RESUMO
Bioactive glasses (BAG) are used as bone-graft substitutes in orthopaedic surgery. A specific BAG scaffold was developed by sintering BAG-S53P4 granules. It is hypothesised that this scaffold can be used as a bone substitute to fill bone defects and induce a bioactive membrane (IM) around the defect site. Beyond providing the scaffold increased mechanical strength, that the initial inflammatory reaction and subsequent IM formation can be enhanced by coating the scaffolds with poly(DL-lactide-co-glycolide) (PLGA) is also hypothesised. To study the immunomodulatory effects, BAG-S53P4 (± PLGA) scaffolds were placed on monolayers of primary human macrophage cultures and the production of various pro- and anti-inflammatory cytokines was assessed using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and ELISA. To study the osteogenic effects, BAG-S53P4 (± PLGA) scaffolds were cultured with rabbit mesenchymal stem cells and osteogenic differentiation was evaluated by RT-qPCR and matrix mineralisation assays. The scaffold ion release was quantified and the BAG surface reactivity visualised. Furthermore, the pH of culture media was measured. BAG-S53P4 scaffolds had both anti-inflammatory and osteogenic properties that were likely attributable to alkalinisation of the media and ion release from the scaffold. pH change, ion release, and immunomodulatory properties of the scaffold could be modulated by the PLGA coating. Contrary to the hypothesis, the coating functioned by attenuating the BAG surface reactions and subsequent anti-inflammatory properties, rather than inducing an elevated inflammatory response compared to BAG-S53P4 alone. These results further validated the use of BAG-S53P4 (± PLGA) scaffolds as bone substitutes and indicate that scaffold properties can be tailored to a specific clinical need.
Assuntos
Substitutos Ósseos , Células-Tronco Mesenquimais , Osteonecrose , Animais , Terapia Baseada em Transplante de Células e Tecidos , Osteogênese , Coelhos , Alicerces TeciduaisRESUMO
Although women reportedly have a higher prevalence of medial tibial stress syndrome (MTSS) than men, the possible role of gender-based anatomical differences has not been investigated. The aim of the present study was to investigate the presence of gender-based differences in the range of muscle attachments along the entire medial tibia, the proportion of muscle attachment at the middle and distal thirds of the medial margin of the tibia, the structure of the crural fascia, and chiasm position. The specimens were 100 legs of 55 Japanese cadavers. Statistical analysis was carried out using a chi-square test to compare anatomical features between the sexes. The flexor digitorum longus (FDL) had a higher proportion of attachment to the middle and distal thirds of the medial margin of the tibia than the soleus (SOL; P < 0.001). The proportion of the SOL attachment to the middle and distal thirds of the medial margin of the tibia was 33.3% in men and 72.5% in women (P < 0.001). The soleal aponeurosis was not observed in any specimen. In all specimens the FDL formed the top layer of both chiasms. These results suggest that the higher prevalence of MTSS reported among women may be the result of gender-based anatomical differences.
Assuntos
Aponeurose/anatomia & histologia , Perna (Membro)/anatomia & histologia , Síndrome do Estresse Tibial Medial/epidemiologia , Músculo Esquelético/anatomia & histologia , Fatores Sexuais , Tíbia/anatomia & histologia , Idoso , Idoso de 80 Anos ou mais , Cadáver , Fáscia/anatomia & histologia , Feminino , Humanos , Masculino , Caracteres Sexuais , Distribuição por SexoRESUMO
The demand for the use of mice as animal models for elucidating the pathophysiologies and pathogeneses of oral motor disorders has been increasing in recent years, as more and more kinds of genetically modified mice that express functional disorders of the stomatognathic system become available. However, the fundamental characteristics of mouse jaw movements during mastication have yet to be fully elucidated. The purpose of this study was to investigate the roles of the masseter and temporalis muscles, and the mechanisms of motor coordination of these muscles for increasing masticatory efficiency in the closing phase in mice. Twenty-two male Jcl:ICR mice were divided into control (n = 8), masseter-hypofunction (n = 7) and temporalis-hypofunction groups (n = 7). Botulinum neurotoxin type A (BoNT/A) was used to induce muscle hypofunction. The masticatory movement path in the horizontal direction during the occlusal phase became unstable after BoNT/A injection into the masseter muscle. BoNT/A injection into the temporalis muscle decreased antero-posterior excursion of the late-closing phase corresponding to the power phase of the chewing cycle. These results suggest that the masseter plays an important role in stabilizing the grinding path, where the food bolus is ground by sliding the posterior teeth from back to front during the occlusal phase. The temporalis plays a major role in retracting the mandible more posteriorly in the early phase of closing, extending the grinding path. Masticatory efficiency is thus increased based on the coordination of activities by the masseter and temporalis muscles.
