RESUMO
A new 15-valent pneumococcal conjugate vaccine (PCV15) against serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F, and 23F has been developed using aluminum phosphate as an adjuvant. Using the rabbit model, immunogenicity of each serotype was evaluated by measuring antigen specific antibodies and functional antibody titers and comparing them to a control vaccine, Prevnar13®. Among the shared serotypes in both PCV15 and Prevnar13®, Type 3 and 23F in PCV15 exhibited a lower opsonic index than Prevnar13®. Conversely, the other types showed greater or nearly the same immunogenic effects. Type 11A and 22F are two additional serotypes included in PCV15, and only 22F showed a reasonable opsonic index compared with other types. Type 11A exhibited a basal level fold-increase in OPA; thus, we further optimized 11A as well as 3 and 23F by controlling the polysaccharide-to-protein conjugation ratio as a variable. Antibody levels and functional antibody activities were evaluated by ELISA and OPA, and improved levels of immunogenic activities were observed for all three serotypes. In this study, we propose a new PCV15 candidate, in which the common 13 serotypes and a licensed control vaccine have equivalent efficacy while two additional serotypes showed adequate immunogenicity in the rabbit model.
Assuntos
Anticorpos Antibacterianos/imunologia , Imunogenicidade da Vacina , Vacinas Pneumocócicas , Streptococcus pneumoniae/imunologia , Animais , Avaliação Pré-Clínica de Medicamentos , Humanos , Vacinas Pneumocócicas/imunologia , Vacinas Pneumocócicas/farmacologia , Coelhos , Vacinas ConjugadasRESUMO
Cross-reactive material 197 (CRM197) is a non-toxic mutant of diphtheria toxin containing a single amino acid substitution of glycine 52 with glutamic acid. CRM197 has been used as a carrier protein for poorly immunogenic polysaccharide antigens to improve immune responses. In this study, to develop a sandwich ELISA that can detect CRM197 and CRM197 conjugate vaccines, we generated a human anti-CRM197 monoclonal antibody (mAb) 3F9 using a phage-displayed human synthetic Fab library and produced mouse anti-CRM197 polyclonal antibody. The affinity (KD) of 3F9 for CRM197 was 3.55 nM, based on Bio-Layer interferometry, and it bound specifically to the B fragment of CRM197. The sandwich ELISA was carried out using 3F9 as a capture antibody and the mouse polyclonal antibody as a detection antibody. The detection limit of the sandwich ELISA was ï¼1 ng/ml CRM197. In addition, the 3F9 antibody bound to the CRM197-polysaccharide conjugates tested in a dose-dependent manner. This ELISA system will be useful for the quantification and characterization of CRM197 and CRM197 conjugate vaccines. To our knowledge, this study is the first to generate a human monoclonal antibody against CRM197 and to develop a sandwich ELISA for CRM197 conjugate vaccines.
Assuntos
Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/imunologia , Técnicas de Visualização da Superfície Celular/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Vacinas Conjugadas/imunologia , Animais , Anticorpos Antibacterianos , Anticorpos Monoclonais/isolamento & purificação , Formação de Anticorpos , Reações Antígeno-Anticorpo , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Mapeamento de Epitopos , Humanos , Imunoglobulina G/imunologia , Limite de Detecção , Camundongos , Modelos MolecularesRESUMO
Cultivation of the smooth colony Mycobacterium abscessus at the sub-minimum inhibitory concentration (MIC) of amikacin changed its growth pattern including its colony morphology (smooth to rough) and cell arrangement (dispersed to cord formation). In addition, reduced sliding motility and biofilm formation were observed. The amount of glycogpetidolipid (GPL) and mRNA expression of key genes involved in GPL synthesis were decreased in the amikacin-treated M. abscessus strain. An in vitro infection assay revealed that the amikacin-treated smooth M. abscessus strain induced more pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6) than that of the smooth strain in murine macrophage cells. These results suggest that long-term exposure to a low concentration of amikacin causes a physical change in the cell wall which may increase its virulence.
