RESUMO
Mitochondrial dysfunction in skeletal muscle has been implicated in the development of type 2 diabetes. However, whether these changes are a cause or a consequence of insulin resistance is not clear. We investigated the structure and function of muscle mitochondria during the development of insulin resistance and progression to diabetes in mice fed a high-fat, high-sucrose diet. Although 1 month of high-fat, high-sucrose diet feeding was sufficient to induce glucose intolerance, mice showed no evidence of mitochondrial dysfunction at this stage. However, an extended diet intervention induced a diabetic state in which we observed altered mitochondrial biogenesis, structure, and function in muscle tissue. We assessed the role of oxidative stress in the development of these mitochondrial abnormalities and found that diet-induced diabetic mice had an increase in ROS production in skeletal muscle. In addition, ROS production was associated with mitochondrial alterations in the muscle of hyperglycemic streptozotocin-treated mice, and normalization of glycemia or antioxidant treatment decreased muscle ROS production and restored mitochondrial integrity. Glucose- or lipid-induced ROS production resulted in mitochondrial alterations in muscle cells in vitro, and these effects were blocked by antioxidant treatment. These data suggest that mitochondrial alterations do not precede the onset of insulin resistance and result from increased ROS production in muscle in diet-induced diabetic mice.
Assuntos
Diabetes Mellitus Tipo 2/complicações , Mitocôndrias Musculares/metabolismo , Doenças Mitocondriais/etiologia , Músculo Esquelético/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Animais , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Dieta , Gorduras na Dieta/administração & dosagem , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Musculares/ultraestrutura , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/ultraestrutura , Espécies Reativas de Oxigênio/análise , Sacarose/administração & dosagemRESUMO
Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by Staphylococcus aureus that has recently been associated with necrotizing pneumonia. In the present study, we report that in vitro, PVL induces polymorphonuclear cell death by necrosis or by apoptosis, depending on the PVL concentration. PVL-induced apoptosis was associated with a rapid disruption of mitochondrial homeostasis and activation of caspase-9 and caspase-3, suggesting that PVL-induced apoptosis is preferentially mediated by the mitochondrial pathway. Polymorphonuclear cell exposure to PVL leads to mitochondrial localization of the toxin, whereas Bax, 1 of the 2 essential proapoptotic members of the Bcl-2 family, was still localized in the cytosol. Addition of PVL to isolated mitochondria induced the release of the apoptogenic proteins cytochrome c and Smac/DIABLO. Therefore, we suggest that PVL, which belongs to the pore-forming toxin family, could act at the mitochondrion level by creating pores in the mitochondrial outer membrane. Furthermore, LukS-PV, 1 of the 2 components of PVL, was detected in lung sections of patients with necrotizing pneumonia together with DNA fragmentation, suggesting that PVL induces apoptosis in vivo and thereby is directly involved in the pathophysiology of necrotizing pneumonia.
Assuntos
Apoptose/efeitos dos fármacos , Leucocidinas/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Proteína X Associada a bcl-2/fisiologia , Proteínas Reguladoras de Apoptose , Toxinas Bacterianas , Membrana Celular/fisiologia , Células Cultivadas , Citocromos c/metabolismo , Exotoxinas , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinética , Pulmão/microbiologia , Pulmão/patologia , Mitocôndrias/fisiologia , Proteínas Mitocondriais/metabolismo , Necrose , Neutrófilos/microbiologia , Pneumonia Estafilocócica/microbiologia , Pneumonia Estafilocócica/patologia , Staphylococcus aureus/fisiologiaRESUMO
Microdissected rat proximal straight tubules (PST) and inner medullary collecting ducts (IMCD) highly produce urea from l-arginine, supporting the expression of the mitochondrial arginase II. However, IMCD contain a very low density of mitochondria compared with PST. Recently, arginase II has been localized by immunohistochemistry in rat PST but not IMCD. This study was designed to verify whether rat IMCD express arginase II and to identify its subcellular localization. We developed an antibody raised against arginase II that allowed the detection of a band of 38 kDa corresponding to arginase II on immunoblots. In male and female rat kidneys, Western blot analyses revealed that arginase II was highly expressed in the inner medulla (IM), the outer stripe of the outer medulla (osOM), and the deep cortex. Immunocytochemistry demonstrated that arginase II was homogeneously expressed in IMCD. Proteins of the cytosolic and mitochondrial fractions extracted from osOM and IM and analyzed by Western blot showed that 86% of arginase II was associated with mitochondria. The molecular weight of arginase II was similar in the cytosolic and mitochondrial fractions. Immunoelectron microscopy confirmed the presence of arginase II in the mitochondria of IMCD. In conclusion, arginase II is expressed in mitochondria of male and female rat IMCD.