Assuntos
Toxinas Botulínicas Tipo A/farmacologia , Deglutição/fisiologia , Mastigação/fisiologia , Músculos da Mastigação/patologia , Fármacos Neuromusculares/farmacologia , Articulação Temporomandibular/patologia , Animais , Fenômenos Biomecânicos , Modelos Animais de Doenças , Eletromiografia , Masculino , Camundongos , Camundongos Endogâmicos ICRRESUMO
It has been suggested that feeding a soft diet could possibly inhibit normal development of the masticatory function. However, the consequences of such changes in the alimentary habits have yet to be fully clarified. Therefore, the aim of this study was to determine whether a soft diet prevents the development of masticatory function and whether a critical period for programming the masticatory system exists. To examine these hypotheses, we used a three-dimensional jaw-movement tracking device and jaw muscle electromyography (EMG) to analyse masticatory function changes in mice. Jcl:ICR mice were divided into three groups, with the normal group fed a hard diet, the hypofunctional group fed a soft diet, and the rehabilitation group first fed a soft diet that was then changed to a hard diet. Our results showed that the excursion and duration of late-closing phase (occlusal phase) of the chewing cycle and EMG activity in the masseter muscle were not only reduced in the hypofunctional but also in the rehabilitation group as compared with the normal group. These results suggest that optimisation of the chewing pattern and acquisition of appropriate masticatory function are impeded by feeding a soft diet during the animal's growth period and that no catch-up effect of the masticatory function is observed when there is a prolonged period of time prior to changing the diet from soft to hard. In conclusion, masticatory function can only be fully developed through a learning process such as exposure to chewing various kinds of foods with different food textures.
Assuntos
Alimentos , Músculo Masseter/fisiologia , Mastigação/fisiologia , Animais , Eletromiografia/métodos , Masculino , Camundongos , Camundongos Endogâmicos ICRRESUMO
Midventricular obstruction (MVO) is a rare form of hypertrophic cardiomyopathy (HCM). While surgical treatment for HCM is among the most technically challenging cardiac operations for acquired disease, surgery for MVO is rarely reported. A 38-year-old man was admitted to our hospital with a cough and dyspnea. Transthoracic and transesophageal echography and computed tomography revealed extensive left ventricular hypertrophy, extending from the anteroseptal wall to the apex, and marked papillary muscle hypertrophy. We underwent septal myectomy via aortotomy (Morrow procedure) and apical surgery. Extended myectomy provides the best exposure to the hypertrophied septum and improves the functional status of patients.
RESUMO
The interactions between fibroblast growth factors (FGF) and their receptors have important roles in mediating mesenchymal-epithelial cell interactions during embryogenesis. In particular, Fgf10 is predicted to function as a regulator of brain, lung and limb development on the basis of its spatiotemporal expression pattern in the developing embryo. To define the role of Fgf10, we generated Fgf10-deficient mice. Fgf10-/- mice died at birth due to the lack of lung development. Trachea was formed, but subsequent pulmonary branching morphogenesis was disrupted. In addition, mutant mice had complete truncation of the fore- and hindlimbs. In Fgf10-/- embryos, limb bud formation was initiated but outgrowth of the limb buds did not occur; however, formation of the clavicles was not affected. Analysis of the expression of marker genes in the mutant limb buds indicated that the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA) did not form. Thus, we show here that Fgf10 serves as an essential regulator of lung and limb formation.