Assuntos
Amicacina/farmacologia , Antibacterianos/farmacologia , Parede Celular/química , Glicolipídeos/análise , Glicopeptídeos/análise , Mycobacterium abscessus/efeitos dos fármacos , Proteolipídeos/análise , Animais , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Linhagem Celular , Citocinas/metabolismo , Locomoção/efeitos dos fármacos , Macrófagos/microbiologia , Camundongos , Mycobacterium abscessus/fisiologia , Virulência/efeitos dos fármacosAssuntos
Colo do Útero/patologia , Inflamação/complicações , Nascimento Prematuro/patologia , Animais , Colo do Útero/cirurgia , Modelos Animais de Doenças , Feminino , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Gravidez , Nascimento Prematuro/induzido quimicamente , Distribuição AleatóriaRESUMO
PURPOSE: Dickkopf 1 (DKK1) has been extensively investigated in mouse models of multiple myeloma, which results in osteolytic bone lesions. Elevated DKK1 levels in bone marrow plasma and serum inhibit the differentiation of osteoblast precursors. Present pharmaceutical approaches to target bone lesions are limited to antiresorptive agents. In this study, we developed a cyclized oligopeptide against DKK1-low density lipoprotein receptor-related protein (LRP) 5/6 interaction and tested the effects of the oligopeptide on tumor burden. MATERIALS AND METHODS: A cyclized oligopeptide based on DKK1-LRP5/6 interactions was synthesized chemically, and its nuclear magnetic resonance structure was assessed. Luciferase reporter assay and mRNA expressions of osteoblast markers were evaluated after oligopeptide treatment. MOPC315.BM.Luc cells were injected into the tail vein of mice, after which cyclized oligopeptide was delivered subcutaneously 6 days a week for 4 weeks. RESULTS: The cyclized oligopeptide containing NXI motif bound to the E1 domain of LRP5/6 effectively on surface plasmon resonance analysis. It abrogated the Wnt-ß-catenin signaling inhibited by DKK1, but not by sclerostin, dose dependently. RT-PCR and alkaline phosphatase staining showed increased expressions of osteoblast markers according to the treatment concentrations. Bioluminescence images showed that the treatment of cyclized oligopeptide reduced tumor burden more in oligopeptide treated group than in the vehicle group. CONCLUSION: The cyclized oligopeptide reported here may be another option for the treatment of tumor burden in multiple myeloma.
Assuntos
Medula Óssea/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mieloma Múltiplo/complicações , Mieloma Múltiplo/fisiopatologia , Oligopeptídeos/farmacologia , Osteoblastos/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Camundongos , Mieloma Múltiplo/patologia , Osteoblastos/patologia , Transdução de Sinais , Proteínas Wnt/metabolismo , beta CateninaRESUMO
The PDZ domain-containing scaffold protein, syntenin-1, binds to the transmembrane proteoglycan, syndecan-4, but the molecular mechanism/function of this interaction are unknown. Crystal structure analysis of syntenin-1/syndecan-4 cytoplasmic domains revealed that syntenin-1 forms a symmetrical pair of dimers anchored by a syndecan-4 dimer. The syndecan-4 cytoplasmic domain is a compact intertwined dimer with a symmetrical clamp shape and two antiparallel strands forming a cavity within the dimeric twist. The PDZ2 domain of syntenin-1 forms a direct antiparallel interaction with the syndecan-4 cytoplasmic domain, inhibiting the functions of syndecan-4 such as focal adhesion formation. Moreover, C-terminal region of syntenin-1 reveals an essential role for enhancing the molecular homodimerization. Mutation of key syntenin-1 residues involved in the syndecan-4 interaction or homodimer formation abolishes the inhibitory function of syntenin-1, as does deletion of the homodimerization-related syntenin-1 C-terminal domain. Syntenin-1, but not dimer-formation-incompetent mutants, rescued the syndecan-4-mediated inhibition of migration and pulmonary metastasis by B16F10 cells. Therefore, we conclude that syntenin-1 negatively regulates syndecan-4 function via oligomerization and/or syndecan-4 interaction, impacting cytoskeletal organization and cell migration.