Assuntos
Arginase/biossíntese , Medula Renal/enzimologia , Túbulos Renais Coletores/enzimologia , Animais , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Immunoblotting , Masculino , Microscopia Imunoeletrônica , Ratos , Ratos Sprague-DawleyRESUMO
Elastic fiber formation involves the secretion of tropoelastin which is converted to insoluble elastin by cross-linking, initiated by the oxidative deamination of lysine residues by lysyl oxidase. Five lysyl oxidase genes have been discovered. This study deals with the expression of two isoforms, LOX and LOX-like (LOXL), in human foreskin and in a human skin-equivalent (SE) model that allows the formation of elastic fibers. In this model, keratinocytes are added to a dermal equivalent made of fibroblasts grown on a chitosan-cross-linked collagen-GAG matrix. LOX and LOXL were detected by immunohistochemistry in the dermis and the epidermis of both normal skin and in a SE. This expression was confirmed by in situ hybridization on the SE. LOX and LOXL expression patterns were confirmed in human skin. The ultrastructural localization of LOXL was indicative of its association with elastin-positive materials within the SE and human skin, though interaction with collagen could not be discarded. LOX was found on collagen fibers and could be associated with elastin-positive materials in the SE and human skin. LOXL and LOX were detected in keratinocytes where LOX was mainly expressed by differentiating keratinocytes, in contrast to LOXL that can be found in both proliferating and differentiating fibroblasts. These data favor a role for LOXL in elastic fiber formation, together with LOX, and within the epidermis where both enzymes should play a role in post-translational modification of yet unknown substrates.
Assuntos
Aminoácido Oxirredutases/análise , Derme/enzimologia , Tecido Elástico/fisiologia , Epiderme/enzimologia , Proteína-Lisina 6-Oxidase/análise , Pele/enzimologia , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/fisiologia , Células Cultivadas , Colágeno/biossíntese , Humanos , Imuno-Histoquímica , Queratinócitos/enzimologia , Microscopia Imunoeletrônica , Proteína-Lisina 6-Oxidase/genética , Proteína-Lisina 6-Oxidase/fisiologiaRESUMO
Replicative senescence appears after a finite number of cell divisions. After proliferation has ceased, senescent cells remain viable for long periods and metabolic modifications are observed such as lipofuscin accumulation. In order to understand this phenomenon, we examined the emergence of subcellular modifications corresponding to autophagy in MRC5 normal human fibroblasts. An increase of monodansylcadaverine fluorescence, a specific marker of autophagy, in aging compared to young fibroblasts was observed (p<0.0001). The increase of autophagic vacuoles in aging fibroblasts was confirmed by electron microscopy. We compared young versus senescent fibroblasts and showed that autophagic vacuoles, already present in young cells, became larger in senescent fibroblasts with a significant relative increase of inclusion area with respect to measured cell area (p=0.0041). However, autophagy-associated-gene expression remained stable in senescent compared to young fibroblasts, suggesting that the autophagy process per se is not enhanced. In parallel, transmission electron microscopy analysis showed that beta-galactosidase activity distribution was modified by aging: beta-galactosidase (an enzyme linked to lysosome) was scattered in young fibroblasts, but clustered at the level of autophagic vacuoles in senescent fibroblasts, suggesting a predominance of autolysosomes at this stage. These results support the hypothesis that, during fibroblast aging, the increase of autophagic vacuoles, as well as that of beta-galactosidase activity, may be associated to an increase of lysosomal mass and to an accumulation of degradative autolysosomes with lipofuscin. This phenomenon could be involved in the death of senescent fibroblasts.