Assuntos
Extremidades/embriologia , Fatores de Crescimento de Fibroblastos/fisiologia , Pulmão/embriologia , Proteínas com Domínio T , Transativadores , Animais , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/genética , Feminino , Fator 10 de Crescimento de Fibroblastos , Fator 8 de Crescimento de Fibroblasto , Fatores de Crescimento de Fibroblastos/genética , Proteínas Hedgehog , Proteínas de Homeodomínio/genética , Proteínas com Homeodomínio LIM , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/genética , Proteínas Wnt , Proteína Wnt2RESUMO
Hypertrophic cardiomyopathy (HCM), the most common cause of sudden death in the young, is an autosomal dominant disease characterized by ventricular hypertrophy accompanied by myofibrillar disarrays. Linkage studies and candidate-gene approaches have demonstrated that about half of the patients have mutations in one of six disease genes: cardiac beta-myosin heavy chain (c beta MHC), cardiac troponin T (cTnT), alpha-tropomyosin (alpha TM), cardiac myosin binding protein C (cMBPC), ventricular myosin essential light chain (vMLC1) and ventricular myosin regulatory light chain (vMLC2) genes. Other disease genes remain unknown. Because all the known disease genes encode major contractile elements in cardiac muscle, we have systematically characterized the cardiac sarcomere genes, including cardiac troponin I (cTnI), cardiac actin (cACT) and cardiac troponin C (cTnC) in 184 unrelated patients with HCM and found mutations in the cTnI gene in several patients. Family studies showed that an Arg145Gly mutation was linked to HCM and a Lys206Gln mutation had occurred de novo, thus strongly suggesting that cTnI is the seventh HCM gene.
Assuntos
Cardiomiopatia Hipertrófica/genética , Mutação , Troponina I/genética , Actinas/genética , Sequência de Aminoácidos , Animais , Arginina , Sequência de Bases , Proteínas de Transporte/genética , DNA Complementar , Éxons , Feminino , Ligação Genética , Glicina , Humanos , Masculino , Dados de Sequência Molecular , Miocárdio/metabolismo , Linhagem , Polimorfismo Genético , Troponina C/genéticaRESUMO
BACKGROUND: Glutathione redox status, changes in intracellular reduced (GSH) or oxidized (GSSG) glutathione, plays a significant role in various aspects of cellular function. In this study, we examined whether intracellular glutathione redox status in human dendritic cells (DCs) regulates the polarization of Th1/Th2 balance. METHODS: Human monocyte-derived DCs (MD-DCs) treated with glutathione reduced form ethyl ester (GSH-OEt) or L-buthionine-(S,R)-sulfoximine (BSO) were stimulated by lipopolysaccharide (LPS), and the levels of polarization cytokines were measured. Next, DCs matured by LPS or thymic stromal lymphopoietin (TSLP) were cocultured with allogeneic CD4(+) naive T cells and Th1/Th2 balance was evaluated by cytokine production from the primed T cells. RESULTS: Monocyte-derived DCs exposed to GSH-OEt and BSO had increased and decreased intracellular GSH contents, respectively. Lipopolysaccharide-induced interleukin (IL)-27 production was enhanced by GSH-OEt and suppressed by BSO, but neither GSH-OEt nor BSO affected the expression of HLA-DR, CD80, CD83, or CD86. Mature GSH-OEt-treated MD-DCs enhanced interferon (IFN)-γ production from CD4(+) T cells compared with nontreated MD-DCs, and small interfering RNA (siRNA) against IL-27 suppressed the effect of GSH-OEt on IFN-γ production. Additionally, although human myeloid DCs activated by TSLP (TSLP-DCs) prime naïve CD4(+) T cells to differentiate into Th2 cells, treatment of TSLP-DCs with GSH-OEt reduced IL-13 production and enhanced IFN-γ production by CD4(+) T cells. Interleukin-27 siRNA attenuated the inhibitory effect of GSH-OEt on Th2 polarization. CONCLUSION: Our results reveal that Th1 and Th2 responses are controlled by intracellular glutathione redox status in DCs through IL-27 production.