Assuntos
Sindecana-4/química , Sinteninas/química , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Movimento Celular , Cristalografia por Raios X , Humanos , Metástase Linfática , Melanoma Experimental/metabolismo , Melanoma Experimental/secundário , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Ratos , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Sindecana-4/fisiologia , Sinteninas/fisiologiaRESUMO
The folding mechanism of typical proteins has been studied widely, while our understanding of the origin of the high stability of thermophilic proteins is still elusive. Of particular interest is how an atypical thermophilic protein with a novel fold maintains its structure and stability under extreme conditions. Folding-unfolding transitions of MTH1880, a thermophilic protein from Methanobacterium thermoautotrophicum, induced by heat, urea, and GdnHCl, were investigated using spectroscopic techniques including circular dichorism, fluorescence, NMR combined with molecular dynamics (MD) simulations. Our results suggest that MTH1880 undergoes a two-state N to D transition and it is extremely stable against temperature and denaturants. The reversibility of refolding was confirmed by spectroscopic methods and size exclusion chromatography. We found that the hyper-stability of the thermophilic MTH1880 protein originates from an extensive network of both electrostatic and hydrophobic interactions coordinated by the central ß-sheet. Spectroscopic measurements, in combination with computational simulations, have helped to clarify the thermodynamic and structural basis for hyper-stability of the novel thermophilic protein MTH1880.
Assuntos
Proteínas Arqueais/química , Dobramento de Proteína , Desdobramento de Proteína , Termodinâmica , Proteínas Arqueais/genética , Dicroísmo Circular , Modelos Moleculares , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína/efeitos dos fármacos , Desdobramento de Proteína/efeitos dos fármacos , Temperatura , Ureia/farmacologiaRESUMO
Mitogen inducible gene-6 (Mig-6) is a feedback inhibitor of epidermal growth factor receptor (EGFR) signaling pathway. The liver-specific knockout mice of the Mig-6 gene (Mig-6 d/d ) showed hepatomegaly and increased hypercholesterolemia. In this study, the biomarkers of insulin resistance and the effects of high-fat diets in the wild (Mig-6 f/f ) and Mig-6 d/d mice were analyzed. The fasting plasma concentrations of glucose, triglyceride, cholesterols, free fatty acids, and HOMA-IR were measured and the glucose tolerance and insulin resistance tests were performed in the 25-week-old Mig-6 f/f and the Mig-6 d/d mice. The protein levels of active insulin receptor, glucose 6-phosphatase, and phosphoenolpyruvate carboxykinase were analyzed in the liver and fat. The fasting plasma cholesterol and glucose concentration were higher in the Mig-6 d/d mice than the Mig-6 f/f mice with increased fat deposition in the liver. But the Mig-6 d/d mice had the improved glucose intolerance and insulin resistance without increased amount of phosphoinsulin receptor after insulin infusion in the liver. The hepatic concentration of phosphoenolpyruvate carboxykinase was increased in fasting Mig-6 d/d mice. The feeding of high-fat diet accelerated the plasma lipids profiles and HOMA-IR in the Mig-6 d/d mice but had no differential effects in oral glucose tolerance test and insulin tolerance test in both genotypes. These results suggest that the activated EGFR signaling might increase the fasting plasma glucose concentration through inducing the hepatic steatosis and the improved whole-body insulin resistance in the KO mice be caused by decreased adipogenesis in fat tissues.
