SDF‐1/CXCR4 signalling is involved in blood vessel growth and remodelling by intussusception

The precise mechanisms of SDF‐1 (CXCL12) in angiogenesis are not fully elucidated. Recently, we showed that Notch inhibition induces extensive intussusceptive angiogenesis by recruitment of mononuclear cells and it was associated with increased levels of SDF‐1 and CXCR4. In the current study, we demonstrated SDF‐1 expression in liver sinusoidal vessels of Notch1 knockout mice with regenerative hyperplasia by means of intussusception, but we did not detect any SDF‐1 expression in wild‐type mice with normal liver vessel structure. In addition, pharmacological inhibition of SDF‐1/CXCR4 signalling by AMD3100 perturbs intussusceptive vascular growth and abolishes mononuclear cell recruitment in the chicken area vasculosa. In contrast, treatment with recombinant SDF‐1 protein increased microvascular density by 34% through augmentation of pillar number compared to controls. The number of extravasating mononuclear cells was four times higher after SDF‐1 application and two times less after blocking this pathway. Bone marrow‐derived mononuclear cells (BMDC) were recruited to vessels in response to elevated expression of SDF‐1 in endothelial cells. They participated in formation and stabilization of pillars. The current study is the first report to implicate SDF‐1/CXCR4 signalling in intussusceptive angiogenesis and further highlights the stabilizing role of BMDC in the formation of pillars during vascular remodelling.     

Hyperthermophilic Thermotoga arginine repressor binding to full-length cognate and heterologous arginine operators and to half-site targets

The degree of sequence conservation of arginine repressor proteins (ArgR) and of the cognate operators (tandem pairs of 18 bp imperfect palindromes, ARG boxes) in evolutionarily distant bacteria is unusually high, and the global mechanism of ArgR-mediated regulation appears to be similar. However, here we demonstrate that the arginine repressor from the hyperthermophilic bacterium Thermotoga neapolitana (ArgR(Tn)) exhibits characteristics that clearly distinguish this regulator from the well-studied homologues from Escherichia coli, Bacillus subtilis and B.stearothermophilus. A high-resolution contact map of ArgR(Tn) binding to the operator of the biosynthetic argGHCJBD operon of Thermotoga maritima indicates that ArgR(Tn) establishes all of its strong contacts with a single ARG box-like sequence of the operator only. Protein array and electrophoretic mobility-shift data demonstrate that ArgR(Tn) has a remarkable capacity to bind to arginine operators from Gram-negative and Gram-positive bacteria, and to single ARG box-bearing targets. Moreover, the overall effect of L-arginine on the apparent K(d) of ArgR(Tn) binding to various cognate and heterologous operator fragments was minor with respect to that observed with diverse bacterial arginine repressors. We demonstrate that this unusual behaviour for an ArgR protein can, to a large extent, be ascribed to the presence of a serine residue at position 107 of ArgR(Tn), instead of the highly conserved glutamine that is involved in arginine binding in the E.coli repressor. Consistent with these results, ArR(Tn) was found to behave as a superrepressor in E.coli, inhibiting growth in minimal medium, even supplemented with arginine, whereas similar constructs bearing the S107Q mutant allele did not inhibit growth. We assume that ArgR(Tn), owing to its broad target specificity and its ability to bind single ARG box sequences, might play a more general regulatory role in Thermotoga     

SANE,a novel LEM domain protein,regulates bone morphogenetic protein signaling through interaction with Smad1

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) superfamily that play important roles in bone formation, embryonic patterning, and epidermal-neural cell fate decisions. BMPs signal through pathway specific mediators such as Smads1 and 5, but the upstream regulation of BMP-specific Smads has not been fully characterized. Here we report the identification of SANE (Smad1 Antagonistic Effector), a novel protein with significant sequence similarity to nuclear envelop proteins such as MAN1. SANE binds to Smad1/5 and to BMP type I receptors and regulates BMP signaling. SANE specifically blocks BMP-dependent signaling in Xenopus embryos and in a mammalian model of bone formation but does not inhibit the TGF-beta/Smad2 pathway. Inhibition of BMP signaling by SANE requires interaction between SANE and Smad1, because a SANE mutant that does not bind Smad1 does not inhibit BMP signaling. Furthermore, inhibition appears to be mediated by inhibition of BMP-induced Smad1 phosphorylation, blocking ligand-dependent nuclear translocation of Smad1. These studies define a new mode of regulation for intracellular BMP/Smad1 signaling.     

