RESUMEN
AIM: To demonstrate a capacity for producing exopolysaccharides (EPSs) and an ability to form biofilm on abiotic materials of Actinomyces oris strain K20. METHODOLOGY: The productivity of EPSs and the ability to form biofilm of strain K20 were evaluated by measuring viscosity of spent culture media and by scanning electron microscopy (SEM) and the biofilm assay on microtitre plates, respectively. High-performance liquid chromatography was used to determine the chemical composition of the viscous materials. To examine the role of the viscous materials attributable to the pathogenicity in this organism, the ability of strain K20 to induce abscess formation was compared in mice to that of ATCC 27044. RESULTS: The viscosity of the spent culture media of K20 was significantly higher than that of ATCC 27044. Strain K20 showed dense meshwork structures around the cells and formed biofilms on microtitre plates, whereas ATCC 27044 did not. Chemical analysis of the viscous materials revealed that they were mainly composed of neutral sugars with mannose constituting 77.5% of the polysaccharides. Strain K20 induced persistent abscesses in mice lasting at least 5 days at a concentration of 10(8) cells mL(-1), whereas abscesses induced by ATCC 27044 healed and disappeared or decreased in size at day 5. CONCLUSIONS: Strain K20 produced EPSs, mainly consisting of mannose, and formed biofilms. This phenotype might play an important role for A. oris to express virulence through the progression of apical periodontitis.
Asunto(s)
Actinomyces/patogenicidad , Infecciones por Actinomycetales/microbiología , Absceso Periapical/microbiología , Polisacáridos Bacterianos , Actinomyces/clasificación , Actinomyces/aislamiento & purificación , Animales , Biopelículas , Medios de Cultivo , Masculino , Ratones , Ratones Endogámicos BALB C , Filogenia , Especificidad de la Especie , Virulencia , ViscosidadRESUMEN
BACKGROUND AND OBJECTIVE: Gingival crevicular fluid is a bodily fluid transuded from periodontal tissues into the gingival crevice and periodontal pocket, and contains many species of components. Proteins in gingival crevicular fluid have been studied as markers for periodontal diseases. Mass spectrometric analysis is used for the analyses of proteins, lipids, saccharides and metals, and expected as an approach for disease diagnosis. For better analysis of the protein components in gingival crevicular fluid, we investigated proteins in gingival crevicular fluid samples from the healthy gingival crevice and periodontal pocket using mass spectrometry. MATERIAL AND METHODS: Gingival crevicular fluid samples were collected from subjects who gave their informed consent and were periodontally healthy or had diseased pockets. These samples were electrophoretically separated, and each fraction on the gels was analysed by nano liquid chromatography coupled with tandem mass spectrometry. Antimicrobial peptides detected in gingival crevicular fluid were confirmed by western blotting. RESULTS: One hundred and four proteins were detected in gingival crevicular fluid samples from both healthy sites and sites of periodontitis; 64 proteins were contained only in gingival crevicular fluid from healthy sites and 63 proteins were observed only in gingival crevicular fluid from periodontitis sites. These proteins were blood-, cytoskeleton-, immunity-, inflammation- and lipid-related proteins and enzymes. Some proteins, including ceruloplasmin, glycogen phosphorylase, glutathione S-transferase, phosphoglycerate mutase, psoriasin, S100A11 and resistin, were identified for the first time in gingival crevicular fluid. Antimicrobial peptides, such as lactoferrin, α1-antitrypsin, lipocalin, S100A7, S100A8, S100A9 and cathelicidin, were observed by mass spectrometry and western blotting. CONCLUSION: Multiple protein components in gingival crevicular fluid were analysed at the same time using mass spectrometry, and this approach may be useful for the diagnosis of periodontal diseases.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/análisis , Líquido del Surco Gingival/química , Bolsa Periodontal/metabolismo , Periodontitis/diagnóstico , Proteínas/análisis , Espectrometría de Masas en Tándem/métodos , Adulto , Anciano , Western Blotting , Estudios de Casos y Controles , Ceruloplasmina/análisis , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Femenino , Líquido del Surco Gingival/enzimología , Glutatión Transferasa/análisis , Glucógeno Fosforilasa/análisis , Humanos , Masculino , Persona de Mediana Edad , Bolsa Periodontal/enzimología , Fosfoglicerato Mutasa/análisis , Resistina/análisis , Proteína A7 de Unión a Calcio de la Familia S100 , Proteínas S100/análisisRESUMEN
"Johkasou" is a small sewage treatment apparatus commonly used in Japan which can effectively treat domestic wastewater in places where a public sewage system is difficult to supply. The behaviour of enterohaemorrhagic E. coli O157 and Salmonella enteritidis in a "Johkasou" was studied. Their reduction rates depended significantly on the water temperature in the "Johkasou" with minimal decrease in numbers at 10 degrees C within 48 h. The reduction rates increased at 20 degrees C and 30 degrees C where 4 log reduction could be expected. The reduction rates were influenced by the BOD of the solutions that contained the pathogens with the lower the BOD the higher the reduction rate. The reduction rates were about the same between both pathogens. The result showed that it was necessary to disinfect the effluent as some pathogens can pass through the apparatus when some users of the apparatus excrete pathogens.
