RESUMEN
Alzheimer's disease is a progressively dementing illness characterized by the extracellular accumulation and deposition of beta-amyloid. Early onset Alzheimer's disease is linked to mutations in three genes, all of which lead to increased beta-amyloid production. Inflammatory changes and gliosis may also play a role in the disease process, but the importance of these reactive events remains unclear. We recently reported that chronic cortical gliosis in heterotopic fetal rat cortical transplants is associated with significant changes in the levels of some of the proteins implicated in the pathogenesis of Alzheimer's disease. Because rodent beta-amyloid does not form extracellular amyloid deposits, we have now extended this model of chronic cortical gliosis to transgenic mice expressing the Swedish mutant form of human amyloid precursor protein. In addition, apolipoprotein E knockout mice were used to elucidate the role of this protein in reactive gliosis. The expression of mutant and murine proteins was assayed 6 or 10 months after transplantation using immunohistochemical and western blot methods. Heterotopic transplantation of fetal cortex onto the midbrain of neonatal mice consistently resulted in reactive gliosis, independent of apolipoprotein E status. In contrast, in homotopic cortex-to-cortex grafts there was little alteration in glial reactivity, a result similar to that obtained previously in rats. By 10 months post-transplantation the level of presenilin-1 expression was lower in heterotopic grafts than in host cortex and there was increased expression of transgenic amyloid precursor protein, but only in the gliotic cortex-to-midbrain grafts. Most importantly, increased levels of beta-amyloid, and particularly its precursor, C-99, were selectively found in these heterotopic transplants. Our results show that chronic gliosis is associated with altered processing of the amyloid precursor protein in vivo and thus may initiate or exacerbate pathological changes associated with Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Gliosis/metabolismo , Procesamiento Proteico-Postraduccional , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/genética , Animales , Animales Recién Nacidos , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Trasplante de Tejido Encefálico/patología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Corteza Cerebral/trasplante , Enfermedad Crónica , Modelos Animales de Enfermedad , Embrión de Mamíferos , Femenino , Trasplante de Tejido Fetal/patología , Gliosis/genética , Gliosis/patología , Humanos , Mesencéfalo/metabolismo , Mesencéfalo/patología , Mesencéfalo/trasplante , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , EmbarazoRESUMEN
The modified fatty acids, (Z,Z,Z)-(octadeca-6,9,12-trienyloxy)acetic acid, (Z,Z,Z)-(octadeca-9,12,15-trienyloxy)acetic acid, (all-Z)-(eicosa-5,8,11,14-tetraenyloxy)acetic acid, (all-Z)-(eicosa-5,8,11,14-tetraenylthio)acetic acid, 3-[(all-Z)-(eicosa-5,8,11,14-tetraenylthio)]propionic acid, (all-Z)-(eicosa-5,8,11,14-tetraenylthio)succinic acid, N-[(all-Z)-(eicosa-5,8,11,14-tetraenoyl)]glycine and N-[(all-Z)-(eicosa-5,8,11,14-tetraenoyl)]aspartic acid, all react with soybean 15-lipoxygenase. The products were treated with triphenylphosphine to give alcohols, which were isolated using HPLC. Analysis of the alcohols using negative ion tandem electrospray mass spectrometry, and by comparison with compounds obtained by autoxidation of arachidonic acid, shows that each enzyme-catalysed oxidation occurs at the omega-6 position of the substrate. In a similar fashion, it has been found that (Z,Z,Z)-(octadeca-6,9,12-trienyloxy)acetic acid, (Z,Z,Z)-(octadeca-9,12,15-trienyloxy)acetic acid, (all-Z)-(eicosa-5,8,11,14-tetraenylthio)acetic acid and 3-[(all-Z)-(eicosa-5,8,11,14-tetraenylthio)]propionic acid each undergoes regioselective oxidation at the carboxyl end of the polyene moiety on treatment with potato 5-lipoxygenase. Neither (all-Z)-(eicosa-5,8,11,14-tetraenylthio)succinic acid nor N-[(all-Z)-(eicosa-5,8,11,14-tetraenoyl)]aspartic acid reacts in the presence of this enzyme, while N-[(all-Z)-(eicosa-5,8,11,14-tetraenoyl)]glycine affords the C11' oxidation product. The alcohol derived from (Z,Z,Z)-(octadeca-6,9,12-trienyloxy)acetic acid using the 15-lipoxygenase reacts at the C6' position with the 5-lipoxygenase.