Assuntos
Células Dendríticas/imunologia , Glutationa/metabolismo , Interleucina-17/biossíntese , Linfócitos T/imunologia , Diferenciação Celular/imunologia , Citocinas/biossíntese , Células Dendríticas/efeitos dos fármacos , Glutationa/análogos & derivados , Glutationa/farmacologia , Humanos , Espaço Intracelular/metabolismo , Lipopolissacarídeos/imunologia , Oxirredução , Linfócitos T/citologia , Células Th1/imunologia , Células Th2/imunologiaRESUMO
AIMS: The study aimed to combine a metagenomics approach with complementary genetics to identify novel bacterial genes with orthologous functions, with the identification of novel RNase H genes as a test case. METHODS AND RESULTS: A metagenomic DNA library was prepared from leaf-and-branch compost and used to screen for the RNase H genes by their abilities to complement the temperature-sensitive growth phenotype of the rnhA mutant Escherichia coli strain MIC3001. Determination of the nucleotide sequences of the cloned DNA fragments allowed us to identify 12 different genes encoding type 1 RNases H. Eleven of them encode novel RNases H, which show 40-72% amino acid sequence identities to those available from database. One of them lacks a typical DEDD/E active-site motif, which is almost fully conserved in various RNases H. CONCLUSIONS: Functional screening of environmental DNA without cultivation of microbes is a useful procedure to isolate novel RNase H genes. SIGNIFICANCE AND IMPACT OF THE STUDY: One of the identified RNase H genes had no sequence similarity to a previously assumed conserved motif, suggesting multiple catalytic mechanisms exist. This test case illustrates that metagenomics combined with complementary genetics can identify novel genes that are orthologous without sequence similarity to those from cultivated bacteria.
Assuntos
Metagenoma , Ribonuclease H/química , Ribonuclease H/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência de Bases , Domínio Catalítico , Clonagem Molecular , DNA Bacteriano/química , Escherichia coli/genética , Biblioteca Gênica , Genes Bacterianos , Metagenômica , Dados de Sequência Molecular , Filogenia , Ribonuclease H/classificação , Alinhamento de SequênciaRESUMO
BACKGROUND: Oligosaccharides may have beneficial properties of the prevention of atopic dermatitis (AD). Kestose, a fructo-oligosaccharide, stimulates the activity of bifidobacteria. OBJECTIVE: To assess the clinical effect of kestose on the treatment of AD in infants. METHODS: A randomized, double-blind, placebo-controlled trial was carried out using 15 and 14 infants with AD in the kestose group and placebo groups, respectively. One to 2 g kestose and maltose were administered to the subjects in the kestose and placebo groups, respectively, everyday for 12 weeks. Clinical evaluations of AD using Severity Scoring of Atopic Dermatitis (SCORAD) and the enumeration of bifidobacteria in the feces using real-time PCR were performed at Weeks 0, 6, and 12. RESULTS: The medians of the SCORAD score were significantly lower in the kestose group than in the placebo group on both Week 6 (25.3 vs. 36.4; P=0.004) and Week 12 (19.5 vs. 37.5; P<0.001). No significant correlation was found between the improvement of the SCORAD score and the count of bifidobacteria. CONCLUSION: Kestose was found to exert a beneficial effect on the clinical symptoms in infants with AD. The mechanism how does kestose improve the symptoms of AD remains to be elucidated.
Assuntos
Dermatite Atópica/tratamento farmacológico , Trissacarídeos/administração & dosagem , Bifidobacterium , Pré-Escolar , Dermatite Atópica/microbiologia , Método Duplo-Cego , Fezes/microbiologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Maltose/administração & dosagem , Edulcorantes/administração & dosagem , Fatores de TempoRESUMO
When oral tolerance was induced in either specific pathogen-free (SPF) or germ-free (GF) mice, ovalbumin (OVA) feeding before immunization induced oral tolerance successfully in SPF mice. On the other hand, OVA-specific immunoglobulin G1 (IgG1) and IgE titres in OVA-fed GF mice were comparable to those in phosphate-buffered saline-fed GF mice, thus demonstrating that oral tolerance could not be induced in GF mice. The frequencies of CD25(+) CD4(+)/CD4(+) cells in the mesenteric lymph node (MLN) and the absolute number of CD25(+) CD4(+) cells in the Peyer's patches and MLN of naive GF mice were significantly lower than those in naive SPF mice. In an in vitro assay, the CD25(+) CD4(+) cells from the naive SPF mice suppressed more effectively the proliferation of responder cells in a dose-dependent manner than those from the GF mice. In addition, the CD25(+) CD4(+) regulatory T (T(reg)) cells from the naive SPF mice produced higher amounts of interleukin (IL)-10 and transforming growth factor (TGF)-beta than those from the GF mice. When anti-TGF-beta neutralizing antibody, but not anti-IL-10 neutralizing antibody, was added to the in vitro proliferation assay, the suppressive effect of the CD25(+) CD4(+) T(reg) cells from the SPF mice was attenuated to the same level as that of the CD25(+) CD4(+) cells from the GF mice. In conclusion, the TGF-beta-producing CD25(+) CD4(+) T(reg) cells from the MLN of SPF mice played a major role in oral tolerance induction. In addition, as the regulatory function of the CD25(+) CD4(+) cells from the naive GF mice was much lower than that of the CD25(+) CD4(+) T(reg) cells from the SPF mice, indigenous microbiota are thus considered to contribute to the induction and maintenance of CD25(+) CD4(+) T(reg) cells.