Asunto(s)
Escherichia coli , Salmonella enteritidis , Aguas del Alcantarillado/microbiología , Eliminación de Residuos Líquidos/métodos , Oxígeno/metabolismo , Dinámica Poblacional , Análisis de Supervivencia , Temperatura , Purificación del Agua , Abastecimiento de AguaRESUMEN
Orexins (hypocretins) are a pair of neuropeptides implicated in energy homeostasis and arousal. Recent reports suggest that loss of orexin-containing neurons occurs in human patients with narcolepsy. We generated transgenic mice in which orexin-containing neurons are ablated by orexinergic-specific expression of a truncated Machado-Joseph disease gene product (ataxin-3) with an expanded polyglutamine stretch. These mice showed a phenotype strikingly similar to human narcolepsy, including behavioral arrests, premature entry into rapid eye movement (REM) sleep, poorly consolidated sleep patterns, and a late-onset obesity, despite eating less than nontransgenic littermates. These results provide evidence that orexin-containing neurons play important roles in regulating vigilance states and energy homeostasis. Orexin/ataxin-3 mice provide a valuable model for studying the pathophysiology and treatment of narcolepsy.
Asunto(s)
Proteínas Portadoras/metabolismo , Trastornos de Alimentación y de la Ingestión de Alimentos/genética , Hipotálamo/fisiopatología , Péptidos y Proteínas de Señalización Intracelular , Narcolepsia/genética , Proteínas del Tejido Nervioso/genética , Neuronas/fisiología , Neuropéptidos/metabolismo , Neurotransmisores/metabolismo , Obesidad/genética , Fases del Sueño/genética , Animales , Ataxina-3 , Trastornos de Alimentación y de la Ingestión de Alimentos/fisiopatología , Femenino , Humanos , Hipotálamo/patología , Enfermedad de Machado-Joseph/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Narcolepsia/fisiopatología , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Neuronas/patología , Proteínas Nucleares , Obesidad/fisiopatología , Orexinas , Péptidos/genética , Proteínas Represoras , Eliminación de Secuencia , Fases del Sueño/fisiología , Sueño REM/genética , Factores de TranscripciónRESUMEN
The purpose of this report is to assess clinically acceptable compression ratios on the detection of brain lesions at magnetic resonance imaging (MRI). Four consecutive T2-weighted and the corresponding T1-weighted images obtained in 20 patients were studied for 109 anatomic sites including 50 with lesions and 59 without lesions. The images were obtained on a 1.5-T MR unit with a pixel size of 0.9 to 1.2 x 0.47 mm and a section thickness of 5 mm. The image data were compressed by wavelet-based algorithm at ratios of 20:1, 40:1, and 60:1. Three radiologists reviewed these images on an interactive workstation and rated the presence or absence of a lesion with a 50 point scale for each anatomic site. The authors also evaluated the influence of pixel size on the quality of image compression. At receiver operating characteristic (ROC) analysis, no statistically significant difference was detected at a compression ratio of 20:1. A significant difference was observed with 40:1 compressed images for one reader (P = .023), and with 60:1 for all readers (P = .001 to .012). A root mean squared error (RMSE) was higher in 0.94- x 0.94-mm pixel size images than in 0.94- x 0.47-mm pixel size images at any compression ratio, indicating compression tolerance is lower for the larger pixel size images. The RMSE, subjective image quality, and error images of 10:1 compressed 0.94- x 0.94-mm pixel size images were comparable with those of 20:1 compressed 0.94- x 0.47-mm pixel size images. Wavelet compression can be acceptable clinically at ratios as high as 20:1 for brain MR images when a pixel size at image acquisition is around 1.0 x 0.5 mm, and as high as 10:1 for those with a pixel size around 1.0 x 1.0 mm.
