, originating from the same area as strain A5, extended outside the catabolic genes and covered the putative transfer region of Tn4371. On the other hand, none of the (chloro)biphenyl degraders hybridized with the outer left part of Tn4371 containing the int gene. The bph catabolic determinant of the two strains displaying homology to the Tn4371 transfer genes and a third strain isolated from the A5 area could be mobilized to a R. eutropha recipient, after insertion into an endogenous or introduced IncP1 plasmid. The mobilized DNA of those strains included all Tn4371 homologous sequences previously identified in their genome. Our observations show that the bph genes present on Tn4371 are highly conserved between different (chloro)biphenyl-degrading hosts, isolated globally but belonging mainly to the β-proteobacteria. On the other hand, Tn4371-related mobile elements carrying bph genes are apparently only found in isolates from the environment that provided the Tn4371-bearing isolate A5.     

Impaired mitophagy in Sanfilippo a mice causes hypertriglyceridemia and brown adipose tissue activation

Lysosomal storage diseases result in various developmental and physiological complications, including cachexia. To study the causes for the negative energy balance associated with cachexia, we assessed the impact of sulfamidase deficiency and heparan sulfate storage on energy homeostasis and metabolism in a mouse model of type IIIa mucopolysaccharidosis (MPS IIIa, Sanfilippo A syndrome). At 12-weeks of age, MPS IIIa mice exhibited fasting and postprandial hypertriglyceridemia compared with wildtype mice, with a reduction of white and brown adipose tissues. Partitioning of dietary [³H]triolein showed a marked increase in intestinal uptake and secretion, whereas hepatic production and clearance of triglyceride-rich lipoproteins did not differ from wildtype controls. Uptake of dietary triolein was also elevated in brown adipose tissue (BAT), and notable increases in beige adipose tissue occurred, resulting in hyperthermia, hyperphagia, hyperdipsia, and increased energy expenditure. Furthermore, fasted MPS IIIa mice remained hyperthermic when subjected to low temperature but became cachexic and profoundly hypothermic when treated with a lipolytic inhibitor. We demonstrated that the reliance on increased lipid fueling of BAT was driven by a reduced ability to generate energy from stored lipids within the depot. These alterations arose from impaired autophagosome–lysosome fusion, resulting in increased mitochondria content in beige and BAT. Finally, we show that increased mitochondria content in BAT and postprandial dyslipidemia was partially reversed upon 5-week treatment with recombinant sulfamidase. We hypothesize that increased BAT activity and persistent increases in energy demand in MPS IIIa mice contribute to the negative energy balance observed in patients with MPS IIIa.