Arfophilins are dual Arf/Rab 11 binding proteins that regulate recycling endosome distribution and are related to Drosophila nuclear fallout

Arfophilin is an ADP ribosylation factor (Arf) binding protein of unknown function. It is identical to the Rab11 binding protein eferin/Rab11-FIP3, and we show it binds both Arf5 and Rab11. We describe a related protein, arfophilin-2, that interacts with Arf5 in a nucleotide-dependent manner, but not Arf1, 4, or 6 and also binds Rab11. Arfophilin-2 localized to a perinuclear compartment, the centrosomal area, and focal adhesions. The localization of arfophilin-2 to the perinuclear compartment was selectively blocked by overexpression of Arf5-T31N. In contrast, a green fluorescent protein-arfophilin-2 chimera or arfophilin-2 deletions were localized around the centrosome in a region that was also enriched for transferrin receptors and Rab11 but not early endosome markers, suggesting that the distribution of the endosomal recycling compartment was altered. The arfophilins belong to a conserved family that includes Drosophila melanogaster nuclear fallout, a centrosomal protein required for cellularization. Expression of green fluorescent protein-nuclear fallout in HeLa cells resulted in a similar phenotype, indicative of functional homology and thus implicating the arfophilins in mitosis/cytokinesis. We suggest that the novel dual GTPase-binding capacity of the arfophilins could serve as an interface of signals from Rab and Arf GTPases to regulate membrane traffic and integrate distinct signals in the late endosomal recycling compartment.     

Modulation of PAI-1 and proMMP-9 syntheses by soluble TNFalpha and its receptors during differentiation of the human monocytic HL-60 cell line

During phorbol ester-induced differentiation of HL-60 monocytic cells, tumor necrosis factoralpha (TNFalpha) synthesis and secretion are increased, which contributes to the autocrine regulation of TNFalpha-responsive genes. We investigated how, during phorbol ester-induced differentiation of HL-60 cells, the secreted TNFalpha modulated plasminogen activator inhibitor type I (PAI-1) and gelatinase B (MMP-9) syntheses, two proteins involved in pericellular proteolysis. The differentiation-induced release of TNFalpha, was abolished by the hydroxamate-based matrix metalloproteinase (MMP) inhibitor, RU36156. RU36156 or a neutralizing anti-TNFalpha significantly down-regulated PAI-1 synthesis exclusively during the early phases of differentiation (from promyelocyte to monocytic-like cells), which underlined the activating role of autocrine TNFalpha during this time range. As cells progressed to monocyte/macrophage phenotype, they still released TNFalpha, but RU36156 or anti-TNFalpha no longer had an effect on PAI-1 synthesis. This lack of effect was not due to a default of TNFalpha signaling since PAI-1 synthesis was still stimulated in response to exogenous TNFalpha. TNFalpha receptor RI was also actively released and was shown to reduce TNFalpha activity which may account for the inability of soluble TNFalpha to up-regulate PAI-1 synthesis. In later mature stage, cells became susceptible to exogenous TNFalpha-induced apoptosis and rapidly lost their ability to respond to TNFalpha. The MMP-9 synthesis followed similar regulation as PAI-1. Isolated human blood monocytes-derived macrophages behave like HL-60-derived macrophages. In conclusion, these results show that during leukocyte differentiation, time windows exist during which the autocrine TNFalpha is active and then down-regulated by RI, which may temper a continuous up-regulation of the synthesis of proteins involved in pericellular proteolysis.     

The loss of culturability by Escherichia coli cells in seawater depends on availability of phosphate ions and phosphate transport systems

Using strains with or without the PhoE porin or different components of the phosphate regulon, we determined that maintenance of the culturability of Escherichia coli in seawater depended significantly on the presence of structures allowing access of phosphate ions to the periplasm, then to the cytoplasm of cells. Cells totally deprived of the two main phosphate transport systems (Pit, Pst) exhibited the highest loss of culturability. Most of this effect resulted from the loss of the high-affinity Pst system, and more specifically that of the periplasmic phosphate-binding protein PhoS. Survival was enhanced in seawater supplemented with phosphate (0.5 mm), whether or not these structures were present. From an ecological point of view, it is assumed that the presence of phosphate ions, even at low concentrations, can influence the behavior of E. coli cells in seawater. Offprint requests to: M.J. Gauthier     

