RESUMO
Rheumatoid arthritis (RA) has an inflammatory milieu in the synovial compartment, which is regulated by a complex cytokine and chemokine network that induces continuously degenerative and inflammatory reactions. The secreted osteoclastogenic factor of activated T cells (SOFAT) is a unique cytokine and represents an alternative pathway for osteoclast activation. In this study, we examined whether SOFAT is able to induce joint pain and investigated the presence of SOFAT in a Collagen-induced Arthritis (CIA) model and in human subjects. Here, we found that an intra-articular stimulation with SOFAT (1, 10, 100, or 1,000 ng/10 µl) in the knee joint significantly decreases the mechanical threshold in the hind paw of mice (p < 0.05). Moreover, after a second injection of SOFAT, the mechanical threshold decrease was sustained for up to 8 days (p < 0.05). In the CIA model, the immunohistochemical assay of knee joint showed positivity stained for SOFAT, and the mRNA and protein expression of SOFAT were significantly higher in the affected-group (p < 0.05). Besides, the mRNA of RANKL, IL-1ß, IL-6, and IL-15 were significantly higher in the affected-group (p < 0.05). Finally, SOFAT was detected in the synovial fluid of RA patients, but not in OA patients (p < 0.05). In conclusion, SOFAT is up regulated in inflammatory milieu such as RA but not in non-inflammatory OA. SOFAT may be a novel molecule in the complex inflammatory phenotype of RA.
Assuntos
Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Carbono-Oxigênio Liases/metabolismo , Citocinas/metabolismo , Articulações/fisiologia , Dor/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos DBA , Osteoclastos/fisiologia , Osteogênese , Transdução de Sinais , Regulação para CimaRESUMO
Enzymes can be very useful on exopolysaccharides (EPS) research, can be used at elucidation and also to modify the polysaccharides' structure in order to alter their physical properties. Thus, the reduction of the molecular mass could increase applications of these biopolymers. Therefore, the EPS production of different rhizobia and the action of xanthan lyase on its structures were evaluated. The strains produced significant amounts of EPS, and it was noticed that are heteropolysaccharides, composed galactose and glucose. Both EPS and xanthan were modified on ß-glycosidic bonds, the mannose was removed of xanthan had but the EPS was affected in the CO stretching vibration, where the glucuronic acid removed from of your structure. The ester/carboxylic acid portions affected functional groups of the acetate/succinate, methyl carbons of the O-acetyl and pyruvate methyl groups in addition to affect the carbons the main pyranoid. The Resistance to temperature increase of the EPS was observed, made possible by the activity of the lyase. EPS has the ability to form stable gels at higher temperatures and anionic feature can be used on solubilization and controlled release of substances. Modified EPS knowledge will presently facilitate future investigations relating the structure of the rhizobia polysaccharide against rheological properties.
Assuntos
Carbono-Oxigênio Liases/metabolismo , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/química , Rhizobium/metabolismo , Concentração de Íons de Hidrogênio , Monossacarídeos/análise , Polissacarídeos Bacterianos/metabolismo , Rhizobium/crescimento & desenvolvimentoRESUMO
Orthodontic tooth movement is based on mechanical forces inducing bone remodeling, and several methods have been proposed to increase tooth movement, including photobiomodulation. This study evaluated, in an animal model, the effects of photobiomodulation on SOFAT-a secreted osteoclastogenic factor of activated T cells and RANK-L during tooth movement. The results showed that tooth displacement, RANK-L and SOFAT levels were significantly greater compared to Control group. SOFAT may play an important role in bone remodeling during orthodontic movement, possibly increasing the osteoclast cells at the compression area and bone remodeling activity.
Assuntos
Citocinas/metabolismo , Luz , Linfócitos T/metabolismo , Animais , Remodelação Óssea , Carbono-Oxigênio Liases , Planejamento de Prótese Dentária , Masculino , Ligante RANK/metabolismo , Ratos Wistar , Técnicas de Movimentação DentáriaRESUMO
O-Acetylserine (thiol)-lyase (cysteine synthase) was purified from Azospirillum brasilense Sp7. After hydrolysis of the purified protein, amino acid sequences of five peptides were obtained, which permitted the cloning and sequencing of the cysK gene. The deduced amino acid sequence of cysteine synthase exhibited homology with several putative proteins from Alpha- and Gammaproteobacteria. Azospirillum brasilense Sp7 cysK exhibited 58% identity (72% similarity) with Escherichia coli K12 and Salmonella enterica serovar Typhimurium cysteine synthase proteins. An E. coli auxotroph lacking cysteine synthase loci could be complemented with A. brasilense Sp7 cysK. The 3.0-kb HindIII-EcoRI fragment bearing cysK contained two additional ORFs encoding a putative transcriptional regulator and dUTPase. Insertional disruption of the cysK gene did not produce a cysteine auxotroph, indicating that gene redundancy in the cysteine biosynthetic or other biosynthetic pathways exists in Azospirillum, as already described in other bacteria. Nitrogen fixation was not altered in the mutant strain as determined by acetylene reduction. However, this strain showed an eight-fold reduction in tellurite resistance as compared to the wild-type strain, which was only observed during growth in minimal medium. These data confirm earlier observations regarding the importance of cysteine metabolism in tellurite resistance.