Assuntos
Vida Livre de Germes , Intestinos/microbiologia , Linfonodos/imunologia , Organismos Livres de Patógenos Específicos , Linfócitos T Reguladores/imunologia , Animais , Proliferação de Células , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Fatores de Transcrição Forkhead/análise , Tolerância Imunológica , Interleucina-10/análise , Interleucina-2/análise , Intestinos/imunologia , Mesentério , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina , Baço/imunologia , Fator de Crescimento Transformador beta/análiseRESUMO
AIMS: CD10+ colorectal carcinomas have a high risk of giving rise to liver metastasis. The aim was to examine phenotypic expression in colorectal neoplasia and to elucidate changes in such expression through the adenoma-carcinoma sequence. METHODS AND RESULTS: We examined the expression of various proteins immunohistochemically in 111 flat [non-polypoid growth (NPG)] colorectal neoplasms, categorized into 28 low-grade (NPG-LGN), 44 high-grades (NPG-HGN) and 39 cases of invasive neoplasia (NPG-IN), as well as in 96 polypoid [polypoid growth (PG)] neoplasms, categorized into 26 PG-LGN, 39 PG-HGN and 31 PG-IN according to the Vienna classification. CD10 was more frequently expressed in NPG than in PG neoplasia. MUC2 and MUC5AC were more frequently expressed in PG than in NPG neoplasias. Nuclear beta-catenin was more frequently expressed in NPG-LGN than in PG-LGN. No difference in p53 expression was found between NPG and PG neoplasia. CONCLUSIONS: From the viewpoint of the expression of CD10 and beta-catenin, it would appear that NPG-LGN differs significantly from PG-LGN, thereby indicating that NPG-LGN is a precursor of CD10+ carcinoma. It is important to ensure that NPG neoplasia is not overlooked if cases of CD10+ carcinoma are to be detected at an early stage.
Assuntos
Adenocarcinoma/patologia , Adenoma/patologia , Pólipos do Colo/patologia , Neoplasias Colorretais/patologia , Neprilisina/metabolismo , beta Catenina/metabolismo , Adenocarcinoma/metabolismo , Adenoma/metabolismo , Idoso , Biomarcadores Tumorais/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Pólipos do Colo/metabolismo , Neoplasias Colorretais/metabolismo , Progressão da Doença , Diagnóstico Precoce , Feminino , Humanos , Imuno-Histoquímica , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Invasividade NeoplásicaRESUMO
A 6-year-old girl was referred to our institute for cardiac evaluation. She had been diagnosed as pulmonary atresia with intact ventricular septum (PAIVS) at 16 days after birth and she had underwent balloon atrial septostomy and bilateral Blalock-Taussig shunts. A cardiac catheterization at 5 months showed that her right ventricular end diastolic volume was 58% of normal, the Z value (standard deviation units) of the diameter of the tricuspid valve was -3.3, and a biventricular repair was performed. After the operation, she suffered from severe congestive heart failure for 10 months. A cardiac catheterization at the age of 6 years demonstrated that the pulmonary blood flow was generated during the diastolic phase like Fontan circulation. Although biventricular repair had been performed at 5 months, the circulation may be less advantageous for long term survival than if the patient had undergone the staged Fontan procedure. Careful and continuous hemodynamic assessment is essential for surgical therapy of PAIVS.
