RESUMO
Cupriavidus necator UYPR2.512 is a rhizobial strain that belongs to the Beta-subclass of proteobacteria, able to establish successful symbiosis with Mimosoid legumes. The initial steps of rhizobium-legumes symbioses involve the reciprocal recognition by chemical signals, being luteolin one of the molecules involved. However, there is a lack of information on the effect of luteolin in beta-rhizobia. In this work, we used long-read sequencing to complete the genome of UYPR2.512 providing evidence for the existence of four closed circular replicons. We used an RNA-Seq approach to analyse the response of UYPR2.512 to luteolin. One hundred and forty-five genes were differentially expressed, with similar numbers of downregulated and upregulated genes. Most repressed genes were mapped to the main chromosome, while the upregulated genes were overrepresented among pCne512e, containing the symbiotic genes. Induced genes included the nod operon and genes implicated in exopolysaccharides and flagellar biosynthesis. We identified many genes involved in iron, copper and other heavy metals metabolism. Among repressed genes, we identified genes involved in basal carbon and nitrogen metabolism. Our results suggest that in response to luteolin, C. necator strain UYPR2.512 reshapes its metabolism in order to be prepared for the forthcoming symbiotic interaction.
Assuntos
Cupriavidus necator , Cupriavidus , Fabaceae , Rhizobium , Cupriavidus/genética , Cupriavidus necator/genética , Fabaceae/microbiologia , Genômica , Luteolina/metabolismo , Luteolina/farmacologia , Fixação de Nitrogênio , Filogenia , Rhizobium/genética , Simbiose/genética , TranscriptomaRESUMO
Here, we report the genome sequence of Proteus mirabilis Pr2921, a uropathogenic bacterium that can cause severe complicated urinary tract infections. After gene annotation, we identified two additional copies of ucaA, one of the most studied fimbrial protein genes, and other fimbriae related-proteins that are not present in P. mirabilis HI4320.
RESUMO
The presence of Myosin Va (an actin-based molecular motor) in the peripheral nervous system was examined and its subcellular distribution within the axons of the sciatic nerve was demonstrated via immunocytochemistry. Myosin Va (M-Va) in the nerve was detected by using SDS-PAGE and Western blot techniques with a polyclonal antibody specifically raised against the M-Va globular tail domain. In addition, purification of M-Va from the rat sciatic nerve prior to immunoblotting yielded a M-Va standard band. Likewise, optical immunocytochemical procedures revealed the presence of M-Va, particularly in the cortical axoplasmic territory, but also in the Schwann cell soma. The above experiments were carried out both on intact as well as on severed sciatic nerves with similar results. The proximal stumps of severed sciatic nerves (from 0 to 72 h after injury) were labelled in vivo with (35)S-methionine. SDS-PAGE autoradiography of the immunoabsorbed M-Va from the radiolabelled homogenized nerve tissue showed a significant increment of the radioactive intensity of M-Va heavy chain band through time. Moreover, a significant increment of transcripts coding for M-Va heavy chain was detected through time using RT-PCR after nerve injury and compared to intact nerves. This data suggest that M-Va is up-regulated in a time-dependent manner. The latter suggests a possible involvement of M-Va in nerve regeneration processes.
Assuntos
Actinas/fisiologia , Miosina Tipo V/fisiologia , Regeneração Nervosa/fisiologia , Nervo Isquiático/lesões , Actinas/biossíntese , Animais , Axônios/metabolismo , Western Blotting , Imuno-Histoquímica , Miosina Tipo V/biossíntese , Ratos , Ratos Wistar , Nervo Isquiático/fisiologiaRESUMO
Local protein synthesis within axons has been studied on a limited scale. In the present study, several techniques were used to investigate this synthesis in sciatic nerve, and to show that it increases after damage to the axon. Neurofilament (NF) mRNAs were probed by RT-PCR, Northern blot and in situ hybridization in axons of intact rat sciatic nerve, and in proximal or distal stumps after sciatic nerve transection. RT-PCR demonstrated the presence of NF-L, NF-M and NF-H mRNAs in intact sciatic nerve, as well as in proximal and distal stumps of severed nerves. Northern blot analysis of severed nerve detected NF-L and NF-M, but not NF-H. This technique did not detect the three NFs mRNAs in intact nerve. Detection of NF-L and NF-M mRNA in injured nerve, however, indicated that there was an up-regulation in response to nerve injury. In situ hybridization showed that NF-L mRNA was localized in the Schwann cell perinuclear area, in the myelin sheath, and at the boundary between myelin sheath and cortical axoplasm. RNA and protein synthesizing activities were always greater in proximal as compared to distal stumps. NF triplet proteins were also shown to be synthesized de novo in the proximal stump. The detection of neurofilament mRNAs in nerves, their possible upregulation during injury and the synthesis of neurofilament protein triplet in the proximal stumps, suggest that these mRNAs may be involved in nerve regeneration, providing a novel point of view of this phenomenon.
Assuntos
Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Biossíntese de Proteínas/genética , RNA Mensageiro/metabolismo , Nervo Isquiático/metabolismo , Animais , Autorradiografia , Axotomia , Northern Blotting , Hibridização In Situ , Masculino , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Ribossômicas/metabolismo , Células de Schwann/citologia , Células de Schwann/metabolismo , Nervo Isquiático/cirurgia , Regulação para CimaRESUMO
The ATP dependent Ca2+ uptake of platelet vesicles was inhibited by the two hydrophobic drugs trifluoperazine (TFP) and propranolol (PROP). Inhibition was significantly lowered when Pi was used instead of oxalate as a precipitant agent. When the ATPase ligands substrate (Mg2+ and Pi) were absent of the efflux medium, a slow release of Ca2+ which did not couple with ATP synthesis (passive Ca2+ efflux) was observed. Both, TFP and PROP enhanced the passive Ca2+ efflux. This enhanced efflux was partially inhibited only when Mg2+ and Pi were added together to the efflux reaction media, but it was not affected by spermidine, ruthenium red or thapsigargin (TG). The Ca2+ ionophores A23187 and ionomycin, also enhanced passive Ca2+ efflux. However, in this case, Ca2+ efflux was inhibited just by inclusion of Mg2+ to the medium. Ca2+ efflux promoted by Triton X-100 was not affected by either Mg2+ or Pi, included together or separately into the efflux medium. The ATP <==> Pi measured in the presence of Triton X-100 and millimolar Ca2+ concentrations was inhibited by both TFP and PROP, but not by Ca2+ ionophores up to 4 microM. The data suggest that the observed enhancement of passive Ca2+ efflux promoted by TFP and PROP could be attributed to a direct effect of these drugs over the platelet Ca2+ pump isoforms (Sarco Endoplasmic Reticulum Calcium ATPase, SERCA2b and SERCA3) themselves, as it was reported for the sarcoplasmic reticulum Ca2+ ATPase (SERCA1).