Assuntos
Azospirillum brasilense/genética , Carbono-Oxigênio Liases/genética , Carbono-Oxigênio Liases/isolamento & purificação , Telúrio/metabolismo , Telúrio/farmacologia , Azospirillum brasilense/efeitos dos fármacos , Azospirillum brasilense/enzimologia , Sequência de Bases , Carbono-Oxigênio Liases/química , Carbono-Oxigênio Liases/metabolismo , Cromatografia , Clonagem Molecular , Farmacorresistência Fúngica , Teste de Complementação Genética , Mutação , Oxirredução , FenótipoRESUMO
The synthesis of L-cysteine, the major mechanism by which sulfur is incorporated into organic compounds in microorganisms, occupies a significant fraction of bacterial metabolism. In Bacillus subtilis the cysH operon, encoding several proteins involved in cysteine biosynthesis, is induced by sulfur starvation and tightly repressed by cysteine. We show that a null mutation in the cysK gene encoding an O-acetylserine-(thiol)lyase, the enzyme that catalyzes the final step in cysteine biosynthesis, results in constitutive expression of the cysH operon. Using DNA microarrays we found that, in addition to cysH, almost all of the genes required for sulfate assimilation are constitutively expressed in cysK mutants. These results indicate that CysK, besides its enzymatic role in cysteine biosynthesis, is a global negative regulator of genes involved in sulfur metabolism.
Assuntos
Bacillus subtilis/enzimologia , Cisteína Sintase/metabolismo , Regulação Bacteriana da Expressão Gênica , Enxofre/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbono-Oxigênio Liases/genética , Carbono-Oxigênio Liases/metabolismo , Cisteína/biossíntese , Cisteína Sintase/genética , Escherichia coli , Fusão Gênica , Genes Reporter , Análise de Sequência com Séries de Oligonucleotídeos , Óperon , beta-Galactosidase/análise , beta-Galactosidase/genéticaRESUMO
The enzymatic properties and the physiological function of the type IV apurinic/apyrimidinic (AP)-endonuclease homolog of Bacillus subtilis, encoded by yqfS, a gene specifically expressed in spores, were studied here. To this end, a recombinant YqfS protein containing an N-terminal His6 tag was synthesized in Escherichia coli and purified to homogeneity. An anti-His6-YqfS polyclonal antibody exclusively localized YqfS in cell extracts prepared from B. subtilis spores. The His6-YqfS protein demonstrated enzymatic properties characteristic of the type IV family of DNA repair enzymes, such as AP-endonucleases and 3'-phosphatases. However, the purified protein lacked both 5'-phosphatase and exonuclease III activities. YqfS showed not only a high level of amino acid identity with E. coli Nfo but also a high resistance to inactivation by EDTA, in the presence of DNA containing AP sites (AP-DNA). These results suggest that YqfS possesses a trinuclear Zn center in which the three metal atoms are intimately coordinated by nine conserved basic residues and two water molecules. Electrophoretic mobility shift assays demonstrated that YqfS possesses structural properties that permit it to bind and scan undamaged DNA as well as to strongly interact with AP-DNA. The ability of yqfS to genetically complement the DNA repair deficiency of an E. coli mutant lacking the major AP-endonucleases Nfo and exonuclease III strongly suggests that its product confers protection to cells against the deleterious effects of oxidative promoters and alkylating agents. Thus, we conclude that YqfS of B. subtilis is a spore-specific protein that has structural and enzymatic properties required to participate in the repair of AP sites and 3' blocking groups of DNA generated during both spore dormancy and germination.
Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/metabolismo , Carbono-Oxigênio Liases/metabolismo , Desoxirribonuclease IV (Fago T4-Induzido) , Endonucleases/metabolismo , Proteínas de Escherichia coli , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Carbono-Oxigênio Liases/genética , DNA/metabolismo , Reparo do DNA/fisiologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Endonucleases/genética , Ativação Enzimática , Escherichia coli/genética , Teste de Complementação Genética , Histidina/genética , Dados de Sequência Molecular , Família Multigênica , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Esporos Bacterianos/fisiologia , Zinco/metabolismoRESUMO
The temporal and spatial expression of the yqfS gene of Bacillus subtilis, which encodes a type IV apurinic/apyrimidinic endonuclease, was studied. A reporter gene fusion to the yqfS opening reading frame revealed that this gene is not transcribed during vegetative growth but is transcribed during the last steps of the sporulation process and is localized to the developing forespore compartment. In agreement with these results, yqfS mRNAs were mainly detected by both Northern blotting and reverse transcription-PCR, during the last steps of sporulation. The expression pattern of the yqfS-lacZ fusion suggested that yqfS may be an additional member of the Esigma(G) regulon. A primer extension product mapped the transcriptional start site of yqfS, 54 to 55 bp upstream of translation start codon of yqfS. Such an extension product was obtained from RNA samples of sporulating cells but not from those of vegetatively growing cells. Inspection of the nucleotide sequence lying upstream of the in vivo-mapped transcriptional yqfS start site revealed the presence of a sequence with good homology to promoters preceding genes of the sigma(G) regulon. Although yqfS expression was temporally regulated, neither oxidative damage (after either treatment with paraquat or hydrogen peroxide) nor mitomycin C treatment induced the transcription of this gene.
Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/fisiologia , Carbono-Oxigênio Liases/genética , Carbono-Oxigênio Liases/metabolismo , Reparo do DNA , Regulação Bacteriana da Expressão Gênica , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Sequência de Bases , Clonagem Molecular , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Dados de Sequência Molecular , Estresse Oxidativo , Resposta SOS em Genética , Fator sigma , Esporos Bacterianos/enzimologia , Esporos Bacterianos/genética , Superóxidos/farmacologia , Transcrição GênicaRESUMO
Batimastat (BB-94), a synthetic hydroxamate peptidomimetic matrix metalloproteinase inhibitor, was tested for its ability to inhibit proteolytic and toxic effects induced by BaP1, a 24-kDa hemorrhagic metalloproteinase isolated from the venom of Bothrops asper, the medically most important snake species in Central America and southern Mexico. Batimastat inhibited proteolytic activity on biotinylated casein, with anIC(50) of 80 nM. In addition, batimastat was effective in inhibiting hemorrhagic, dermonecrotic, and edema-forming activities of this metalloproteinase if incubated with the enzyme prior to the assays. When the inhibitor was administered i.m. at the site of the toxin injection without preincubation, rapidly after metalloproteinase administration, it totally abrogated the hemorrhagic and dermonecrotic effects of BaP1. Inhibition was less effective as the time lapse between toxin and batimastat injection increased, due to the extremely rapid development of BaP1-induced local tissue damage in this experimental model. On the other hand, batimastat was ineffective if administered by the i.p. route immediately after toxin injection. It is concluded that batimastat, and probably other synthetic metalloproteinase inhibitors, may become useful therapeutic tools aimed at the in situ inhibition of venom metalloproteinases, when injected at the site of the bite rapidly after envenomation.
Assuntos
Bothrops , Carbono-Oxigênio Liases/antagonistas & inibidores , Venenos de Crotalídeos/enzimologia , Inibidores de Metaloproteinases de Matriz , Metaloendopeptidases/antagonistas & inibidores , Fenilalanina/análogos & derivados , Tiofenos/farmacologia , Animais , Carbono-Oxigênio Liases/toxicidade , Venenos de Crotalídeos/antagonistas & inibidores , Venenos de Crotalídeos/toxicidade , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Desoxirribonuclease IV (Fago T4-Induzido) , Interações Medicamentosas , Edema/prevenção & controle , Hemorragia/induzido quimicamente , Hemorragia/prevenção & controle , Metaloendopeptidases/toxicidade , Camundongos , Fenilalanina/farmacologia , Fenilalanina/uso terapêutico , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Tiofenos/uso terapêuticoRESUMO
Retinal cell differentiation leads to resistance to apoptosis induced by inhibition of protein synthesis, suggesting the accumulation of anti-apoptotic proteins. The redox factor/AP endonuclease Ref-1 (APE, APEX, HAP1) affects both DNA repair and the activity of various transcription factors, and controls sensitivity to genotoxic insults. We studied the expression of Ref-1 in the retina and brain of developing rats. Ref-1 immunoreactivity increased progressively within the nucleus of differentiating retinal cells, whereas it decreased in the developing hippocampal formation. During both natural and experimentally-induced cell death, Ref-1 disappeared from the nucleus of apoptotic cells. Degradation of Ref-1 in axotomized ganglion cells preceded the morphological characteristics of apoptosis. The sensitivity to apoptosis triggered by either thapsigargin or okadaic acid was the highest in photoreceptors, that contain the least Ref-1 among differentiated retinal cells. In both these differentiated cell types, inhibition of protein synthesis prevented the loss of Ref-1 and rescued the neurons. The data suggest that Ref-1 is an anti-apoptotic protein associated with cell differentiation in the retina.