SELDI-TOF MS detection of urinary hepcidin

Hepcidin is a 25-residue hepatic peptide that regulates iron absorption from the diet and tissue iron distribution. Inappropriately low Hepcidin expression is implicated in the pathogenesis of hereditary hemochromatosis and iron-loading anemias, like the thalassemias. Increased hepcidin expression mediates iron retention in the anemias of inflammation and plays a pathogenic role in iron-refractory iron-deficiency anemia (IRIDA). Because of its clinical importance, Hepcidin is expected to be a useful biomarker for diagnosis and management of iron-related disorders. So far an ELISA for human hepcidin and SELDI-TOF-MS based approaches have been applied to monitor urinary and/or serum hepcidin levels. Here we report a modified protocol for SELDI-TOF based detection of human, urinary hepcidin. We show that CM10 Proteinchips are superior to NP20 Proteinchips commonly used in previously reported protocols to sensitively and accurately detect urinary hepcidin. Application of this modified hepcidin assay accurately detects increased hepcidin levels in the urine of sepsis patients.     

A tandem repeats database for bacterial genomes: application to the genotyping of Yersinia pestis and Bacillus anthracis

Background  

Some pathogenic bacteria are genetically very homogeneous, making strain discrimination difficult. In the last few years, tandem repeats have been increasingly recognized as markers of choice for genotyping a number of pathogens. The rapid evolution of these structures appears to contribute to the phenotypic flexibility of pathogens. The availability of whole-genome sequences has opened the way to the systematic evaluation of tandem repeats diversity and application to epidemiological studies.     

Nest Predation Deviates from Nest Predator Abundance in an Ecologically Trapped Bird

In human-modified environments, ecological traps may result from a preference for low-quality habitat where survival or reproductive success is lower than in high-quality habitat. It has often been shown that low reproductive success for birds in preferred habitat types was due to higher nest predator abundance. However, between-habitat differences in nest predation may only weakly correlate with differences in nest predator abundance. An ecological trap is at work in a farmland bird (Lanius collurio) that recently expanded its breeding habitat into open areas in plantation forests. This passerine bird shows a strong preference for forest habitat, but it has a higher nest success in farmland. We tested whether higher abundance of nest predators in the preferred habitat or, alternatively, a decoupling of nest predator abundance and nest predation explained this observed pattern of maladaptive habitat selection. More than 90% of brood failures were attributed to nest predation. Nest predator abundance was more than 50% higher in farmland, but nest predation was 17% higher in forest. Differences between nest predation on actual shrike nests and on artificial nests suggested that parent shrikes may facilitate nest disclosure for predators in forest more than they do in farmland. The level of caution by parent shrikes when visiting their nest during a simulated nest predator intrusion was the same in the two habitats, but nest concealment was considerably lower in forest, which contributes to explaining the higher nest predation in this habitat. We conclude that a decoupling of nest predator abundance and nest predation may create ecological traps in human-modified environments.     

Anatomically Discrete Sex Differences in Neuroplasticity in Zebra Finches as Reflected by Perineuronal Nets

Large morphological sex differences in the vertebrate brain were initially identified in song control nuclei of oscines. Besides gross differences between volumes of nuclei in males and females, sex differences also concern the size and dendritic arborization of neurons and various neurochemical markers, such as the calcium-binding protein parvalbumin (PV). Perineuronal nets (PNN) of the extracellular matrix are aggregates of different compounds, mainly chondroitin sulfate proteoglycans, that surround subsets of neurons, often expressing PV. PNN develop in zebra finches song control nuclei around the end of the sensitive period for song learning and tutor deprivation, known to delay the end of the song learning sensitive period, decreases the numbers of PNN in HVC. We demonstrate here the existence in zebra finches of a major sex difference (males > females) affecting the number of PNN (especially those surrounding PV-positive cells) in HVC and to a smaller extent the robust nucleus of the arcopallium, RA, the two main nuclei controlling song production. These differences were not present in Area X and LMAN, the lateral magnocellular nucleus of the anterior nidopallium. A dense expression of material immunoreactive for chondroitin sulfate was also detected in several nuclei of the auditory and visual pathways. This material was often organized in perineuronal rings but quantification of these PNN did not reveal any sex difference with the exception that the percentage of PNN surrounding PV-ir cells in the dorsal lateral mesencephalic nucleus, MLd, was larger in females than in males, a sex difference in the opposite direction compared to what is seen in HVC and RA. These data confirm and extend previous studies demonstrating the sex difference affecting PNN in HVC-RA by showing that this sex difference is anatomically specific and does not concern visual or auditory pathways.     

