The histone-like protein HupB influences biofilm formation and virulence in Xanthomonas citri ssp. citri through the regulation of flagellar biosynthesis

Citrus canker is an important disease of citrus, whose causal agent is the bacterium Xanthomonas citri ssp. citri (Xcc). In previous studies, we found a group of Xcc mutants, generated by the insertion of the Tn5 transposon, which showed impaired ability to attach to an abiotic substrate. One of these mutants carries the Tn5 insertion in hupB, a gene encoding a bacterial histone-like protein, homologue to the β-subunit of the Heat-Unstable (HU) nucleoid protein of Escherichia coli. These types of protein are necessary to maintain the bacterial nucleoid organization and the global regulation of gene expression. Here, we characterized the influence of the mutation in hupB regarding Xcc biofilm formation and virulence. The mutant strain hupB was incapable of swimming in soft agar, whereas its complemented strain partially recovered this phenotype. Electron microscope imaging revealed that impaired motility of hupB was a consequence of the absence of the flagellum. Comparison of the expression of flagellar genes between the wild-type strain and hupB showed that the mutant exhibited decreased expression of fliC (encoding flagellin). The hupB mutant also displayed reduced virulence compared with the wild-type strain when they were used to infect Citrus lemon plants using different infection methods. Our results therefore show that the histone-like protein HupB plays an essential role in the pathogenesis of Xcc through the regulation of biofilm formation and biosynthesis of the flagellum.     

Gastrulation of Gastrotheca riobambae in comparison with other frogs

Blastopore formation, the embryonic disk, archenteron and notochord elongation, and Brachyury expression in the marsupial frog Gastrotheca riobambae was compared with embryos of Xenopus laevis and of the dendrobatids Colostethus machalilla and Epipedobates anthonyi. In contrast with X. laevis embryos, the blastopore closes before elongation of the archenteron and notochord in the embryos of G. riobambae and of the dendrobatid frogs. Moreover, the circumblastoporal collar (CBC) thickens due to the accumulation of involuted cells. An embryonic disk, however, is formed only in the G. riobambae gastrula. We differentiate three gastrulation patterns according to the speed of development: In X. laevis, elongation of the archenteron and notochord begin in the early to mid gastrula, whereas in the dendrobatids C. machalilla and E. anthonyi the archenteron elongates at mid gastrula and the notochord elongates after gastrulation. In G. riobambae, only involution takes place during gastrulation. Archenteron and notochord elongation occur in the post gastrula. In the non-aquatic reproducing frogs, the margin of the archenteron expands anisotropically, resulting in an apparent displacement of the CBC from a medial to a posterior location, resembling the displacement of Hensen's node in the chick and mouse. The differences detected indicate that amphibian gastrulation is modular.     

Modulation of auxin signalling through DIAGETROPICA and ENTIRE differentially affects tomato plant growth via changes in photosynthetic and mitochondrial metabolism

Auxin modulates a range of plant developmental processes including embryogenesis, organogenesis, and shoot and root development. Recent studies have shown that plant hormones also strongly influence metabolic networks, which results in altered growth phenotypes. Modulating auxin signalling pathways may therefore provide an opportunity to alter crop performance. Here, we performed a detailed physiological and metabolic characterization of tomato (Solanum lycopersicum) mutants with either increased (entire) or reduced (diageotropica—dgt) auxin signalling to investigate the consequences of altered auxin signalling on photosynthesis, water use, and primary metabolism. We show that reduced auxin sensitivity in dgt led to anatomical and physiological modifications, including altered stomatal distribution along the leaf blade and reduced stomatal conductance, resulting in clear reductions in both photosynthesis and water loss in detached leaves. By contrast, plants with higher auxin sensitivity (entire) increased the photosynthetic capacity, as deduced by higher V_cmax and J_max coupled with reduced stomatal limitation. Remarkably, our results demonstrate that auxin‐sensitive mutants (dgt) are characterized by impairments in the usage of starch that led to lower growth, most likely associated with decreased respiration. Collectively, our findings suggest that mutations in different components of the auxin signalling pathway specifically modulate photosynthetic and respiratory processes.     

Abnormal osteogenesis in osteoporotic patients is reflected by altered mesenchymal stem cells dynamics.

Bone marrow contains a population of mesenchymal stem cells with the ability to differentiate into cells that form bone, cartilage, adipose, and other connective tissues. Stem cells can be isolated from bone marrow aspirates and expanded in vitro. Presently, most stem cells studies have been performed in cells obtained from "healthy" control subjects. The goal of this study was to compare the functional characteristics of mesenchymal stem cells derived from "healthy" control and osteoporotic postmenopausal women to better understand the mechanisms involved in the pathogenesis of this disease. Osteoporotic and control stem cells have similar morphology and size and express similar cell surface antigens as evidenced by their reactivity with cell specific monoclonal antibodies. Mesenchymal stem cells from osteoporotic women differ from controls in having a lower growth rate than control cells, being refractory to the mitogenic effect of IGF-1, and exhibiting a deficient ability to differentiate into the osteogenic linage as evidenced by the alkaline phosphatase activity and calcium phosphate deposition. We conclude that in osteoporosis stem cell growth, proliferative response and osteogenic differentiation are significantly affected. Also, the study of mesenchymal stem cells from osteoporotic postmenopausal women may provide a better understanding of the mechanisms involved in the pathogenesis of the osteoporosis. It may also serve to test in vitro in rapid manner novel new therapeutic strategies.     