Assuntos
Atresia Pulmonar/cirurgia , Circulação Pulmonar , Criança , Feminino , Técnica de Fontan , Comunicação Interventricular/complicações , Comunicação Interventricular/cirurgia , Ventrículos do Coração/cirurgia , Humanos , Atresia Pulmonar/etiologia , Atresia Pulmonar/fisiopatologiaRESUMO
Tumor suppressor p53 is known to play a crucial role in chemosensitivity in colorectal cancer. We previously demonstrated that an apoptosis-associated speck-like protein, ASC, is a p53-target gene which regulates p53-Bax mitochondrial apoptotic pathway. ASC is also known to be a target of methylation-induced gene silencing. An inactivation of ASC might thus cause resistance to chemotherapy, and if this is the case, then the expression of ASC would restore the chemosensitivity. The aim of this study was to clarify this hypothesis. ASC was methylated in 25% of all resected specimens in patients with colorectal cancer; however, ASC methylation did not always correspond to a lack of ASC protein. When expressed in colon cancer cells, in which ASC is absent due to methylation, ASC was found to enhance the chemosensitivity in a p53-dependent manner. In p53-null cells, ASC increased the p53-mediated cell death induced by p53-expressing adenovirus infection. Our data suggest that the methylation-induced silencing of ASC might cause resistance to p53-mediated chemosensitivity in colorectal cancer. The gene introduction of ASC may thus restore such chemosensitivity, and this modality may therefore be a useful new treatment strategy for colorectal cancer.
Assuntos
Neoplasias Colorretais/genética , Proteínas do Citoesqueleto/genética , Metilação de DNA , Resistencia a Medicamentos Antineoplásicos/genética , Inativação Gênica , Proteína Supressora de Tumor p53/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas Adaptadoras de Sinalização CARD , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Ilhas de CpG/genética , Proteínas do Citoesqueleto/metabolismo , Fluoruracila/farmacologia , Humanos , Reação em Cadeia da PolimeraseRESUMO
Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis is synthesized in a prepro-form (prepro-Tk-subtilisin), secreted in a pro-form (pro-Tk-subtilisin), and matured to an active form (mat-Tk-subtilisin*; a Ca(2+)-bound active form of matured domain of Tk-subtilisin) upon autoprocessing and degradation of the propeptide [Tk-propeptide; propeptide of Tk-subtilisin (Gly1-Leu69)]. Pro-Tk-subtilisin exhibited halo-forming activity only at 80 degrees C, but not at 70 and 60 degrees C, because Tk-propeptide is not effectively degraded by mat-Tk-subtilisin* and forms an inactive complex with mat-Tk-subtilisin* at <80 degrees C. Random mutagenesis in the entire prepro-Tk-subtilisin gene, followed by screening for mutant proteins with halo-forming activity at 70 and 60 degrees C, allowed us to identify single Gly56 --> Ser mutation in the propeptide region responsible for low-temperature adaptation of pro-Tk-subtilisin. SDS-PAGE analyses and mat-Tk-subtilisin* activity assay of pro-G56S-subtilisin indicated more rapid maturation than pro-Tk-subtilisin. The resultant active form was indistinguishable from mat-Tk-subtilisin* in activity and stability, indicating that Gly56 --> Ser mutation does not seriously affect the folding of the mature domain. However, this mutation greatly destabilized the propeptide, making it unstructured in an isolated form. As a result, Tk-propeptide with Gly56 --> Ser mutation (G56S-propeptide) was more susceptible to proteolytic degradation and less effectively inhibited mat-Tk-subtilisin* activity than Tk-propeptide. These results suggest that pro-G56S-subtilisin is more effectively matured than pro-Tk-subtilisin at lower temperatures, because autoprocessed G56S-propeptide is unstructured upon dissociation from mat-Tk-subtilisin* and is therefore effectively degraded by mat-Tk-subtilisin*.