Susceptibility and adaptive response to bile salts in Propionibacterium freudenreichii: physiological and proteomic analysis

Tolerance to digestive stresses is one of the main factors limiting the use of microorganisms as live probiotic agents. Susceptibility to bile salts and tolerance acquisition in the probiotic strain Propionibacterium freudenreichii SI41 were characterized. We showed that pretreatment with a moderate concentration of bile salts (0.2 g/liter) greatly increased its survival during a subsequent lethal challenge (1.0 g/liter, 60 s). Bile salts challenge led to drastic morphological changes, consistent with intracellular material leakage, for nonadapted cells but not for preexposed ones. Moreover, the physiological state of the cells during lethal treatment played an important role in the response to bile salts, as stationary-phase bacteria appeared much less sensitive than exponentially growing cells. Either thermal or detergent pretreatment conferred significantly increased protection toward bile salts challenge. In contrast, some other heterologous pretreatments (hypothermic and hyperosmotic) had no effect on tolerance to bile salts, while acid pretreatment even might have sensitized the cells. Two-dimensional electrophoresis experiments revealed that at least 24 proteins were induced during bile salts adaptation. Identification of these polypeptides suggested that the bile salts stress response involves signal sensing and transduction, a general stress response (also triggered by thermal denaturation, oxidative toxicity, and DNA damage), and an alternative sigma factor. Taken together, our results provide new insights into the tolerance of P. freudenreichii to bile salts, which must be taken into consideration for the use of probiotic strains and the improvement of technological processes.     

Microsporidian Parasites Found in the Hemolymph of Four Baikalian Endemic Amphipods

At present, approximately 187 genera and over 1300 species of Microsporidia have been described, among which almost half infect aquatic species and approximately 50 genera potentially infect aquatic arthropods. Lake Baikal is the deepest and one of the oldest lakes in the world, and it has a rich endemic fauna with a predominance of arthropods. Among the arthropods living in this lake, amphipods (Crustacea) are the most dominant group and are represented by more than 350 endemic species. Baikalian amphipods inhabit almost all depths and all types of substrates. The age and geographical isolation of this group creates excellent opportunities for studying the diversity, evolution and genetics of host-parasite relationships. However, despite more than 150 years of study, data investigating the microsporidia of Lake Baikal remain incomplete. In this study, we used molecular genetic analyses to detect microsporidia in the hemolymph of several endemic species of amphipods from Lake Baikal. We provide the first evidence that microsporidian species belonging to three genera (Microsporidium, Dictyocoela and Nosema) are present in the hemolymph of Baikalian endemic amphipods. In the hemolymph of Eulimnogammarus verrucosus, we detected SSU rDNA of microsporidia belonging to the genus Nozema. In the hemolymph of Pallasea cancellous, we found the DNA of Microsporidium sp. similar to that in other Baikalian endemic amphipods; Dictyocoela sp. was found in the hemolymph of Eulimnogammarus marituji and Acanthogammarus lappaceus longispinus.     

PLP/DM20 ratio is regulated by hnRNPH and F and a novel G-rich enhancer in oligodendrocytes

Alternative splicing of competing 5′ splice sites is regulated by enhancers and silencers in the spliced exon. We have characterized sequences and splicing factors that regulate alternative splicing of PLP and DM20, myelin proteins produced by oligodendrocytes (OLs) by selection of 5′ splice sites in exon 3. We identify a G-rich enhancer (M2) of DM20 5′ splice site in exon 3B and show that individual G triplets forming M2 are functionally distinct and the distal group plays a dominant role. G-rich M2 and a G-rich splicing enhancer (ISE) in intron 3 share similarities in function and protein binding. The G-rich sequences are necessary for binding of hnRNPs to both enhancers. Reduction in hnRNPH and F expression in differentiated OLs correlates temporally with increased PLP/DM20 ratio. Knock down of hnRNPH increased PLP/DM20 ratio, while hnRNPF did not. Silencing hnRNPH and F increased the PLP/DM20 ratio more than hnRNPH alone, demonstrating a novel synergistic effect. Mutation of M2, but not ISE reduced the synergistic effect. Replacement of M2 and all G runs in exon 3B abolished it almost completely. We conclude that developmental changes in hnRNPH/F associated with OLs differentiation synergistically regulate PLP alternative splicing and a G-rich enhancer participates in the regulation.     