Assuntos
Fígado Gorduroso/complicações , Fígado Gorduroso/genética , Resistência à Insulina/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Animais , Biomarcadores/metabolismo , Peso Corporal , Receptores ErbB/metabolismo , Fígado Gorduroso/patologia , Genótipo , Teste de Tolerância a Glucose , Hepatomegalia/genética , Hipercolesterolemia/genética , Insulina/sangue , Camundongos , Camundongos Knockout , Receptor de Insulina/metabolismo , Transdução de SinaisRESUMO
Syndesmos, nucleoside diphosphate linked moiety X (nudix)-type motif 16-like 1 (Nudt16l1), is evolutionarily divergent from the Nudt16 family. Syndesmos, which is co-localized with syndecan-4 cytoplasmic domain (Syn4(cyto)) in focal contacts, interacts with various cell adhesion adaptor proteins to control cell signaling. We determined the X-ray crystal structure of syndesmos; it is composed of seven α-helices and seven ß-strands. Although syndesmos has a molecular topology similar to that of nudix hydrolase proteins, the structure of the nudix motif differs from that of X29. The dimeric interface of syndesmos is composed of α-helix 4, 7 and ß-strand 2, 7, which primarily form hydrophobic interactions. The binding interaction between syndesmos and syn4(cyto) was characterized as a low-affinity interaction (Kd = 62 µM) by surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR). The NMR resonances of Lys (177, 178, 179), Gly182, and Ser183 in the C1 region and Lys193 and Lys194 in the V region of syndecan-4 are perturbed upon syndesmos binding. Our results provide structural insight into the molecular function of syndesmos in the regulation of cell signaling via binding to syndecan-4.
Assuntos
Pirofosfatases/metabolismo , Sindecana-4/metabolismo , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Ligação Proteica , Pirofosfatases/química , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície , Sindecana-4/químicaRESUMO
Telomere homeostasis is regulated by telomere-associated proteins, and the Myb domain is well conserved for telomere binding. AtTRB2 is a member of the SMH (Single-Myb-Histone)-like family in Arabidopsis thaliana, having an N-terminal Myb domain, which is responsible for DNA binding. The Myb domain of AtTRB2 contains three α-helices and loops for DNA binding, which is unusual given that other plant telomere-binding proteins have an additional fourth helix that is essential for DNA binding. To understand the structural role for telomeric DNA binding of AtTRB2, we determined the solution structure of the Myb domain of AtTRB2 (AtTRB21-64) using nuclear magnetic resonance (NMR) spectroscopy. In addition, the inter-molecular interaction between AtTRB21-64 and telomeric DNA has been characterized by the electrophoretic mobility shift assay (EMSA) and NMR titration analyses for both plant (TTTAGGG)n and human (TTAGGG)n telomere sequences. Data revealed that Trp28, Arg29, and Val47 residues located in Helix 2 and Helix 3 are crucial for DNA binding, which are well conserved among other plant telomere binding proteins. We concluded that although AtTRB2 is devoid of the additional fourth helix in the Myb-extension domain, it is able to bind to plant telomeric repeat sequences as well as human telomeric repeat sequences.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , DNA/química , Proteínas de Ligação a Telômeros/química , Telômero/química , Sequência de Aminoácidos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arginina/química , Arginina/metabolismo , Sítios de Ligação , Sequência Conservada , DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Triptofano/química , Triptofano/metabolismo , Valina/química , Valina/metabolismoRESUMO
An extracellular metalloprotease (vEP) secreted by Vibrio vulnificus ATCC29307 is a 45-kDa proteolytic enzyme that has prothrombin activation and fibrinolytic activities during bacterial infection. The action of vEP could result in clotting that could serve to protect the bacteria from the host defense machinery. Very recently, we showed that the C-terminal propeptide (C-ter100), which is unique to vEP, is involved in regulation of vEP activity. To understand the structural basis of this function of vEP C-ter100, we have determined the solution structure and backbone dynamics using multidimensional nuclear magnetic resonance spectroscopy. The solution structure shows that vEP C-ter100 is composed of eight anti-parallel ß-strands with a unique fold that has a compact ß-barrel formation which stabilized by hydrophobic and hydrogen bonding networks. Protein dynamics shows that the overall structure, including loops, is very rigid and stabilized. By structural database analysis, we found that vEP C-ter100 shares its topology with that of the collagen-binding domain of collagenase, despite low sequence homology between the two domains. Fluorescence assay reveals that vEP C-ter100 interacts strongly with iron (Fe(3+)). These findings suggest that vEP protease might recruit substrate molecules, such as collagen, by binding at C-ter100 and that vEP participates in iron uptake from iron-withholding proteins of the host cell during infection.