Assuntos
Autofagia/fisiologia , Cadaverina/análogos & derivados , Senescência Celular/fisiologia , Fibroblastos/fisiologia , Corpos de Inclusão/ultraestrutura , beta-Galactosidase/análise , Biomarcadores/análise , Cadaverina/análise , Células Cultivadas , Fibroblastos/química , Humanos , Lipofuscina/análise , Microscopia Eletrônica , Microscopia de FluorescênciaRESUMO
As autophagic inclusions accumulate in senescent fibroblasts, we wondered whether an increase in cellular fragility during in vitro lymphocyte aging may be related to an autolysosome accumulation. We established that, during long-term cultures, repeatedly stimulated T-lymphocytes acquired characteristics of replicative senescence and became progressively intolerant to activation. Cell death following stimulations: (i) corresponded to apoptosis, associated with necrosis at the end of the culture; (ii) was not, for its main part, mediated through CD95/CD178 or TNFRII/TNF alpha interactions; and (iii) occurred in spite of bcl-2 increased expression. After 14 weeks of culture, the percentage of lymphocytes containing at least one autophagic inclusion (p<0.0001) and the lipofuscin autofluorescence in lymphocytes (p<0.0001) were significantly increased. The expression of several genes regulating autophagy did not significantly vary with the age of the culture. Forty-eight hours after each stimulation, the percentage of induced cell death rose while, in the remaining living cells, the percentage of lymphocytes with autophagic vacuoles (p<0.05), with beta-galactosidase activity and the lipofuscin autofluorescence (p<0.001) significantly decreased, suggesting the preferential death of cells with autophagy. Our data support the view that the accumulation of autolysosomes in senescent lymphocytes might aggravate cellular fragility, leading to apoptosis and necrosis mainly induced by lymphocyte activation.
Assuntos
Linfócitos T CD8-Positivos/fisiologia , Morte Celular/fisiologia , Senescência Celular/fisiologia , Lisossomos/fisiologia , Apoptose/fisiologia , Autofagia/fisiologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD4-Positivos/ultraestrutura , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/ultraestrutura , Morte Celular/efeitos dos fármacos , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Expressão Gênica/fisiologia , Humanos , Lipofuscina/biossíntese , Microscopia Eletrônica/métodos , Necrose , Fito-Hemaglutininas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/análise , Fator de Necrose Tumoral alfa/fisiologia , Vacúolos/fisiologia , Receptor fas/fisiologiaRESUMO
Vascular Ehlers-Danlos syndrome (vEDS) results from a mutation in the gene encoding alpha-1, type III pro-collagen (COL3A1) and confers fragility to skin, ligament and vascular tissue. We tested the value of skin biopsy for diagnosis of vEDS through an ultrastructure scoring procedure. Study design was a multicentric, case-control, blinded trial consisting of two phases: phase 1 was to identify an ultra-structure score providing the best discriminative value for vEDS and phase 2 was to replicate this result in a different population. We enrolled 103 patients, 66 cases defined through the revised nosology for Ehlers-Danlos syndromes and 37 control subjects selected from patients referred for other pathologies. Ultrastructure of extracellular matrix was read by three to five experienced pathologists blinded for diagnosis. We used the receiver operating curves and logistic regression analysis for ranking ultrastructure scores. We created a detailed description of lesions observed in vEDS patients with 27 items (coded 0 or 1). In the phase 1 (17 cases and 20 controls), abnormal fibroblast shape, presence of lysosomes in the fibroblast and abnormal basal lamina were found to be independent discriminative items. Addition of these three items (defining an ultrastructure score) had the best diagnosis value (area under the curve (AUC) = 0.96). In the phase 2 (49 cases, 17 controls), ultrastructure score provided odds ratio of 9.76 (95 % CI 2.91-32.78), and AUC of 0.90. The ultrastructure score of skin biopsy has predictive value for the diagnosis of vEDS. Presence of two or more signs (either abnormal fibroblast, presence of lysosomes in the fibroblast or abnormal basal lamina) is very evocative of vEDS.