Assuntos
Substituição de Aminoácidos , Evolução Molecular Direcionada , Subtilisina/genética , Thermococcus/enzimologia , Precursores de Proteínas , Subtilisina/fisiologia , Temperatura , Thermococcus/genéticaRESUMO
4 wk after intraperitoneal inoculation of 0.2 LD50 (50% lethal dose) of murine cytomegalovirus (MCMV) in adult BALB/c mice, MCMV remained detectable in the salivary glands, but not in the lungs or other organs. When the T cells of these mice were activated in vivo by a single injection of anti-CD3 monoclonal antibody, interstitial pneumonitis was induced in the lungs that were free of the virus with an excessive production of the cytokines. In the lungs of such mice persistently infected with MCMV, the mRNA of the cytokines such as IL-2, IL-6, TNF-alpha, and IFN-gamma were abundantly expressed 3 h after the anti-CD3 injection, and the elevated levels continued thereafter. A marked expression of inducible nitric oxide synthetase (iNOS) was then noted in the lungs, suggesting that such cytokines as TNF-alpha and IFN-gamma may have induced iNOS. Although the increase in NO formation was demonstrated by the significant elevation of the serum levels of nitrite and nitrate, the interstitial pneumonitis was not associated with either increased superoxide formation or peroxynitrite-induced tyrosine nitration. Nevertheless, the administration of an NO antagonist also alleviated the interstitial pneumonitis provoked by anti-CD3 mAb. Based on these findings, it was concluded that MCMV-associated pneumonitis is mediated by a molecule of cytokine-induced NO other than peroxynitrite.
Assuntos
Infecções por Citomegalovirus/complicações , Doenças Pulmonares Intersticiais/etiologia , Pulmão/metabolismo , Muromegalovirus , Óxido Nítrico/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Líquido da Lavagem Broncoalveolar , Complexo CD3/imunologia , Óxidos N-Cíclicos , Citocinas/biossíntese , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/patologia , Pulmão/patologia , Doenças Pulmonares Intersticiais/induzido quimicamente , Doenças Pulmonares Intersticiais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , NG-Nitroarginina Metil Éster/farmacologia , Nitratos/sangue , Óxido Nítrico Sintase/biossíntese , Nitritos/sangue , Óxidos de Nitrogênio/farmacologia , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Espécies Reativas de Oxigênio , Tirosina/análise , Xantina Desidrogenase/análise , Xantina Oxidase/análiseRESUMO
At 4 wk after intraperitoneal inoculation of murine cytomegalovirus (MCMV) in adult BALB/c mice, MCMV remained detectable only in the salivary glands. When T cells of these mice were activated by a single injection of anti-CD3 epsilon monoclonal antibody, mice died of interstitial pneumonitis at 24-48 h after injection, accompanied by elevation of serum levels of TNF-alpha and IFN-gamma. However, MCMV remained undetectable in the lungs during the period. Simultaneous injection of cyclosporin A reduced such effects of anti-CD3. In conclusion, although the presence of MCMV in the host may be required, MCMV-associated pneumonitis is not mediated by virus in the lung but probably by the cytokines released from T cells, of which responsiveness to stimulation via CD3 molecule has been presumably modified by MCMV infection.
Assuntos
Infecções por Citomegalovirus/patologia , Citomegalovirus/isolamento & purificação , DNA Viral/análise , Pulmão/patologia , Pneumonia Viral/microbiologia , Reação em Cadeia da Polimerase/métodos , Animais , Anticorpos Monoclonais/farmacologia , Sequência de Bases , Complexo CD3/imunologia , Ciclosporina/farmacologia , Citomegalovirus/genética , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/sangue , Infecções por Citomegalovirus/microbiologia , Primers do DNA , Interferon gama/sangue , Pulmão/microbiologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Pneumonia Viral/sangue , Pneumonia Viral/patologia , Glândulas Salivares/microbiologia , Glândulas Salivares/patologia , Baço/patologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Timo/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
To examine the possible involvement of cytokines in reperfusion injury, we have studied production of IL-1 by human vascular cells, including smooth muscle and mononuclear phagocytes. Exposure of cells to hypoxia (pO2 approximately 14 torr) followed by reoxygenation led to significant release of IL-1 only from the mononuclear phagocytes. Elaboration of IL-1 was dependent on the oxygen tension and duration of hypoxia (optimal at lower pO2s, approximately 14-20 torr, and after 9 h), as well as the time in reoxygenation (maximal IL-1 release at 6-9 h). Although a period of hypoxia was necessary for subsequent IL-1 production during reoxygenation of either peripheral blood monocytes or cultured monocyte-derived macrophages, no IL-1 release occurred during the hypoxic exposure. IL-1 released during reoxygenation was newly synthesized, and its production was triggered by the generation of oxygen free radicals, as it could be blocked by the addition of either allopurinol or free radical scavengers to cultures and could be stimulated in part by low concentrations of hydrogen peroxide or xanthine/xanthine oxidase. The potential pathophysiological effects of IL-1-containing supernatants from reoxygenated macrophages was shown by their induction of endothelial tissue factor and enhancement of endothelial adhesiveness for neutrophils, both of which could be blocked by anti-IL-1 antibody. The relevance of IL-1 to hypoxia/reoxygenation in vivo was suggested by the presence of circulating nanogram amounts of this cytokine in the plasma of mice during the reoxygenation period following a hypoxia.