Assuntos
Proteínas de Bactérias/química , Metaloproteases/química , Vibrio vulnificus/enzimologia , Fluorescência , Ferro/química , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: Pyrazinamide (PZA) is an effective antitubercular drug that becomes toxic to Mycobacterium tuberculosis when converted to pyrazinoic acid by pyrazinamidase (PZase), encoded by mycobacterial pncA. A strong association was noted between the loss of PZase activity and PZA resistance. The causative organisms in extrapulmonary tuberculosis are rarely cultured and isolated. To detect pncA mutations in specimens from extrapulmonary tuberculosis as confirmative diagnosis of mycobacterial infection and alternative susceptibility test to PZA. METHODS: Specimens were collected from clinically proven extrapulmonary tuberculosis. pncA was sequenced and compared with wild-type pncA. RESULTS: pncA from 30 specimens from 23 donors were successfully amplified (56.6% in specimens, 59% in donors). Six mutations in pncA were detected (20.0% in amplified specimens, 26.1% in specimen donors) at nucleotide positions of 169, 248 and 419. The mutation at position 169 results in substitution of aspartic acid for histidine, a possible allelic variation of M. bovis that have intrinsic PZA resistance. The mutation at position 248 changes proline into arginine and that at position 419, arginine into histidine. CONCLUSION: DNA-based diagnosis using pncA may be simultaneously useful for the early diagnosis of mycobacterial infection and the rapid susceptibility to PZA in extrapulmonary tuberculosis. A potential implication of pncA allelic variation at 169 might be suggested as a rapid diagnostic test for M. bovis infection or Bacille Calmette-Guérin (BCG) reactivation.
RESUMO
The population structure of Korean (150 strains) and Japanese (92 strains) Legionella pneumophila isolates along with 18 reference strains were investigated using hsp60 sequence (1647 bp) analysis. Twelve clonal subgroups (hsP-I to hsP-X and hsF-I and hsF-II) were designated on the hsp60 tree, inferred from representative sequences using the neighbor-joining method. Some of the isolates showed unique subgroups depending on the source of isolates, including hsP-I, hsF-I, and hsF-II from cooling tower water, and subgroups hsP-VIII and hsP-X from circulating hot water bath. These subgroups may be useful for epidemiological studies to chase or specify sources of infection in Korea and Japan.
Assuntos
Chaperonina 60/genética , Microbiologia Ambiental , Variação Genética , Legionella pneumophila/classificação , Legionella pneumophila/isolamento & purificação , Doença dos Legionários/microbiologia , Proteínas de Bactérias/genética , Análise por Conglomerados , Humanos , Japão/epidemiologia , Coreia (Geográfico)/epidemiologia , Legionella pneumophila/genética , Doença dos Legionários/epidemiologia , Epidemiologia Molecular , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de SequênciaAssuntos
Proteínas de Bactérias/química , RNA Polimerases Dirigidas por DNA/química , Methanobacterium/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de Sequência , Soluções/químicaRESUMO
The effects of leptin on rosiglitazone-induced adipocyte differentiation were investigated in the primary adipocytes prepared from subcutaneous fat of TallyHO/Jng (TallyHO) mouse, a recently developed model animal for type 2 diabetes mellitus (T2DM). The treatment of leptin inhibited the rosiglitazone-induced adipocyte differentiation with a decreased expression of peroxisome proliferator-activated receptor γ (PPARγ) a key adipogenic transcription factor, both in mRNA and protein levels. Leptin (10 nM) was sufficient to inhibit the adipocyte differentiation, which seemed to come from increased expression of leptin receptor genes in the fat of TallyHO mice. The inhibition of adipogenesis by leptin was restored by the treatment of inhibitors for extracellular-signal-regulated kinase (ERK) (PD98059) and signal transducer and activator of transcription-1 (STAT1) (fludarabine). Furthermore, in vivo intraperitoneal administration of PD98059 and fludarabine increased the PPARγ expression in the subcutaneous fat of TallyHO mice. These data suggest that leptin could inhibit the PPARγ expression and adipocyte differentiation in its physiological concentration in TallyHO mice.
Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Leptina/farmacologia , PPAR gama/antagonistas & inibidores , Adipócitos/citologia , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Flavonoides/farmacologia , Camundongos , Camundongos Endogâmicos , PPAR gama/biossíntese , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Receptores para Leptina/biossíntese , Rosiglitazona , Gordura Subcutânea/metabolismo , Tiazolidinedionas/farmacologiaRESUMO
Erythromycin ribosome methyltransferase gene (erm) sequences of Mycobacterium massiliense and Mycobacterium bolletii isolates were newly investigated. Forty nine strains of M. massiliense that were analyzed in the present study had a deleted erm(41). Due to a frame-shift mutation, large deletion, and truncated C-terminal region, the Erm(41) of M. massiliense had only 81 amino acids encoded by 246 nucleotides. Corresponding to these findings, most of the M. massiliense isolates (89.8%) were markedly clarithromycin susceptible, but resistant strains invariably had a point mutation at the adenine (A(2058) or A(2059)) in the peptidyltransferase region of the 23S rRNA gene, which is quite different from Mycobacterium abscessus and M. bolletii. In addition, erm(41) sequences of M. massiliense were more conserved than those of M. abscessus and M. bolletii. The results of species identification using erm(41) showed concordant results with those of multi-locus sequence analysis (rpoB, hsp65, sodA and 16S-23S ITS) where there were originally inconsistent results between rpoB and hsp65 sequence analysis in previous research. Therefore, erm(41) PCR that was used in the present study can be efficiently used to simply differentiate M. massiliense from M. abscessus and M. bolletii.
Assuntos
Antibacterianos/farmacologia , Claritromicina/farmacologia , Metiltransferases/genética , Mycobacterium/classificação , Sequência de Bases , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mycobacterium/efeitos dos fármacos , Mycobacterium/genética , Reação em Cadeia da PolimeraseRESUMO
Partial RNA polymerase ß-subunit gene (rpoB) sequences (315 bp) were determined and used to differentiate the type strains of 23 species of the genus Bifidobacterium. The sequences were compared with those of the partial hsp60 (604 bp) and 16S rRNA genes (1475 or 1495 bp). The rpoB gene sequences showed nucleotide sequence similarities ranging from 84.1â% to 99.0â%, while the similarities of the hsp60 sequences ranged from 78.5â% to 99.7â% and the 16S rRNA gene sequence similarities ranged from 89.4â% to 99.2â%. The phylogenetic trees constructed from the sequences of these three genes showed similar clustering patterns, with the exception of several species. The Bifidobacterium catenulatum-Bifidobacterium pseudocatenulatum, Bifidobacterium pseudolongum subsp. pseudolongum-Bifidobacterium pseudolongum subsp. globosum and Bifidobacterium gallinarum-Bifidobacterium pullorum-Bifidobacterium saeculare groups were more clearly differentiated in the partial rpoB and hsp60 gene sequence trees than they were in the 16S rRNA gene tree. Based on sequence similarities and tree topologies, the newly determined rpoB gene sequences are suitable molecular markers for the differentiation of species of the genus Bifidobacterium and support various other molecular tools used to determine the relationships among species of this genus.