Identification and Heterologous Expression of Genes Involved in Anaerobic Dissimilatory Phosphite Oxidation by Desulfotignum phosphitoxidans

Desulfotignum phosphitoxidans is a strictly anaerobic, Gram-negative bacterium that utilizes phosphite as the sole electron source for homoacetogenic CO₂ reduction or sulfate reduction. A genomic library of D. phosphitoxidans, constructed using the fosmid vector pJK050, was screened for clones harboring the genes involved in phosphite oxidation via PCR using primers developed based on the amino acid sequences of phosphite-induced proteins. Sequence analysis of two positive clones revealed a putative operon of seven genes predicted to be involved in phosphite oxidation. Four of these genes (ptxD-ptdFCG) were cloned and heterologously expressed in Desulfotignum balticum, a related strain that cannot use phosphite as either an electron donor or as a phosphorus source. The ptxD-ptdFCG gene cluster was sufficient to confer phosphite uptake and oxidation ability to the D. balticum host strain but did not allow use of phosphite as an electron donor for chemolithotrophic growth. Phosphite oxidation activity was measured in cell extracts of D. balticum transconjugants, suggesting that all genes required for phosphite oxidation were cloned. Genes of the phosphite gene cluster were assigned putative functions on the basis of sequence analysis and enzyme assays.Phosphorus (P) is an important nutrient for all living organisms. The predominant forms of phosphorus in biological systems are inorganic phosphate and its organic esters and acid anhydrides in which P is at its highest oxidation state (+V). The P requirements of living cells can be fulfilled with phosphate in various forms, including reduced organic and inorganic phosphorus compounds (23). Several aerobic bacteria were shown to be able to oxidize hypophosphite (+I) and phosphite (+III) to phosphate (+V) and to incorporate the last into their biomass (5, 15-17, 31, 34). Phosphite can also be oxidized under anaerobic conditions, as shown for an anaerobic Bacillus strain (7) and for Pseudomonas stutzeri which can use phosphite under denitrifying conditions (17, 21). The only bacterium known to oxidize phosphite as the sole source of electrons in lithoautotrophic energy metabolism is Desulfotignum phosphitoxidans (24, 25).Three different metabolic pathways for the use of phosphite as a single P source have been characterized so far. Two of them were discovered and characterized with Escherichia coli and one with Pseudomonas stutzeri. The first pathway in E. coli is mediated by the enzyme carbon phosphorus lyase (C-P lyase), and the second one by the alkaline phosphatase encoded by phoA (16, 34). This alkaline phosphatase not only hydrolyzes phosphate esters but also hydrolyzes phosphite to phosphate and molecular hydrogen (32). This is a particular property only of the E. coli alkaline phosphatase and is not observed with alkaline phosphatases of other bacteria. The third pathway is encoded by the ptxABCDE gene cluster in P. stutzeri (17). In this system, phosphite is transported into the cell by a binding protein-dependent phosphite transporter at the expense of ATP (PtxABC). Phosphite is oxidized by a phosphite:NAD⁺ oxidoreductase (encoded by ptxD), a new member of the 2-hydroxy acid dehydrogenases (8). The ptx operon of P. stutzeri is regulated in response to phosphate starvation by the two-component regulatory system phoBR (28, 29). Furthermore, in Alcaligenes faecalis WM2072, another gene cluster involved in hypophosphite and phosphite uptake and oxidation was characterized: the htxABCD-ptxDE locus (31). The htxABCD-ptxDE genes and their products in A. faecalis WM 2072 have high nucleotide and amino acid sequence identities with those found in the htx and ptx operons in P. stutzeri WM88, which are required for the oxidation of hypophosphite and phosphite, respectively. This unique genetic arrangement of hypophosphite- and phosphite-oxidizing genes in A. faecalis WM2072 suggests a horizontal gene transfer and an ancient evolution of phosphite oxidation.The diversity of pathways used for assimilatory phosphite oxidation and the fact that D. phosphitoxidans is so far the only bacterium known to use phosphite as an electron source caused us to investigate the phosphite uptake and oxidation gene cluster of this bacterium. The aims of our study were (i) to establish enzymatic assays for measurement of phosphite oxidation activity in cell extracts, (ii) to identify the genes involved in phosphite uptake and oxidation, and (iii) to characterize these genes physiologically.