Sources of nitrate in rivers draining sixteen watersheds in the northeastern U.S.: Isotopic constraints

The feasibility of using nitrogen and oxygenisotope ratios of nitrate (NO₃ ^–) forelucidating sources and transformations ofriverine nitrate was evaluated in a comparativestudy of 16 watersheds in the northeastern U.S.A. Stream water was sampled repeatedly at theoutlets of the watersheds between January andDecember 1999 for determining concentrations,¹⁵N values, and ¹⁸Ovalues of riverine nitrate.In conjunction with information about land useand nitrogen fluxes,¹⁵N_nitrate and¹⁸O_nitrate values providedmainly information about sources of riverinenitrate. In predominantly forested watersheds,riverine nitrate had mean concentrations ofless than 0.4 mg NO₃ ^–-N L^–1,¹⁵N_nitrate values of lessthan +5, and ¹⁸O_nitratevalues between +12 and +19. This indicatesthat riverine nitrate was almost exclusivelyderived from soil nitrification processes withpotentially minor nitrate contributions fromatmospheric deposition in some catchments. Inwatersheds with significant agricultural andurban land use, concentrations of riverinenitrate were as high as 2.6 mg NO₃ ^–-NL^–1 with ¹⁵N_nitratevalues between +5 and +8 and¹⁸O_nitrate values generallybelow +15. Correlations between nitrateconcentrations, ¹⁵N_nitratevalues, and N fluxes suggest that nitrate inwaste water constituted a major, and nitrate inmanure a minor additional source of riverinenitrate. Atmospheric nitrate deposition ornitrate-containing fertilizers were not asignificant source of riverine nitrate inwatersheds with significant agricultural andurban land use. Although complementary studiesindicate that in-stream denitrification wassignificant in all rivers, the isotopiccomposition of riverine nitrate sampled at theoutlet of the 16 watersheds did not provideevidence for denitrification in the form ofelevated ¹⁵N_nitrate and¹⁸O_nitrate values. Relativelylow isotopic enrichment factors for nitrogenand oxygen during in-stream denitrification andcontinuous admixture of nitrate from theabove-described sources are thought to beresponsible for this finding.     

Development of a lab-made microarray for analyzing the genetic diversity of nitrogen fixing symbionts Sinorhizobium meliloti and Sinorhizobium medicae

Some bacterial species, like nitrogen-fixing Sinorhizobium that interact with Medicago plants, are prone to frequent horizontal gene transfers. Investigation of their genetic structure requires to study polymorphism patterns at many loci. Although DNA microarrays represent a method of choice for high throughput analysis of polymorphisms, this technology yet remains an expensive and heavy approach, thus depriving most of research groups from this powerful tool. In an attempt to overcome this limitation, we have developed a simple genotyping procedure by DNA microarrays, and have evaluated its ability to characterize a Sinorhizobium population. Thirty 18- to 24-mer oligonucleotide probes were designed to target the most frequent mutations in three polymorphic loci of Sinorhizobium meliloti and S. medicae. Probe hybridization efficiency was compared on two spotting surfaces: nylon membranes and epoxy-coated glass slides. Epoxy-coated glass slides revealed more sensitive than nylon membranes and allowed discrimination of single mismatches. Using this procedure, an uncharacterized population consisting of 33 S. meliloti/S. medicae isolates was successfully genotyped.