Asunto(s)
Araquidonato 15-Lipooxigenasa/metabolismo , Araquidonato 5-Lipooxigenasa/metabolismo , Ácidos Grasos Insaturados/metabolismo , Catálisis , Cromatografía Líquida de Alta Presión , Estabilidad de Medicamentos , Ácidos Grasos Insaturados/síntesis química , Oxidación-Reducción , Glycine max/enzimologíaRESUMEN
Poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels have been used in the past as ocular implants. In a recent development, PHEMA sponges have shown suitable properties as materials for the peripheral component of an artificial cornea (keratoprosthesis). However, the propensity of PHEMA to calcify could threaten the long-term stability of the implanted devices. In an attempt to improve the understanding of the calcification mechanism, the dynamics, extent, and nature of calcified deposits within PHEMA sponges implanted in the cornea were investigated in this study, and the possible correlation between necrosis of cells and calcification was critically examined. Samples of a PHEMA sponge were implanted in rabbit corneas and explanted at predetermined time points (2, 4, and 12 weeks). The samples were examined by microscopy (light, transmission, scanning) and energy dispersive analysis of X-rays. Histological assessment and semiquantitative analysis of the amount of calcium deposited was performed using image analysis. An in vitro experiment was also performed by incubating sponge samples for 2 weeks in a solution of calcium and phosphate ions at a ratio similar to that in hydroxyapatite, in the absence of cells. Calcification was not seen in the 2- and 4-week explants, however, small deposits were detected in two of the 12-week explants, both within and on the sponge's constituent polymer particles. The deposit volumes represented 0.094% and 0.21%, respectively, of the total sponge volumes. Calcium deposits were present in large amounts both within the constituent polymer particles and on the surface of the sponges incubated in the abiotic calcifying solution. Cooperative mechanisms are suggested for the calcification of PHEMA sponges in vivo. The initial event may occur at a molecular level, when plasma proteins are adsorbed onto the polymer surface and bound through chelation to the calcium ions present in the medium. After their natural degradation, these structures may act as nucleation sites for calcium phosphate crystallization. Concurrently, the calcium ions can diffuse into the hydrogel particles and then the spontaneous precipitation of calcium phosphate may be caused by supersaturation due to the lower content of water in polymer, an effect which is likely predominant in vitro. The second event is the recruitment of phagocytic cells to clear calcium debris. Degeneration of these cells may then form nucleation sites for secondary calcification.
Asunto(s)
Materiales Biocompatibles , Calcio/metabolismo , Córnea , Hidrogel de Polietilenoglicol-Dimetacrilato/metabolismo , Polihidroxietil Metacrilato , Prótesis e Implantes , Animales , Antraquinonas/farmacología , Colorantes/farmacología , Córnea/patología , Córnea/cirugía , Córnea/ultraestructura , Durapatita/metabolismo , Iones , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Fosfatos/metabolismo , Conejos , Factores de TiempoRESUMEN
The accumulation of tubular aggregates in type II skeletal muscle fibres and fibrillo-granular inclusions in hippocampal protoplasmic astrocytes are characteristic lesions of apolipoprotein E deficient mice. Moreover these inclusions reacted immunocytochemically with an antibody specific to fragment 17-24 of the published sequence of Alzheimer's amyloid peptide. In an effort to evaluate the role of apolipoprotein E in the formation of these abnormal structures, we examined the tibialis anterior muscle and the hippocampus of several groups of animals including: (i) apolipoprotein E "knockout" mice which had been whole body irradiated with 1200 rads and bone marrow replenished with apolipoprotein E sufficient marrow; and (ii) three transgenic murine strains that had been genetically engineered to express either human apolipoprotein E2, E3 or E4 protein on an apoE deficient background. The results of this study showed that the presence of murine apolipoprotein E (even in subnormal levels in the serum) in irradiated bone marrow replenished mice and in all three (E2, E3 or E4) human apoE transgenic strains was sufficient to prevent the aggregation of sarcoplasmic tubules in the tibialis anterior type II muscle fibres. Similarly apolipoprotein E "knockout" bone marrow replenished mice and all three transgenic strains expressing the different human apolipoprotein E alleles reduced the number of the astrocytic inclusions in the hippocampus to levels not significantly different to those observed in control C57Bl6J animals. The data obtained in this study indicate that neurological and neuromuscular abnormalities found in apoE deficient mice are reversed when apoE protein is replaced in the circulation, either by bone marrow transplantation of normal apoE sufficient marrow, or by gene therapy with the apoE gene, albeit of human origin and irrespective of the allele used.