Asunto(s)
Encefalopatías/diagnóstico , Imagen por Resonancia Magnética/métodos , Algoritmos , Encefalopatías/diagnóstico por imagen , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/diagnóstico por imagen , Neoplasias Encefálicas/secundario , Humanos , Procesamiento de Imagen Asistido por Computador , Modelos Teóricos , Curva ROC , Sistemas de Información Radiológica , Tomografía Computarizada por Rayos XRESUMEN
We demonstrated involvement of the ventral tegmental area (VTA) dopaminergic system in orexin-induced hyperlocomotion and stereotypy in rats. In double-label immunohistochemical study of rat brain, we found that tyrosine hydroxylase (TH)-immunoreactive cells in the VTA received innervation from orexin immunoreactive-fibers. Orexin-A induced an increase in [Ca(2+)](i) in isolated A10 dopamine neurons in a dose-dependent manner. In behavioral studies, we found that orexin-A induced hyperlocomotion, stereotypy and grooming behavior when administered centrally in rats, and these effects were abolished by dopamine D(2) (haloperidol and sulpiride) or D(1) (SCH23390) antagonists. These results suggest that the orexin-induced hyperlocomotion, stereotypy and grooming behavior are mediated by the dopaminergic system and this pathway might be involved in orexin-induced emotional responses.
Asunto(s)
Conducta Animal/efectos de los fármacos , Proteínas Portadoras/farmacología , Dopamina/fisiología , Péptidos y Proteínas de Señalización Intracelular , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Neuropéptidos/farmacología , Conducta Estereotipada/fisiología , Animales , Axones/fisiología , Conducta Animal/fisiología , Benzazepinas/farmacología , Proteínas Portadoras/farmacocinética , Antagonistas de Dopamina/farmacología , Haloperidol/farmacología , Masculino , Terminaciones Nerviosas/fisiología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Neuropéptidos/farmacocinética , Orexinas , Ratas , Ratas Wistar , Conducta Estereotipada/efectos de los fármacos , Sulpirida/farmacología , Tegmento Mesencefálico/citología , Tegmento Mesencefálico/fisiología , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
PURPOSE: To determine the earliest findings, subsequent changes, and natural course of osteochondritis dissecans of the humeral capitellum. MATERIALS AND METHODS: Among 95 patients with osteochondritis dissecans of the humeral capitellum, 16 (mean age, 12.5 years) were selected for this retrospective study because they seemed to have early osteochondritis dissecans and had been followed up without any surgical treatment for 6 months or more (mean, 3.5 years). RESULTS: The initial imaging appearances of the 16 patients' lesions were divided into two types: localized subchondral bone flattening without fragments in seven, and nondisplaced fragments in nine. Patients with lesion flattening had younger ages and significantly shorter durations of symptoms, and most had open growth plates. In five of the seven with flattening, new bone formed over the flattened bone, and the fragments united after arm motion reduction. In contrast, patients with nondisplaced fragments at clinical presentation had longer durations of symptoms with continued arm motion, and their nondisplaced fragments failed to unite. CONCLUSION: The earliest feature of osteochondritis dissecans is subchondral bone flattening, over which new bone subsequently forms. The new bone then can unite with the underlying bone. However, if subjected to repetitive forces over a given time, unstable fragments develop. These fragments, even if not yet displaced, are unable to unite.