Assuntos
Interleucina-1/biossíntese , Fagócitos/metabolismo , Animais , Sequência de Bases , Hipóxia Celular , Células Cultivadas , Humanos , Hidróxidos , Radical Hidroxila , Interleucina-6/biossíntese , Camundongos , Dados de Sequência Molecular , Monócitos/metabolismo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Cytoplasts from two unrelated patients with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes) harboring an A----G transition at nucleotide position 3243 in the tRNA(Leu(UUR)) gene of the mitochondrial genome were fused with human cells lacking endogenous mitochondrial DNA (mtDNA) (rho 0 cells). Selected cybrid lines, containing less than 15 or greater than or equal to 95% mutated genomes, were examined for differences in genetic, biochemical, and morphological characteristics. Cybrids containing greater than or equal to 95% mutant mtDNA, but not those containing normal mtDNA, exhibited decreases in the rates of synthesis and in the steady-state levels of the mitochondrial translation products. In addition, NADH dehydrogenase subunit 1 (ND 1) exhibited a slightly altered mobility on polyacrylamide gel electrophoresis. The mutation also correlated with a severe respiratory chain deficiency. A small but consistent increase in the steady-state levels of an RNA transcript corresponding to 16S rRNA + tRNA(Leu(UUR)) + ND 1 genes was detected. However, there was no evidence of major errors in processing of the heavy-strand-encoded transcripts or of altered steady-state levels or ratios of mitochondrial rRNAs or mRNAs. These results provide evidence for a direct relationship between the tRNALeu(UUR) mutation and the pathogenesis of this mitochondrial disease.
Assuntos
Encefalopatias Metabólicas/genética , DNA Mitocondrial/genética , Mitocôndrias/metabolismo , Biossíntese de Proteínas , RNA de Transferência de Leucina/genética , Acidose Láctica/genética , Acidose Láctica/metabolismo , Adolescente , Northern Blotting , Encefalopatias Metabólicas/metabolismo , Linhagem Celular , Transtornos Cerebrovasculares/genética , Transtornos Cerebrovasculares/metabolismo , Criança , Feminino , Humanos , Células Híbridas , Imuno-Histoquímica , Mutação/genética , Consumo de Oxigênio , SíndromeRESUMO
Cytoplasts from patients with myoclonus epilepsy with ragged-red fibers harboring a pathogenic point mutation at either nucleotide 8344 or 8356 in the human mitochondrial tRNA(Lys) gene were fused with human cells lacking endogenous mitochondrial DNA (mtDNA). For each mutation, cytoplasmic hybrid (cybrid) cell lines containing 0 or 100% mutated mtDNAs were isolated and their genetic, biochemical, and morphological characteristics were examined. Both mutations resulted in the same biochemical and molecular genetic phenotypes. Specifically, cybrids containing 100% mutated mtDNAs, but not those containing the corresponding wild-type mtDNAs, exhibited severe defects in respiratory chain activity, in the rates of protein synthesis, and in the steady-state levels of mitochondrial translation products. In addition, aberrant mitochondrial translation products were detected with both mutations. No significant alterations were observed in the processing of polycistronic RNA precursor transcripts derived from the region containing the tRNA(Lys) gene. These results demonstrate that two different mtDNA mutations in tRNA(Lys), both associated with the same mitochondrial disorder, result in fundamentally identical defects at the cellular level and strongly suggest that specific protein synthesis abnormalities contribute to the pathogenesis of myoclonus epilepsy with ragged-red fibers.