Chemical composition and ultrastructure of cellular and extracellular Moraxella glucidolytica lipopolysaccharides

A cellular (LPS I) and extracellular (LPS II) lipopolysaccharide were isolated from Moraxella glucidolytica cells grown on ethanol and from the culture fluid, respectively. Both LPS were toxic when injected to mice and chick embryos. These LPS contained glucose, galactose, glucosamine, galactosamine, 2-keto-3-deoxyoctonate and lipids. By permethylation studies, glucose was found to be linked (16) and (13) in LPS I and only (16) in LPS II. Galactose was the terminal non-reducing sugar. Branching occurred at positions 3 and 4 of galactose residues. LPS I was rich in - and -hydroxylauric and -hydroxymyristic acids and LPS II contained mainly stearic and -hydroxymyristic acids. LPS I was detoxified by mild acid and alkaline treatments. It was also dissociated by sodium deoxycholate and chromatographed on Sephadex G-75. The main fraction was reassociated by removing the surfactant by dialysis. The morphology of LPS I and LPS II was examined by electron microscopy. LPS I (original and reassociated fractions) consisted exclusively of ribbons while LPS II contained ribbons and vesicles.Non-Standard Abbreviations KDO 2-Keto-3-deoxyoctonic acids - LPS Lipopolysaccharide - NaD Sodium deoxycholate     

SUV39H2 epigenetic silencing controls fate conversion of epidermal stem and progenitor cells

Epigenetic histone trimethylation on lysine 9 (H3K9me3) represents a major molecular signal for genome stability and gene silencing conserved from worms to man. However, the functional role of the H3K9 trimethylases SUV39H1/2 in mammalian tissue homeostasis remains largely unknown. Here, we use a spontaneous dog model with monogenic inheritance of a recessive SUV39H2 loss-of-function variant and impaired differentiation in the epidermis, a self-renewing tissue fueled by stem and progenitor cell proliferation and differentiation. Our results demonstrate that SUV39H2 maintains the stem and progenitor cell pool by restricting fate conversion through H3K9me3 repressive marks on gene promoters encoding components of the Wnt/p63/adhesion axis. When SUV39H2 function is lost, repression is relieved, and enhanced Wnt activity causes progenitor cells to prematurely exit the cell cycle, a process mimicked by pharmacological Wnt activation in primary canine, human, and mouse keratinocytes. As a consequence, the stem cell growth potential of cultured SUV39H2-deficient canine keratinocytes is exhausted while epidermal differentiation and genome stability are compromised. Collectively, our data identify SUV39H2 and potentially also SUV39H1 as major gatekeepers in the delicate balance of progenitor fate conversion through H3K9me3 rate-limiting road blocks in basal layer keratinocytes.     

Auxin production and detection of the gene coding for the Auxin Efflux Carrier (AEC) protein in <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis>

Different species of Paenibacillus are considered to be plant growth-promoting rhizobacteria (PGPR) due to their ability to repress soil borne pathogens, fix atmospheric nitrogen, induce plant resistance to diseases and/or produce plant growth-regulating substances such as auxins. Although it is known that indole-3-acetic acid (IAA) is the primary naturally occurring auxin excreted by Paenibacillus species, its transport mechanisms (auxin efflux carriers) have not yet been characterized. In this study, the auxin production of P. polymyxa and P. graminis, which are prevalent in the rhizospheres of maize and sorghum sown in Brazil, was evaluated. In addition, the gene encoding the Auxin Efflux Carrier (AEC) protein from P. polymyxa DSM36(T) was sequenced and used to determine if various strains of P. polymyxa and P. graminis possessed this gene. Each of the 68 P. polymyxa strains evaluated in this study was able to produce IAA, which was produced at concentrations varying from 1 to 17 microg/ml. However, auxin production was not detected in any of the 13 P. graminis strains tested in this study. Different primers were designed for the PCR amplification of the gene coding for the AEC in P. polymyxa, and the predicted protein of 319 aa was homologous to AEC from Bacillus amyloliquefaciens, B. licheniformis, and B. subtilis. However, no product was observed when these primers were used to amplify the genomic DNA of seven strains of P. graminis, which suggests that this gene is not present in this species. Moreover, none of the P. graminis genomes tested were homologous to the gene coding for AEC, whereas all of the P. polymyxa genomes evaluated were. This is the first study to demonstrate that the AEC protein is present in P. polymyxa genome.     

