The invasion-associated type III secretion system of Salmonella enterica serovar Typhimurium is necessary for intracellular proliferation and vacuole biogenesis in epithelial cells

Type III secretion systems (TTSS) are used by Gram-negative pathogens to translocate proteins into eukaryotic host cells. Salmonella enterica serovar Typhimurium (S. Typhimurium) has two of these specialized systems, which are encoded on separate Salmonella pathogenicity islands (SPI-1 and SPI-2) and translocate unique sets of effectors. The specific roles of these systems in Salmonella pathogenesis remain undefined, although SPI-1 is required for bacterial invasion of epithelial cells and SPI-2 for survival/replication in phagocytic cells. However, because SPI-1 TTSS mutants are invasion-incompetent, the role of this TTSS in post-invasion processes has not been investigated. In this study, we have used two distinct methods to internalize a non-invasive SPI-1 TTSS mutant (invA) into cultured epithelial cells: (i) co-internalization with wild-type S. Typhimurium (SPI-1-dependent) and (ii) complementation with the Yersinia pseudotuberculosis invasin (inv) gene (SPI-1-independent). In both cases, internalized invA mutants were unable to replicate intracellularly, indicating that SPI-1 effectors are essential for this process and cannot be complemented by wild-type bacteria in the same cell. Analysis of the biogenesis of SCVs showed that vacuoles containing mutant bacteria displayed abnormal maturation that was dependent on the mechanism of entry. Manipulation of Salmonella-containing vacuole (SCV) biogenesis by pharmacologically perturbing membrane trafficking in the host cell increased intracellular replication of wild-type but not mutant S. Typhimurium This demonstrates a previously unknown role for SPI-1 in vacuole biogenesis and intracellular survival in non-phagocytic cells.     

Influence of age, sex, and sexual activity on trace element levels and antioxidant enzyme activities in field mice (Apodemus sylvaticus and Mus spretus)

The influence of age, gender and sexual activity on both hepatic levels of some trace elements (Fe, Cu, Zn, Mn, Se) and the activities of glutathione-S-transferase (GST) and superoxide dismutase (SOD) was investigated in Wood mice (Apodemus sylvaticus) and Algerian mice (Mus spretus). Animals were taken from a riverside community of an unpolluted area of central Portugal. Adult A. sylvaticus presented the highest hepatic mean concentrations of Cu and Mn, whereas adult M. spretus had the highest Fe concentration in the liver. Moreover, an influence of age on the contents of Fe, Zn, and Mn has been observed in A. sylvaticus, whereas in M. spretus an influence of gender and sexual activity was only detected on Zn levels. In contrast, enzyme activities were not influenced by the studied variables, despite a tendency for an increase in SOD activity in sexually active M. spretus. GST activity was species dependent, whereas SOD activity was similar between species. These findings were analyzed regarding the relationship of both essential trace elements and the two antioxidant enzymes with physiological and metabolic pathways related to life cycles in the two species of mice. Results enhanced the understanding of A. sylvaticus and M. spretus as biological models, allowing their future use as bioindicators of environmental toxicity.     

In vitro culture of Cedrela fissilis Vellozo (Meliaceae)

An efficient micropropagation protocol was developed for Cedrela fissilis (Meliaceae) using nodal segments from juvenile origin for axillary shoot proliferation. Shoot proliferation was significantly affected by salt formulation, explant origin and 6-benzyladenine concentration. Maximum multiplication rates (6–7 new plants were produced in the second subculture cycle per single cotyledonary node cutting) were achieved on Murashige and Skoog media supplemented with 1.25–5.0 M 6-benzyladenine. Addition of -naphthaleneacetic acid to these media caused significant inhibition on shoot proliferation and growth and stimulated callus formation. High frequency callus initiation and synergistic effects on callus growth were achieved on Murashige and Skoog medium supplemented with 6-benzyladenine at either 1.25, 2.5 or 5.0 M combined, respectively, with 2.5, 1.25–5.0 or 5.0 M -naphthaleneacetic acid. Rooting was achieved, after 10–12 days, with 87–100% of the node cuttings on half strength Murashige and Skoog medium either without growth regulators or supplemented with 2.5 M indole-3-butyric acid. Regenerated plants were successfully acclimatized on sterilized sand, for 21 days, but for further plant development the sand:soil (1:1) mixture was the best substrate. The survival rate of plantlets under ex vitro conditions was 100% after 3 months. The optimized micropropagation and callus culture protocols offer the possibility to use the organ/cell culture techniques for vegetative propagation, cryopreservation and secondary metabolism studies.     