Asunto(s)
Húmero/diagnóstico por imagen , Osteocondritis Disecante/diagnóstico por imagen , Adolescente , Niño , Progresión de la Enfermedad , Estudios de Seguimiento , Humanos , Masculino , Radiografía , Estudios Retrospectivos , UltrasonografíaRESUMEN
Orexins (orexin-A and -B) are recently identified neuropeptides, which are thought to be implicated in the regulation of feeding behavior. We used a NPY-Y1 receptor specific antagonist, BIBO3304, to examine whether NPY is involved in orexin-induced feeding behavior. Intracerebroventricular administration of orexin-A (10 nmol) induced food intake in rats (food intake for 3 h; vehicle 0.3+/-0.2 g vs. orexin-A 10 nmol, 4.0+/-0.5 g, n=4). Orexin-induced feeding behavior was partially inhibited by prior administration of BIBO3304 (3 h food intake: orexin-A 10 nmol, 4.0+/-0.5 g vs. BIBO3304 (60 microgram) + orexin-A 10 nmol, 2.2+/-0.2 g, n=4). A low dose of BIBO3304 (30 microgram) did not show a significant inhibitory effect. BIBO3457, an inactive enantiomer, used as a negative control, did not show any inhibitory effect on orexin-A-induced feeding behavior. Fos expression was observed in NPY-containing neurons in the arcuate nucleus 1 h after orexin-A (10 nmol) was administered intracerebroventricularly (control 0.3+/-0.08%, orexin-A 10.2+/-0.8%, n=5 rats/group). These observations suggest that NPY is involved in orexin-induced feeding behavior. However, BIBO3304 did not completely abolish the effect of orexin-A. These results suggest that orexin-A elicits feeding behavior partially via the NPY pathway. The NPY system could be the one of downstream pathways by which orexin-A induces feeding behavior. Another pathway may also be involved in orexin-A-induced feeding behavior, because BIBO3304 did not completely abolish orexin-A-induced feeding behavior.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Portadoras/farmacología , Ingestión de Alimentos/efectos de los fármacos , Ingestión de Alimentos/fisiología , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/metabolismo , Neuropéptido Y/efectos de los fármacos , Neuropéptido Y/metabolismo , Neuropéptidos/metabolismo , Neuropéptidos/farmacología , Receptores de Neuropéptido Y/antagonistas & inhibidores , Receptores de Neuropéptido Y/metabolismo , Animales , Arginina/análogos & derivados , Arginina/farmacología , Hipotálamo/citología , Masculino , Vías Nerviosas/citología , Receptores de Orexina , Orexinas , Ratas , Ratas Wistar , Receptores Acoplados a Proteínas G , Receptores de NeuropéptidoRESUMEN
OBJECTIVE: The purpose of this study was to determine the efficacy of sonography for revealing osteochondritis dissecans of the humeral capitellum. SUBJECTS AND METHODS: Twenty-seven patients with capitellar osteochondritis dissecans (27 males; range, 11-20 years; mean age, 14 years) underwent radiography and sonography performed with a 7.5-MHz mechanical sector probe. Lesions were assessed as stable or unstable. The sonographic assessment was compared with radiographic assessment in 27 patients, MR assessment obtained in 10, and surgical findings in 15. RESULTS: Sonographic assessment agreed with radiographic assessment in 23 of the 27 patients, MR assessment in nine of the 10, and surgical findings in 14 of the 15. Sonography revealed that two lesions, which had been underestimated on radiography, were unstable. CONCLUSION: Sonography facilitates the assessment of capitellar lesions so that treatment can be optimized.
Asunto(s)
Húmero/diagnóstico por imagen , Osteocondritis Disecante/diagnóstico por imagen , Adolescente , Adulto , Niño , Humanos , Masculino , Radiografía , UltrasonografíaRESUMEN
To calibrate magnetic resonance (MR) signal intensity that depends on radio frequency (RF) coil loading, the transmission amplitude (TRA) for the excitation in the transmit-receive RF coil has been used as a good index in the so-called TRA method. As this TRA method needs neither an internal reference nor an additional external reference for the calibration, its accuracy is free from reference measurements. This study elucidated the calibration accuracy of MR signal intensities based on the TRA method. A cylindrical gel phantom was used for accuracy measurements with a 1.5-T MRI unit with conventional T1 imaging as a simple pulse sequence for various loading conditions. The brain parenchyma of eight healthy volunteers also showed calibrated MR signal deviations. The error of the phantom calibration measurements was 2.18% (S.D.%). The background noise intensity of images was theoretically derived to correlate with the impedance mismatching of the RF coil, which is inevitable for fixed tuning, even for automatic tuning that is not always exact. Taking into account this noise intensity, the calibration method was modified to reduce its error to 1.50%. The standard deviations of the calibrated values in the thalamus and frontal white matter were 2.9 and 3.8%, respectively. We suggest that the modified TRA method is a practical and reliable technique to obtain clinical numeric evidence.