The N-terminus of PKR is responsible for the activation of the NF-kappaB signaling pathway by interacting with the IKK complex

The interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) has been shown to activate NF-kappaB independently of its kinase function after interaction with the IKK complex. In order to investigate the mechanism of NF-kappaB activation by PKR, we identified the domain of PKR responsible for stimulating the NF-kappaB pathway in PKR-deficient fibroblasts using an NF-kappaB dependent reporter assay. The N-terminal 1-265 AA of PKR activates NF-kappaB, whereas the 1-180 AA N-terminus restricted to the two dsRNA Binding Domains (DRBD), the third basic domain alone (AA 181-265), or the C-terminus of PKR (AA 266-550) were unable to stimulate the expression of the NF-kappaB dependent reporter gene. Using confocal microscopy, we confirmed that PKR full length as well as PKR N-terminus colocalized with IKKbeta. By GST-pulldown analysis, using different PKR domains, we then revealed the specific ability of the PKR N-terminus 1-265 to bind to and activate IKK and showed that this activation requires the integrity of the IKK complex. This activation is not only due to DRBDs since the DRBD fragment 1-180 failed to inhibit PKR 1-265 induced NF-kappaB activation. Our results therefore demonstrate that the ability of PKR to mediate NF-kappaB activation resides in its full N-terminus, and requires both DRBDs and the third basic domain.     

Targeting human telomerase for cancer therapeutics

The enzyme telomerase is involved in the replication of telomeres, specialized structures that cap and protect the ends of chromosomes. Its activity is required for maintenance of telomeres and for unlimited lifespan, a hallmark of cancer cells. Telomerase is overexpressed in the vast majority of human cancer cells and therefore represents an attractive target for therapy. Several approaches have been developed to inhibit this enzyme through the targeting of its RNA or catalytic components as well as its DNA substrate, the single-stranded 3′-telomeric overhang. Telomerase inhibitors are chemically diverse and include modified oligonucleotides as well as small diffusable molecules, both natural and synthetic. This review presents an update of recent investigations pertaining to these agents and discusses their biological properties in the context of the initial paradigm that the exposure of cancer cells to these agents should lead to progressive telomere shortening followed by a delayed growth arrest response.     

Pertussis toxin reduces the number of splenic Foxp3+ regulatory T cells

Pertussis toxin (PTx) is a bacterial toxin used to enhance the severity of experimental autoimmune diseases such as experimental autoimmune encephalomyelitis. It is known to promote permeabilization of the blood-brain barrier, maturation of APC, activation of autoreactive lymphocytes and alteration of lymphocyte migration. In this study, we show that i.v. injection of PTx in mice induces a decrease in the number of splenic CD4(+)CD25(+) regulatory T cells (Treg cells). Furthermore, PTx not only induces a depletion of the dominant CD4(+)CD25(+)Foxp3(+) subpopulation of splenic Treg cells, but also reduces to a similar extent the CD4(+)CD25(-)Foxp3(+) subpopulation. On a per cell basis, the suppressive properties of the remaining Treg cells are not modified by PTx treatment. The reduction in splenic Treg cells is associated with preferential migration of these cells to the liver. Additionally, Treg cells exhibit a high sensitivity to PTx-mediated apoptosis in vitro. Finally, in vivo depletion of Treg cells by injection of an anti-CD25 Ab, and PTx treatment, present synergistic experimental autoimmune encephalomyelitis exacerbating effects. Therefore, we identify a new effect of PTx and provide an additional illustration of the influence of microbial components on the immune system affecting the balance between tolerance, inflammation and autoimmunity.     

Effects of citrinin on iron-redox cycle

The ability of the mycotoxin citrinin to act as an inhibitor of iron-induced lipoperoxidation of biological membranes prompted us to determine whether it could act as an iron chelating agent, interfering with iron redox reactions or acting as a free radical scavenger. The addition of Fe3+ to citrinin rapidly produced a chromogen, indicating the formation of citrinin-Fe3+ complexes. An EPR study confirms that citrinin acts as a ligand of Fe3+, the complexation depending on the [Fe3+]:[citrinin] ratios. Effects of citrinin on the iron redox cycle were evaluated by oxygen consumption or the o-phenanthroline test. No effect on EDTA-Fe2+-->EDTA-Fe3+ oxidation was observed in the presence of citrinin, but the mycotoxin inhibited, in a dose-dependent manner, the oxidation of Fe2+ to Fe3+ by hydrogen peroxide. Reducing agents such as ascorbic acid and DTT reduced the Fe3+-citrinin complex, but DTT did not cause reduction of Fe3+-EDTA, indicating that the redox potentials of Fe3+-citrinin and Fe3+-EDTA are not the same. The Fe2+ formed from the reduction of Fe3+-citrinin by reducing agents was not rapidly reoxidized to Fe3+ by atmospheric oxygen. Citrinin has no radical scavenger ability as demonstrated by the absence of DPPH reduction. However, a reaction between citrinin and hydrogen peroxide was observed by UV spectrum changes of citrinin after incubation with hydrogen peroxide. It was also observed that citrinin did not induce direct or reductive mobilization of iron from ferritin. These results indicate that the protective effect on iron-induced lipid peroxidation by citrinin occurs due to the formation of a redox inactive Fe3+-citrinin complex, as well as from the reaction of citrinin and hydrogen peroxide.     