Asunto(s)
Apolipoproteínas E/deficiencia , Astrocitos/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/fisiopatología , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/fisiopatología , Animales , Apolipoproteínas E/genética , Astrocitos/patología , Astrocitos/ultraestructura , Hipocampo/metabolismo , Hipocampo/patología , Hipocampo/fisiopatología , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Microscopía Electrónica , Fibras Musculares Esqueléticas/patología , Fibras Musculares Esqueléticas/ultraestructura , Músculo Esquelético/metabolismo , Músculo Esquelético/patologíaRESUMEN
Burkholderia pseudomallei causes melioidosis, a potentially fatal disease whose clinical outcomes include rapid-onset septicemia and relapsing and delayed-onset infections. Like other facultative intracellular bacterial pathogens, B. pseudomallei is capable of survival in human phagocytic cells, but unlike mycobacteria, Listeria monocytogenes, and Salmonella serovar Typhimurium, the species has not been reported to survive as an endosymbiont in free-living amebae. We investigated the consequences of exposing Acanthamoeba astronyxis, A. castellani, and A. polyphaga to B. pseudomallei NCTC 10276 in a series of coculture experiments. Bacterial endocytosis was observed in all three Acanthamoeba species. A more extensive range of cellular interactions including bacterial adhesion, incorporation into amebic vacuoles, and separation was observed with A. astronyxis in timed coculture experiments. Amebic trophozoites containing motile intravacuolar bacilli were found throughout 72 h of coculture. Confocal microscopy was used to confirm the intracellular location of endamebic B. pseudomallei cells. Transmission electron microscopy of coculture preparations revealed clusters of intact bacilli in membrane-lined vesicles inside the trophozoite cytoplasm; 5 x 10(2) CFU of bacteria per ml were recovered from lysed amebic trophozoites after 60 min of coculture. Demonstration of an interaction between B. pseudomallei and free-living acanthamebae in vitro raises the possibility that a similar interaction in vivo might affect environmental survival of B. pseudomallei and subsequent human exposure. Endamebic passage of B. pseudomallei warrants further investigation as a potential in vitro model of intracellular B. pseudomallei infection.
Asunto(s)
Acanthamoeba/fisiología , Burkholderia pseudomallei/fisiología , Fagocitosis , Animales , Técnicas de Cocultivo , Humanos , Microscopía Confocal , Microscopía ElectrónicaRESUMEN
2-Hydroxy-6-keto-nona-2,4-diene 1,9-dioic acid 5,6-hydrolase (MhpC) from Escherichia coli catalyses the hydrolytic cleavage of the extradiol ring fission product on the phenylpropionate catabolic pathway and is a member of the alpha/beta hydrolase family. The catalytic mechanism of this enzyme has previously been shown to proceed via initial ketonization of the dienol substrate (Henderson, I. M. J., and Bugg, T. D. H. (1997) Biochemistry 36, 12252-12258), followed by stereospecific fragmentation. Despite the implication of an active site serine residue in the alpha/beta hydrolase family, attempts to verify a putative acyl enzyme intermediate by radiochemical trapping methods using a (14)C-labeled substrate yielded a stoichiometry of <1% covalent intermediate, which could be accounted for by nonenzymatic processes. In contrast, incorporation of 5-6% of two atoms of (18)O from H(2)(18)O into succinic acid was observed using the natural substrate, consistent with the reversible formation of a gem-diol intermediate. Furthermore, time-dependent incorporation of (18)O from H(2)(18)O into the carbonyl group of a nonhydrolysable analogue 4-keto-nona-1,9-dioic acid was observed in the presence of MhpC, consistent with enzyme-catalyzed attack of water at the ketone carbonyl. These results favor a catalytic mechanism involving base-catalyzed attack of water, rather than nucleophilic attack of an active site serine. The implication of this work is that the putative active site serine in this enzyme may have an alternative function, for example, as a base.