Assuntos
Colágeno Tipo III/genética , Síndrome de Ehlers-Danlos/diagnóstico , Pele/ultraestrutura , Biópsia , Colágeno Tipo III/ultraestrutura , Síndrome de Ehlers-Danlos/genética , Síndrome de Ehlers-Danlos/patologia , HumanosRESUMO
Cutis laxa (CL) is a rare genodermatosis, which is clinically and genetically heterogeneous. It is characterized by redundant, loose, sagging, and inelastic skin. In a consanguineous family from Lebanon with autosomal-recessive transmission, we identified a homozygous missense mutation (c.649T --> C; p.C217R) in the fibulin-5 gene (FBLN5), which was, to our knowledge, previously unreported. Small skin biopsies were performed, which permitted isolation of skin fibroblasts harboring this FBLN5 mutation; they exhibited a deficit in cell growth. A CL skin equivalent (CL-SE) model compared with control SE was successfully developed to define the behavior of CL fibroblasts in a three-dimensional model. There was increased cell death and a global extracellular matrix deficiency in the dermis of this CL-SE model, and a low level of the main elastic fiber expression. There was no basement membrane evident at the ultrastructural level, and type-VII collagen could not be detected at the histological level. This model reproduced some defects of the extracellular matrix and highlighted other defects, which occurred at the time of the basement membrane formation, which were not evident in skin from patients. This CL-SE model could be adapted to screen for therapeutically active molecules.
Assuntos
Cútis Laxa/genética , Proteínas da Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Mutação , Pele/patologia , Membrana Basal/metabolismo , Colágeno Tipo VII/metabolismo , Cútis Laxa/patologia , Análise Mutacional de DNA , Feminino , Fibroblastos/metabolismo , Homozigoto , Humanos , Masculino , Modelos Biológicos , Mutação de Sentido IncorretoRESUMO
Arginase II (AII) has been almost exclusively studied in male mammalian kidneys. Our investigations were conducted to localize AII gene expression in the female mouse kidney, and to analyze the differential expression of AII gene at the transcriptional and translational levels in the kidneys of female and male mice. Total RNAs and soluble proteins extracted from renal zones and whole kidneys were analyzed by Northern and Western blots, respectively. Mitochondrial and cytosolic proteins were analyzed by Western blot. L-[guanidino-14C]arginine hydrolysis by AII was detected in microdissected tubules and the 14CO2 released from [14C]urea hydrolysis was quantified. The results of these experiments showed that: (1) both AII mRNA and protein were highly expressed in the deep cortex and the outer stripe of the outer medulla, (2) urea was produced mainly in the proximal straight tubules (PST), (3) the 38-kDa AII protein was more abundant in the mitochondria than the cytosol, and (4) the renal content of AII mRNA and protein was about three-fold higher in female than in male mice. In conclusion, in both genders, AII gene expression is restricted to the PST and localized into mitochondria. AII gene is differentially expressed in the kidney of female and male mice since higher levels of AII mRNA, protein and activity were observed in the kidneys of the former than those of the latter. Renal AII gene expression was gender-dependent in mice but not in rats. Finally, in the PST of females, L-arginine-derived ornithine may be a precursor for the renal production of L -glutamate and L-glutamine because high levels of AII, ornithine aminotransferase and glutamine synthetase are expressed in this nephron segment.