Adrenoceptors in avian and fish pigment cells

Very little is known about the neurohumoral control of avian pigmentation and about adrenergic subtypes mediating catecholaminergic-controlled color change in nonmelanophore pigment cells of poikilothermic vertebrates. To determine the adrenoceptor subtypes in avian melanocytes and fish GEM-81 competitive binding assays were performed with the following radioactive ligands and their cold ligand counterparts: [3H]prazosin and benoxathian or unlabeled prazosin; [3H]rauwolscine and idazoxan or yohimbine; [3H]propranolol and metoprolol or ICI 118,551 and [125I]iodocyanopindolol and ICI 118,551. Our results suggest that: alpha(1)-adrenoceptors [K(i)=1.38 micro M; maximum displacement (md)=80%, benoxathian), alpha(2)-adrenoceptors (K(i)=0.21 micro M; md=82%, idazoxan), and beta(2)-adrenoceptors (K(i)=7.3 micro M; md=73%, ICI 118,551) are expressed in avian melanocytes, and that alpha(2)-adrenoceptors (K(i)=1.24 nM, idazoxan, K(i)=59 nM, yohimbine, md=65%, idazoxan and yohimbine; K(i)=0.19 nM, md=69%, prazosin), beta(1)-adrenoceptors (K(i)=22.2 micro M, md=75%, metoprolol), and beta(2)-adrenoceptors (K(i)=32.2 micro M, md=92%, ICI 118,551) are expressed in GEM-81 erythrophoroma cells. This may be the first study to show the presence of adrenoceptors in avian melanocytes and one of a few characterizing adrenoceptor subtypes in teleost nonmelanophore pigment cells.     

Binding of haemin by the fish pathogen Photobacterium damselae subsp. piscicida

Whole cells of virulent (DI 21 and B 51) and avirulent (ATCC 29690 and EPOY 8803-II) strains of Photobacterium damselae subsp. piscicida, grown under iron-supplemented or iron-restricted conditions, were able to bind haemin. Iron limitation resulted in an increased binding of haemin by DI 21, B 51 and ATCC 29690 cells but did not affect the haemin-binding ability of the EPOY 8803-II cells. Proteinase K treatment of whole cells markedly reduced the binding of haemin, indicating that protein receptors located at the cell surface are involved in the binding. This was confirmed by the observation that isolated total as well as outer membrane proteins from all the strains, regardless of the iron levels of the media, were able to bind haemin, with the outer membranes showing the strongest binding. Haemin binding by membrane protein extracts was not affected by heat treatment but was almost completely abolished by Proteinase K treatment, suggesting the presence of thermostable protein receptors for haemin. The capsular polysaccharide also appears to play a minor role in binding of haemin. It was concluded that constitutive as well as inducible mechanisms of haemin binding occur in P. damselae subsp. piscicida. These mechanisms would rely mainly upon the direct interaction between the haemin molecules and surface-exposed outer membrane protein receptors.     

Incidence of numerical chromosome aberrations in meningioma tumors as revealed by fluorescence in situ hybridization using 10 chromosome-specific probes

OBJECTIVE: Although information on the cytogenetic characteristics of meningioma tumors has accumulated progressively over the past few decades, information on the genetic heterogeneity of meningiomas is still scanty. The aim of the present study was to analyze by interphase fluorescence in situ hybridization (FISH) the incidence of numerical abnormalities for chromosomes 1, 9, 10, 11, 14, 15, 17, 22, X, and Y in a group of 70 consecutive meningioma tumors. Another goal was to establish the potential associations among the altered chromosomes, as a way to assess both intertumoral and intratumoral heterogeneity. METHODS: For the purpose of the study, 70 patients diagnosed with meningioma were analyzed. Interphase FISH for the detection of numerical abnormalities for chromosomes 1, 9, 10, 11, 14, 15, 17, 22, X, and Y was applied to fresh tumor samples from each of the patients studied. RESULTS: The overall incidence of numerical abnormalities was 76%. Chromosome Y in males and chromosome 22 in the whole series were the most common abnormalities (46% and 61%, respectively). Despite the finding that monosomy of chromosome 22/22q(-) deletions are the most frequent individual abnormality (53%), we have observed that chromosome gains are significantly more common than chromosome losses (60% versus 40%). Chromosome gains corresponded to abnormalities of chromosomes 1 (27%), 9 (25%), 10 (23%), 11 (22%), 14 (33%), 15 (22%), 17 (23%), and X in females (35%) and males (23%) whereas chromosome losses apart from chromosome 22 frequently involved chromosomes 14 (19%), X in males (23%), and Y in males (32%). Although an association was found among most gained chromosomes on one side and chromosome losses on the other side, different association patterns were observed. Furthermore, in the latter group, monosomy 22/22q(-) was associated with monosomy X in females and monosomy 14/14q(-) was associated with nulisomy Y in males. In addition, chromosome losses usually involved a large proportion of the tumor cells whereas chromosome gains were restricted to small tumor cell clones, including tetraploid cells. CONCLUSIONS: Our results show that meningiomas are genetically heterogeneous tumors that display different patterns of numerical chromosome changes, as assessed by interphase FISH.     