Asunto(s)
Imagen por Resonancia Magnética/métodos , Encéfalo/anatomía & histología , Humanos , Imagen por Resonancia Magnética/normas , Imagen por Resonancia Magnética/estadística & datos numéricos , Modelos Teóricos , Fantasmas de Imagen , Ondas de RadioRESUMEN
We isolated the Xenopus gene encoding prepro-orexin to predict the structures of orexins in submammalian chordates. Putative mature Xenopus orexin-A and -B are highly similar to each mammalian counterpart. Especially, the C-terminal 10 residues were highly conserved among these species and isopeptides. Immunohistochemical examination of Xenopus brain revealed that orexin-containing neurons were highly specifically localized in the ventral hypothalamic nucleus. A rich network of immunoreactive fibers was found in various regions of the Xenopus brain. The distribution was similar to that of mammalian orexins. Xenopus orexin-A and -B specifically bind and activate human orexin receptors expressed in Chinese hamster ovary cells. Of interest, Xenopus orexin-B had several-fold higher affinity to human OX2R compared with human orexins. These results suggest that Xenopus orexin-B might be a useful pharmacological tool as an OX2R selective high-affinity agonist.
Asunto(s)
Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Neuropéptidos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/metabolismo , Encéfalo/patología , Células CHO , Proteínas Portadoras/síntesis química , Proteínas Portadoras/química , Proteínas Portadoras/genética , Cricetinae , Humanos , Datos de Secuencia Molecular , Neuropéptidos/síntesis química , Neuropéptidos/química , Neuropéptidos/genética , Receptores de Orexina , Orexinas , Receptores Acoplados a Proteínas G , Receptores de Neuropéptido/metabolismo , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Distribución Tisular , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
Orexin/hypocretins are recently identified neuropeptides which regulate feeding behaviour. We found orexins increased water intake when administrated intracerebroventricularly to rats. The effect of orexin-A was more potent as compared with orexin-B, suggesting the possible involvement of OX(1) receptor. The efficacy of orexin-A was almost comparable with that of angiotensin II, and the effect lasted more than 3 h. Prepro-orexin mRNA level was up-regulated when rats were deprived of water. Orexin-immunoreactive varicose axons were observed in the subfornical organ and area postrema, regions implicated in drinking behaviour. These observations suggest a physiological role for orexin as mediators that regulate drinking behaviour.
Asunto(s)
Proteínas Portadoras/fisiología , Conducta de Ingestión de Líquido/fisiología , Péptidos y Proteínas de Señalización Intracelular , Neuropéptidos/fisiología , Angiotensina II/administración & dosificación , Angiotensina II/farmacología , Animales , Northern Blotting , Proteínas Portadoras/biosíntesis , Ventrículos Cerebrales/metabolismo , Área Hipotalámica Lateral/metabolismo , Área Hipotalámica Lateral/fisiología , Inmunohistoquímica , Inyecciones Intraventriculares , Masculino , Neuropéptidos/biosíntesis , Orexinas , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Estimulación Química , Subtálamo/metabolismo , Subtálamo/fisiología , Regulación hacia Arriba/efectos de los fármacos , Privación de Agua/fisiologíaRESUMEN
Orexin-A and -B (also known as hypocretin-1 and -2) are neuropeptides which stimulate food intake when administered intracerebroventricularly. Orexins are specifically localized in neurons within and around the lateral hypothalamic area (LHA). Previous electrophysiological studies have demonstrated that some neurons in the LHA are activated by hypoglycemia, and are therefore termed 'glucose-sensitive neurons'. In the present study, we examined whether orexin-containing neurons are activated in the hypoglycemic states, using Fos-like immunoreactivity (FLI) as a marker of neuronal activation. We observed that FLI was induced in the LHA by acute insulin treatment. Double staining with anti-Fos and anti-orexin antibodies revealed that up to 33% of the orexin-containing neurons in the LHA also expressed FLI under the hypoglycemic condition. These results suggest that some populations of neurons which contain orexins are activated under hypoglycemic conditions.