Assuntos
Arginase/genética , Arginase/metabolismo , Regulação Enzimológica da Expressão Gênica , Túbulos Renais/enzimologia , Caracteres Sexuais , Animais , Arginina/farmacocinética , Radioisótopos de Carbono , Citosol/enzimologia , Feminino , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Ornitina/metabolismo , Putrescina/metabolismo , RNA Mensageiro/análise , Ureia/metabolismoRESUMO
Purine nucleoside phosphorylase (PNP, EC 2.4.2.1) activity was revealed by enzyme histochemistry in Toxoplasma gondii ME49 strain isolated from murine cerebral cysts and from in vitro cultivation. The activity of the enzyme was revealed by an insoluble electron-opaque precipitate of lead phosphate at the site of the reaction. In bradyzoites and tachyzoites of T. gondii, the enzyme activity could be observed only in the cytoplasm. In bradyzoites, one or two foci of important PNP activity were detected near the nucleus. In tachyzoites, an important PNP activity underlined the plasma membrane. For both bradyzoites and tachyzoites, localization neither in the nucleus nor in cytoplasmic organelles could be detected.
Assuntos
Purina-Núcleosídeo Fosforilase/metabolismo , Purina-Núcleosídeo Fosforilase/ultraestrutura , Toxoplasma/enzimologia , Animais , Transporte Biológico , Encéfalo/ultraestrutura , Linhagem Celular Tumoral , Membrana Celular/ultraestrutura , Núcleo Celular/ultraestrutura , Cistos/ultraestrutura , Citoplasma/ultraestrutura , Camundongos , Microscopia Eletrônica , Purina-Núcleosídeo Fosforilase/análiseRESUMO
HCV particles were isolated from the plasma of chronically infected patients. The virus was analysed by sucrose density gradient centrifugation. The fractions were tested for viral RNA, core antigen and envelope proteins by using a monoclonal antibody directed against the natural E1E2 complex (D32.10). Two populations of particles containing RNA plus core antigen were separated: the first with a density of 1.06-1.08 g/ml did not contain the envelope proteins; the second with a density between 1.17 and 1.21 g/ml expressed both E1 and E2 glycoproteins. Electron microscopy of the enveloped population after immunoprecipitation with D32.10 showed spherical particles with a rather featureless surface and with a diameter around 40 nm. Immuno-gold staining gave evidence that the E1E2 complex was indeed positioned at the surface of these particles.
Assuntos
Hepacivirus/isolamento & purificação , Hepatite C Crônica/virologia , Proteínas do Envelope Viral/análise , Viremia/virologia , Centrifugação com Gradiente de Concentração , Hepacivirus/química , Humanos , Imunoprecipitação , Microscopia Imunoeletrônica , RNA Viral/análise , Proteínas do Core Viral/análiseRESUMO
An antiserum was generated from synthetic peptides highly conserved between different mammalian species to immunolocalise the small leucine-rich proteoglycan osteoadherin (OSAD) in murine teeth. In 19-day-old embryos of rats and mice, a positive staining was found in incisor predentin and alveolar bone surrounding developing incisors and molars. In newborns, OSAD was detected at the tip of the first molar cusp where it accumulated in predentin concomitantly with odontoblast differentiation. In 2-day-old rats and mice, in the first molar, immunostaining revealed positive predentin, enamel matrix close to the apical pole of ameloblasts and a strong signal in dentin. At this stage, OSAD was detected in predentin in the second molar. Ultrastructural immunocytochemistry showed gold particles associated with collagen fibres in predentin and in foci at the dentin mineralisation front. Gold particles were also detected near the secretory pole of ameloblasts where enamel crystallites elongate. No staining was detected in pulp tissue and dental follicle. Restriction of OSAD expression to the extracellular matrix of bone, dentin and enamel suggests a role of this proteoglycan in the organisation of mineralised tissues.