Thermodynamic and hydration effects for the incorporation of a cationic 3-aminopropyl chain into DNA

The introduction of cationic 5-(ω-aminoalkyl)-2′-deoxypyrimidines into duplex DNA has been shown to induce DNA bending. In order to understand the energetic and hydration contributions for the incorporation of a cationic side chain in DNA a combination of spectroscopy, calorimetry and density techniques were used. Specifically, the temperature unfolding and isothermal formation was studied for a pair of duplexes with sequence d(CGTAGUCG TGC)/d(GCACGACTACG), where U represents 2′-deoxyuridine (‘control’) or 5-(3-aminopropyl)-2′-deoxyuridine (‘modified’). Continuous variation experiments confirmed 1:1 stoichiometries for each duplex and the circular dichroism spectra show that both duplexes adopted the B conformation. UV and differential scanning calorimetry melting experiments reveal that each duplex unfolds in two-state transitions. In low salt buffer, the ‘modified’ duplex is more stable and unfolds with a lower endothermic heat and lower release of counterion and water. This electrostatic stabilization is entropy driven and disappears at higher salt concentrations. Complete thermodynamic profiles at 15°C show that the favorable formation of each duplex results from the compensation of a favorable exothermic heat with an unfavorable entropy contribution. However, the isothermal profiles yielded a differential enthalpy of 8.8 kcal/mol, which is 4.3 kcal/mol higher than the differential enthalpy observed in the unfolding profiles. This indicates that the presence of the aminopropyl chain induces an increase in base stacking interactions in the modified single strand and a decrease in base stacking interactions in the modified duplex. Furthermore, the formation of the ‘control’ duplex releases water while the ‘modified’ duplex takes up water. Relative to the control duplex, formation of the modified duplex at 15°C yielded a marginal differential ΔG° term, positive ΔΔH_ITC–Δ(TΔS) compensation, negative ΔΔV and a net release of counterions. The opposite signs of the differential enthalpy–entropy compensation and differential volume change terms show a net uptake of structural water around polar and non-polar groups. This indicates that incorporation of the aminopropyl chain induces a higher exposure of aromatic bases to the solvent, which may be consistent with a small and local bend in the ‘modified’ duplex.     

Human DNA glycosylases of the bacterial Fpg/MutM superfamily: an alternative pathway for the repair of 8-oxoguanine and other oxidation products in DNA

The mild phenotype associated with targeted disruption of the mouse OGG1 and NTH1 genes has been attributed to the existence of back-up activities and/or alternative pathways for the removal of oxidised DNA bases. We have characterised two new genes in human cells that encode DNA glycosylases, homologous to the bacterial Fpg (MutM)/Nei class of enzymes, capable of removing lesions that are substrates for both hOGG1 and hNTH1. One gene, designated HFPG1, showed ubiquitous expression in all tissues examined whereas the second gene, HFPG2, was only expressed at detectable levels in the thymus and testis. Transient transfections of HeLa cells with fusions of the cDNAs to EGFP revealed intracellular sorting to the nucleus with accumulation in the nucleoli for hFPG1, while hFPG2 co-localised with the 30 kDa subunit of RPA. hFPG1 was purified and shown to act on DNA substrates containing 8-oxoguanine, 5-hydroxycytosine and abasic sites. Removal of 8-oxoguanine, but not cleavage at abasic sites, was opposite base-dependent, with 8-oxoG:C being the preferred substrate and negligible activity towards 8-oxoG:A. It thus appears that hFPG1 has properties similar to mammalian OGG1 in preventing mutations arising from misincorporation of A across 8-oxoG and could function as a back-up repair activity for OGG1 in ogg1(-/-) mice.     

Homozygosity mapping of a Weill-Marchesani syndrome locus to chromosome 19p13.3-p13.2

Weill-Marchesani syndrome (WMS) is a rare disease characterized by short stature, brachydactyly, joint stiffness, and characteristic eye abnormalities, including microspherophakia, ectopia lentis, and glaucoma. Both autosomal recessive and autosomal dominant modes of inheritance have been described in association with WMS. We have performed a genome-wide search in two large consanguineous families of Lebanese and Saudian origin consistent with an autosomal recessive mode of inheritance. Here, we report the linkage of the disease gene to chromosome 19p13.3-p13.2 (Zmax=5.99 at theta=0 at locus D19S906). A recombination event between loci D19S905 and D19S901 defines the distal boundary, and a second recombination event between loci D19S221 and D19S840 defines the proximal boundary of the genetic interval encompassing the WMS gene (12.4 cM). We hope that our ongoing studies will lead to the identification of the disease-causing gene.