Pancreatic hepatocytes in hamsters (Mesocricetus auratus) infected with Trypanosoma cruzi

Hepatocytic metaplasia may be induced in hamsters by carcinogens, and associated with aging, diabetes or chronic pancreatitis. By means of histopathologic and immunohistochemic studies, we observed pancreatic hepatocytes in hamsters infected and reinfected with Trypanosoma cruzi. The change was seen in 18 (19%) out of 94 infected animals, and was not found among 53 controls, Normal islet cells were immunoreactive for neuron-specific enolase and not reactive for NCL-HAS. Metaplastic cells were immunoreactive for NCL-HAS and not reactive for islet hormones and enolase. No relationship was observed between number of inoculations and metaplasia; however, the intensity of the inflammatory process and sequels seems to favor the development of metaplastic cells. Hamsters infected with T. cruzi may be useful to study hepatocytic metaplasia, and contribute to clarify aspects of Chagas' disease and pancreatic changes. Our data indicate that aging, in addition to inflammation and atrophy, plays a role in this change.     

Phylogeny and evolution of calcareous sponges: monophyly of calcinea and calcaronea,high level of morphological homoplasy,and the primitive nature of axial symmetry

Because calcareous sponges are triggering renewed interest with respect to basal metazoan evolution, a phylogenetic framework of their internal relationships is needed to clarify the evolutionary history of key morphological characters. Morphological variation was scored at the suprageneric level within Calcispongia, but little phylogenetic information could be retrieved from morphological characters. For the main subdivision of Calcispongia, the analysis of morphological data weakly supports a classification based upon cytological and embryological characters (Calcinea/Calcaronea) rather than the older classification scheme based upon the aquiferous system (Homocoela/Heterocoela). The 18S ribosomal RNA data were then analyzed, both alone and in combination with morphological characters. The monophyly of Calcispongia is highly supported, but the position of this group with respect to other sponge lineages and to eumetazoan taxa is not resolved. The monophyly of both Calcinea and Calcaronea is retrieved, and the data strongly rejected the competing Homocoela/Heterocoela hypothesis. The phylogeny implies that characters of the skeleton architecture are highly homoplastic, as are characters of the aquiferous system. However, axial symmetry seems to be primitive for all Calcispongia, a conclusion that has potentially far-reaching implications for hypotheses of early body plan evolution in Metazoa.     

Leishmania major: Cell type dependent distribution of a 43 kDa antigen related to silent information regulatory-2 protein family

In previous studies we have characterized several Leishmania major polypeptides and showed that one member of this group (LmSIR2rp) shared significant homology to silent information regulator 2 (SIR2) of Saccharomyces cerevisiae, a protein playing a role in both telomeric and mating type loci repression in these organisms. In the present study, by using molecular and immunological approaches, we could identify LmSIR2rp homologues in different Leishmania species and developmental stages (eg logarithmic (LP) and stationary phase promastigotes (SP) and amastigotes). The reactive antigen was also detected in Trypanosoma cruzi extracts. Surprisingly, immunofluorescence assays revealed that LmSIR2rp is associated mainly with cytoplasmic granules of different sizes and numbers depending on the life stage of the parasite used. No reactivity was observed in the nucleus, in agreement with the Western blot showing an absence of immunoreactivity of anti-LmSIR2rp immune serum against parasite nuclear extracts. Furthermore, immunoprecipitation of [³⁵S]methionine-labeled promastigote antigens after pulse chase experiments, using anti-LmSIR2rp fusion protein antibodies, showed that the protein is among parasite excreted-secreted antigens (ESA). Moreover, immunoflurescence assays conducted with short time incubations of either purified LmSIR2rp or viable promastigotes with murine macrophages, revealed that LmSIR2rp could be bound to the macrophage surface. The unexpected cytoplasmic localization of LmSIR2rp and its presence in ESA may suggest a new mode of action for silent information regulatory factor homologues.