Assuntos
Proteínas da Matriz Extracelular/análise , Matriz Extracelular/química , Proteoglicanas/análise , Dente/química , Animais , Animais Recém-Nascidos , Anticorpos/metabolismo , Especificidade de Anticorpos , Matriz Extracelular/ultraestrutura , Imuno-Histoquímica , Camundongos , Microscopia Eletrônica de Varredura , Dente Molar/química , Dente Molar/embriologia , Dente Molar/ultraestrutura , Ratos , Ratos Sprague-Dawley , Dente/embriologia , Dente/ultraestrutura , Germe de Dente/química , Germe de Dente/embriologia , Germe de Dente/ultraestruturaRESUMO
In the kidney, L-ornithine is reabsorbed along the proximal convoluted tubule (PCT), transported by basolateral carriers, and produced by arginase II (AII). Here, the renal metabolic fate of L-ornithine was analyzed in male and female rats. Kidneys and renal zones were dissected and used for Western blot analysis, immunofluorescence, and electron microscopic studies. Ornithine aminotransferase (OAT) and AII were localized using specific antibodies. Ornithine oxidation was determined by incubating microdissected tubules with L-[1-14C] or L-[U-14C]ornithine in the presence or absence of energy-providing substrates. Ornithine decarboxylase (ODC) mRNAs were localized by in situ hybridization. The 48-kDa OAT protein was detected in male and female kidneys, but its level was fourfold higher in the latter. OAT relative distribution increased from the superficial cortex toward the outer medulla to reach its highest level. Almost all OAT protein was localized in cortical and medullary proximal straight tubules (CPST and OSPST, respectively). In proximal straight tubule (PST), AII protein distribution overlapped that of OAT. No gender difference in AII protein level was found. OAT and AII were colocalized within PST mitochondria. L-[1-14C]ornithine decarboxylation occurred in all tubules, but predominantly in proximal tubules. L-[1-14C]ornithine decarboxylation was enhanced when L-[1-14C]ornithine was given to tubules as the sole substrate. The use of L-[U-14C]ornithine demonstrated the complete oxidation of ornithine. In conclusion, the OAT gene was expressed more in female rat proximal tubules than in male. Because OAT and AII proteins overlapped in PST mitochondria, L-arginine-derived ornithine may be preferentially converted to L-glutamate, as proven by ornithine oxidation. However, the coexpression of ODC, glutamate decarboxylase, and glutamine synthetase in PST suggests that L-ornithine can also be metabolized to putrescine, GABA, and L-glutamine. The fate of L-ornithine may depend on the cellular context.
Assuntos
Arginase/metabolismo , Rim/enzimologia , Mitocôndrias/enzimologia , Ornitina-Oxo-Ácido Transaminase/metabolismo , Ornitina/metabolismo , Caracteres Sexuais , Animais , Arginina/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Rim/ultraestrutura , Masculino , Microscopia Eletrônica , Néfrons/enzimologia , Néfrons/ultraestrutura , Ornitina-Oxo-Ácido Transaminase/genética , Oxirredução , RNA Mensageiro/análise , Ratos , Ratos Sprague-DawleyRESUMO
Panton Valentine leukocidin (PVL) may be responsible for pulmonary necrosis in necrotizing Staphylococcus aureus pneumonia, a highly lethal infection. Commercial intravenous immunoglobulin (IVIg) preparations containing antibodies against PVL might have therapeutic value in this setting, as an adjunct to antimicrobial chemotherapy. To test this possibility, we determined anti-PVL antibody titers in commercial IVIg and the capacity of IVIg to prevent the cytopathic effects of PVL in vitro. Specific enzyme-linked immunosorbent assays based on purified recombinant PVL (rPVL) showed that IVIg contained specific anti-PVL antibodies. The cytotoxicity of rPVL and of crude culture supernatants of PVL-producing S. aureus strains were investigated by measuring ethidium-bromide incorporation by polymorphonuclear neutrophils (PMNs) in flow cytometric assays, as well as PMN ultrastructural changes by transmission electron microscopy. IVIg was found to neutralize pore formation and the cytopathic effect of both rPVL and S. aureus culture supernatants.