Asunto(s)
Proteínas Portadoras/metabolismo , Hipoglucemia/inducido químicamente , Hipoglucemia/fisiopatología , Hipoglucemiantes , Área Hipotalámica Lateral/fisiología , Insulina , Péptidos y Proteínas de Señalización Intracelular , Neuronas/fisiología , Neuropéptidos/metabolismo , Animales , Área Hipotalámica Lateral/citología , Área Hipotalámica Lateral/metabolismo , Inmunohistoquímica , Masculino , Neuronas/metabolismo , Orexinas , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas WistarRESUMEN
Orexin (ORX)-A and -B are recently identified neuropeptides, which are specifically localized in neurons within and around the lateral hypothalamic area (LHA) and dorsomedial hypothalamic nucleus (DMH), the regions classically implicated in feeding behavior. Here, we report a further study of the distribution of ORX-containing neurons in the adult rat brain to provide a general overview of the ORX neuronal system. Immunohistochemical study using anti-ORX antiserum showed ORX-immunoreactive (ir) neurons specifically localized within the hypothalamus, including the perifornical nucleus, LHA, DMH, and posterior hypothalamic area. ORX-ir axons and their varicose terminals showed a widespread distribution throughout the adult rat brain. ORX-ir nerve terminals were observed throughout the hypothalamus, including the arcuate nucleus and paraventricular hypothalamic nucleus, regions implicated in the regulation of feeding behavior. We also observed strong staining of ORX-ir varicose terminals in areas outside the hypothalamus, including the cerebral cortex, medial groups of the thalamus, circumventricular organs (subfornical organ and area postrema), limbic system (hippocampus, amygdala, and indusium griseum), and brain stem (locus coeruleus and raphe nuclei). These results indicate that the ORX system provides a link between the hypothalamus and other brain regions, and that ORX-containing LHA and DMH neurons play important roles in integrating the complex physiology underlying feeding behavior.
Asunto(s)
Química Encefálica/fisiología , Proteínas Portadoras/análisis , Péptidos y Proteínas de Señalización Intracelular , Neuronas/química , Neuropéptidos/análisis , Factores de Edad , Amígdala del Cerebelo/citología , Amígdala del Cerebelo/fisiología , Animales , Especificidad de Anticuerpos , Ganglios Basales/citología , Ganglios Basales/fisiología , Tronco Encefálico/citología , Tronco Encefálico/fisiología , Proteínas Portadoras/inmunología , Tamaño de la Célula/fisiología , Cerebelo/citología , Cerebelo/fisiología , Núcleo Hipotalámico Dorsomedial/química , Núcleo Hipotalámico Dorsomedial/fisiología , Conducta Alimentaria/fisiología , Hipocampo/química , Hipocampo/fisiología , Área Hipotalámica Lateral/química , Área Hipotalámica Lateral/fisiología , Masculino , Neuronas/citología , Neuronas/ultraestructura , Neuropéptidos/inmunología , Vías Olfatorias/química , Vías Olfatorias/fisiología , Orexinas , Hipófisis/citología , Hipófisis/fisiología , Terminales Presinápticos/química , Ratas , Ratas Wistar , Núcleos Septales/química , Núcleos Septales/fisiologíaRESUMEN
Because the rod structure of the flagellar basal body crosses the inner membrane, the periplasmic space, and the outer membrane, its formation must involve hydrolysis of the peptidoglycan layer. So far, more than 10 genes have been shown to be required for rod formation in Salmonella typhimurium. Some of them encode the component proteins of the rod structure, and most of the remaining genes are believed to encode proteins involved in the export process of the component proteins. Although FlgJ has also been known to be involved in rod formation, its exact role has not been understood. Recently, it was suggested that the C-terminal half of the FlgJ protein has homology to the active center of some muramidase enzymes from gram-positive bacteria. In this study, we showed that the purified FlgJ protein from S. typhimurium has a peptidoglycan-hydrolyzing activity and that this activity is localized in its C-terminal half. Through oligonucleotide-directed mutagenesis, we constructed flgJ mutants with amino acid substitutions in the putative active center of the muramidase. The resulting mutants produced FlgJ proteins with reduced enzymatic activity and showed poor motility. These results indicate that the muramidase activity of FlgJ is essential for flagellar formation. Immunoblotting analysis with the fractionated cell extracts revealed that FlgJ is exported to the periplasmic space, where the peptidoglycan layer is localized. On the basis of these results, we conclude that FlgJ is the flagellum-specific muramidase which hydrolyzes the peptidoglycan layer to assemble the rod structure in the periplasmic space.