Assuntos
Apoptose , Carbono-Oxigênio Liases/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Endodesoxirribonucleases/metabolismo , Retina/citologia , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Diferenciação Celular , Células Cultivadas , Neurônios/citologia , RatosRESUMO
In Escherichia coli, the repair of lethal DNA damage induced by H(2)O(2) requires exonuclease III, the xthA gene product. Here, we report that both endonuclease IV (the nfo gene product) and exonuclease III can mediate the repair of lesions induced by H(2)O(2) under low-iron conditions. Neither the xthA nor the nfo mutants was sensitive to H(2)O(2) in the presence of iron chelators, while the xthA nfo double mutant was significantly sensitive to this treatment, suggesting that both exonuclease III and endonuclease IV can mediate the repair of DNA lesions formed under such conditions. Sedimentation studies in alkaline sucrose gradients also demonstrated that both xthA and nfo mutants, but not the xthA nfo double mutant, can carry out complete repair of DNA strand breaks and alkali-labile bonds generated by H(2)O(2) under low-iron conditions. We also found indications that the formation of substrates for exonuclease III and endonuclease IV is mediated by the Fpg DNA glycosylase, as suggested by experiments in which the fpg mutation increased the level of cell survival, as well as repair of DNA strand breaks, in an AP endonuclease-null background.
Assuntos
Carbono-Oxigênio Liases/metabolismo , Dano ao DNA , Reparo do DNA , Proteínas de Escherichia coli , Escherichia coli/genética , Peróxido de Hidrogênio/farmacologia , Quelantes de Ferro/farmacologia , N-Glicosil Hidrolases/metabolismo , 2,2'-Dipiridil/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbono-Oxigênio Liases/deficiência , Carbono-Oxigênio Liases/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , DNA-Formamidopirimidina Glicosilase , Desoxirribonuclease IV (Fago T4-Induzido) , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Genoma Bacteriano , Ferro/metabolismo , Cinética , Mutação/genética , N-Glicosil Hidrolases/genética , Plasmídeos/genéticaRESUMO
Programmed cell death by apoptosis plays a major role in neurogenesis. The sensitivity to apoptosis in developing nervous tissue is strongly dependent on cell interactions taking place within a highly structured environment, composed of various cell types at distinct stages of differentiation. In this article, we review evidence gathered both in vivo and in a histotypical retinal explant preparation in vitro that the bifunctional AP endonuclease/redox factor Ref-1 (HAP1, APE, APEX) may be an anti-apoptotic protein associated with cell differentiation in the developing retina.
Assuntos
Apoptose/fisiologia , Carbono-Oxigênio Liases/metabolismo , Diferenciação Celular , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Retina/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Carbono-Oxigênio Liases/genética , Marcação In Situ das Extremidades Cortadas , Técnicas In Vitro , Ratos , Retina/citologia , Retina/embriologia , Retina/metabolismoRESUMO
Comparative gene assignment between the spider monkey species Ateles paniscus chamek (APC) and man (HSA) showed conserved syntenic associations despite extensive karyotypic rearrangement between species. Two HSA 14q genes were allocated to APC 2q, being syntenic to other HSA 14q and HSA 15q markers previously assigned to APC 2q, and to HSA 12q genes previously assigned to APC 2p. These findings were consistent with A. geoffroyi chromosome painting with human whole-chromosome probes, indicating that the genus Ateles is karyotypically very rearranged. On the other hand, three human X-linked markers were assigned to the Ateles X chromosome, indicating that this chromosome is evolutionary stable.
Assuntos
Cebidae/genética , Mapeamento Cromossômico , Cromossomo X , Animais , Sequência de Bases , Carbono-Oxigênio Liases/genética , Sequência Conservada , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Desoxirribonuclease IV (Fago T4-Induzido) , Fator VIII/genética , Marcadores Genéticos , Humanos , Células Híbridas , Cariotipagem , Camundongos , Camundongos Endogâmicos BALB C , Cadeias Pesadas de Miosina/genética , Receptores Androgênicos/genética , alfa-Galactosidase/genéticaRESUMO
Leishmania braziliensis promastigotes incubated anaerobically produce D-lactate from glucose, ribose, and methylglyoxal, but not from glycerol, alanine, or pyruvate, suggesting the presence of glyoxalases I and II but the absence of D-lactate dehydrogenase. Further support for this is shown by: (1) conversion of methylglyoxal to D-lactate in sonicates of promastigotes in the presence of reduced glutathione, (2) utilization of phenylglyoxal at rates comparable to methylglyoxal, (3) lack of utilization of exogenously supplied D-lactate by promastigotes under aerobic conditions. Sonicates of promastigotes catalyze the conversion of dihydroxyacetone phosphate to methylglyoxal, suggesting the presence of methylglyoxal synthase. Whereas the rate of production of D-lactate from glucose is much greater under anaerobic conditions, the rate from methylglyoxal is independent of oxygen tension, indicating that control of flux through the methylglyoxal pathway occurs at, or before, methylglyoxal synthase.