Assuntos
Bifidobacterium/classificação , RNA Polimerases Dirigidas por DNA/genética , Filogenia , Técnicas de Tipagem Bacteriana , Chaperonina 60/genética , DNA Bacteriano/genética , Dados de Sequência Molecular , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Korean isolates of the Mycobacterium chelonae-Mycobacterium abscessus group, which had been isolated from two different hospitals in South Korea, were identified by PCR restriction analysis (PRA) and comparative sequence analysis of 16S rRNA genes, rpoB, and hsp65 to evaluate the proportion of four closely related species (M. chelonae, M. abscessus, M. massiliense, and M. bolletii). Of the 144 rapidly growing mycobacterial strains tested, 127 strains (88.2%) belonged to the M. chelonae-M. abscessus group. In this group, M. chelonae, M. abscessus, M. massiliense, and M. bolletii accounted for 0.8% (n = 1), 51.2% (n = 65), 46.5% (n = 59), and 1.6% (n = 2), respectively. Two isolates which showed discordant results, M. massiliense by rpoB sequence analysis and M. abscessus by hsp65 sequence analysis, were finally identified as M. massiliense based on the additional analysis of sodA and the 16S-23S internal transcribed spacer. M. abscessus group I isolates previously identified by hsp65 PRA were all found to be M. abscessus, whereas group II isolates were further identified as M. massiliense or M. bolletii by sequencing of rpoB and hsp65. Smooth, rough, or mixed colonies of both M. abscessus and M. massiliense isolates were observed. M. massiliense strains that were highly resistant to clarithromycin had a point mutation at the adenine at position 2058 (A(2058)) or 2059 (A(2059)) in the peptidyltransferase region of the 23S rRNA gene.
Assuntos
Mycobacterium/classificação , Mycobacterium/isolamento & purificação , Tuberculose/microbiologia , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Chaperonina 60 , Chaperoninas/genética , Claritromicina/farmacologia , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana , Genes de RNAr , Humanos , Coreia (Geográfico) , Dados de Sequência Molecular , Filogenia , Mutação Puntual , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Superóxido Dismutase/genéticaRESUMO
Twelve strains of a rapidly growing Mycobacterium species were isolated from an outbreak associated with intramuscular injections of an antimicrobial agent and were identified by comparative sequence analysis of rpoB and hsp65. These isolates were identified as Mycobacterium massiliense (100% similarity).
Assuntos
Surtos de Doenças , Injeções Intramusculares/efeitos adversos , Infecções por Mycobacterium/epidemiologia , Infecções por Mycobacterium/microbiologia , Mycobacterium/isolamento & purificação , Ribostamicina/efeitos adversos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/administração & dosagem , Antibacterianos/efeitos adversos , Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Chaperonina 60 , Chaperoninas/genética , Criança , Pré-Escolar , DNA Bacteriano/química , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mycobacterium/genética , Filogenia , Ribostamicina/administração & dosagem , Ribostamicina/uso terapêutico , Análise de Sequência de DNARESUMO
The pathophysiology of TallyHo mouse, a recently established animal model for type 2 diabetes mellitus, was analyzed at prediabetic state to examine the inherent defects which contribute to the development of diabetes. At 4 weeks of age, the TallyHo mice already revealed glucose intolerance while their peripheral tissues exhibited normal insulin sensitivity. On the other hand, decreased plasma insulin concentration was observed with little differences in pancreatic insulin contents, indicating the impaired insulin secretion. Such defect, however, was not found in the isolated islets, which suggests a role of endocrine factor in impaired insulin secretion of TallyHo mice. Treatment of leptin inhibited the glucose-stimulated insulin secretion from the isolated islets of TallyHo mice, while in vivo administration of anti-leptin antibody lowered plasma glucose concentration with increased insulin level in TallyHo mice. Expression of glucokinase mRNA was decreased both in whole pancreas and leptin treated islets of TallyHo mice compared with whole pancreas in C57BL/6 mice and untreated islets of TallyHo mice, respectively. These results suggest that elevated plasma leptin can, through the inhibition of insulin secretion, induce glucose intolerance in TallyHo mice.