Salicylate decreases production of AmpC type beta-lactamases and increases susceptibility to beta-lactams in a Morganella morganii clinical isolate

The effect of salicylate, a marRAB inducer, on the resistance to beta-lactams was characterized in an AmpC beta-lactamase hyperproducer Morganella morganii clinical isolate (the M1 strain). Results were compared with those of the effect of salicylate in a wild-type M. morganii strain. Salicylate induced a decreased susceptibility to nalidixic acid, norfloxacin and tetracycline and simultaneously increased the susceptibility to beta-lactams apparently due to the repression of AmpC beta-lactamase synthesis in the M1 strain. Likewise, salicylate only repressed 46 kDa outer membrane protein expression in the wild-type strain, since the clinical isolate M1 did not express it.     

Effects of buthionine sulfoximine nifurtimox and benznidazole upon trypanothione and metallothionein proteins in Trypanosoma cruzi

Proteins rich in sulfhydryl groups, such as metallothionein, are present in several strains of the parasite Trypanosoma cruzi, the etiological agent of Chagas' disease. Metallothionein-like protein concentrations ranged from 5.1 to 13.2 pmol/mg protein depending on the parasite strain and growth phase. Nifurtimox and benznidazole, used in the treatment of Chagas' disease, decreased metallothionein activity by approximately 70%. T. cruzi metallothionein was induced by ZnCl2. Metallothionein from T. cruzi was partially purified and its monobromobimane derivative showed a molecular weight of approximately 10,000 Da by SDS-PAGE analysis. The concentration of trypanothione, the major glutathione conjugate in T. cruzi, ranged from 3.8 to 10.8 nmol/mg protein, depending on the culture phase. The addition of buthionine sulfoximine to the protozoal culture considerably reduced the concentration of trypanothione and had no effect upon the metallothionein concentration. The possible contribution of metallothionein-like proteins to drug resistance in T. cruzi is discussed.     

Photo-induced processes in vitamin B6 compounds

In this work, we applied multi-wavelength stopped-flow spectroscopy (MSFS) to study the chemical equilibria between tautomeric or hydrated forms of various vitamin B6 compounds and the Schiff base formed by epsilon-aminocaproic acid (= 6-aminohexanoic acid) with pyridoxal 5'-phosphate at 25 degrees and variable pH. Since some of these compounds are photosensitive, we analyzed the possible occurrence of any secondary photo-induced processes under the conditions of irradiation in the MSFS equipment (continuous irradiation with light from a 75-W Xe lamp spanning the wavelength range of 200-700 nm). To determine the tautomeric composition of these compounds, the electronic absorption spectra were analyzed by means of log-normal curves. Continuous irradiation of pyridoxamine and pyridoxal 5'-phosphate over the wavelength range of 200-700 nm displaces the chemical equilibrium between the tautomeric or hydrated forms of these compounds. However, the Schiff base of epsilon-aminocaproic acid with pyridoxal 5'-phosphate is insensitive to the radiation used. The photo-induced processes detected in pyridoxamine and pyridoxal 5'-phosphate should be taken into account in examining vitamers by MSFS. In fact, these additional processes should be considered in studying the mechanism of action of vitamin B6-dependent enzymes by the MSFS technique, whenever some free vitamer may be present in solution.     

Regulation of heptaspanning-membrane-receptor function by dimerization and clustering

G-protein-coupled receptors form homomers and heteromers; agonist-induced conformational changes within interacting receptors of the oligomer modify their pharmacology, signalling and/or trafficking. When these receptors are activated, the oligomers rearrange and cluster and a novel mechanism of receptor-operation regulation by oligomer intercommunication is possible. This intercommunication would be assisted by components of the plasma membrane and by scaffolding proteins. Receptor cross-sensitization, cross-desensitization and novel, integrated receptor responses can then develop between oligomeric receptor complexes of the cluster without direct contact between them. This concept gives a new perspective to the understanding of neurotransmission and neuronal plasticity.     

Metabotropic glutamate type 1alpha receptor localizes in low-density caveolin-rich plasma membrane fractions

Recent evidence suggest that many G protein-coupled receptors (GPCR) and signalling molecules localize in microdomains of the plasma membrane. In this study, flotation gradient analysis in the absence of detergents demonstrated the presence of the metabotropic glutamate receptor type 1alpha (mGlu1alpha) in low-density caveolin-enriched membrane fractions (CEMF) in permanently transfected BHK cells. BHK-1alpha cells exhibit a similar pattern of staining for caveolin-1 and caveolin-2, and these two proteins show a high degree of co-localization with mGlu1alpha receptor as demonstrated by immunogold and confocal laser microscopy. The presence of mGlu1alpha in CEMF was also demonstrated by co-immunoprecipitation of mGlu1alpha receptor using antibodies against caveolin proteins. Activation of the mGlu1alpha receptor by agonist increased extracellular signal-regulated kinases phosphorylation in CEMF and not in high-density membrane fractions (HDMF), suggesting that mGlu1alpha receptor-mediated signal transduction could occur in caveolae-like domains. Overall, these results clearly show a molecular and functional association of mGlu1alpha receptor with caveolins.