Asunto(s)
Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , Radioisótopos de Carbono/metabolismo , Catálisis , Escherichia coli/enzimología , Hidrólisis , Cetonas/química , Cetonas/metabolismo , Ácidos Nicotínicos/química , Ácidos Nicotínicos/metabolismo , Isótopos de Oxígeno , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The hippocampal region of apolipoprotein E-deficient mice of varying ages was examined for any morphological changes by light and electron microscopy. Unusual periodic acid-Schiff-positive granules were seen in the hippocampal area of these animals as early as the fourth week of life and their numbers increased gradually with age. These granules were never found in control C57BL/6J (B6) mice before six months-of-age and their numbers were invariable low. They were strongly congophilic when stained with a modified Congo Red technique and reacted with a monoclonal antibody specific to amino acids 17-24 and 35-43 of the beta-amyloid peptide. The immunostaining of these granules with the beta-amyloid peptide was lost after specific adsorption with the appropriate synthetic peptide. These granules were identified ultrastructurally as non-membrane-bound fibrillogranular material in the cytoplasm of protoplasmic astrocytes. The data indicate that an amyloid-like protein accumulates in the protoplasmic astrocytes of the hippocampus of apolipoprotein E-deficient mice, especially in the brains of old animals.
Asunto(s)
Envejecimiento/patología , Apolipoproteínas E/deficiencia , Encéfalo/patología , Péptidos beta-Amiloides/metabolismo , Animales , Anticuerpos Monoclonales , Encéfalo/crecimiento & desarrollo , Colorantes , Rojo Congo , Femenino , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes Neurológicos , Microscopía Electrónica , Degeneración Nerviosa/patologíaRESUMEN
The tibialis anterior muscle and soleus muscle of apolipoprotein-E-deficient mice were examined by light and electron microscopy. By light microscopy, sarcoplasmic inclusions were seen in tibialis anterior muscle and 40% of type 2 myofibers were affected in all animals over 8 months of age. These inclusions reacted for nonspecific esterase, cytochrome oxidase, and myoadenylate deaminase and were also periodic acid Schiff positive and stained basophilic with hematoxylin. Moreover, they reacted immunocytochemically with an antibody specific to fragment 17 to 24 of the published sequence of Alzheimer's cerebrovascular amyloid peptide. Immunoreactivity was lost when the antibody was adsorbed with the appropriate synthetic peptide. Ultrastructurally, the inclusions consisted of tubular arrays and were similar to those observed in human muscle in several pathological conditions. In type 1 myofibers of both tibialis anterior and soleus muscle, however, mitochondrial abnormalities including an increase in their number and size were detected, but tubular aggregates were not seen. These large mitochondria possessed an electron-dense inner chamber with an increased number of tightly packed cristae. The results obtained suggest that in these mice there is a disturbed lipid metabolism in skeletal muscle fibers that manifests itself with an accumulation of phospholipid in the form of sarcoplasmic reticulum tubules in the type 2 fibers and enlarged mitochondria with tightly packed cristae in the type 1 fibers. In addition, beta-amyloid protein was closely associated with the accumulated tubules and vesicles of sarcoplasmic reticulum and may represent dysregulation of amyloid precursor protein metabolism.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Apolipoproteínas E/deficiencia , Cuerpos de Inclusión/metabolismo , Músculo Esquelético/metabolismo , Animales , Femenino , Miembro Posterior , Histocitoquímica , Humanos , Inmunohistoquímica , Cuerpos de Inclusión/ultraestructura , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microscopía Electrónica , Músculo Esquelético/ultraestructuraRESUMEN
Monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) are potent chemokines which attract circulating monocytes and neutrophils respectively to inflamed tissues. JE/MCP-1 gene expression has been previously studied in rabbit aortae after endothelial denudation and the rapid appearance of this transcript was thought to precede emigration of phagocytes. We now report MCP-1 gene expression following de-endothelialization of iliac arteries in the pig, a species which can develop spontaneous atherosclerosis. Using Northern blot analysis, we demonstrated that MCP-1 mRNA was rapidly induced in pig arteries at 2 h and continued to increase to reach a maximum at 8 h before returning to low levels at 16-24 h after injury. The increase seen for MCP-1 mRNA at 8 h was also observed for IL-8 mRNA but was not apparent for growth-related gene expressions, urokinase-type plasminogen activator (u-PA), and plasminogen activator inhibitor-1 (PAI-1). Since smooth muscle cells, endothelial cells, and phagocytes are all capable of expressing MCP-1, we examined pig arteries for immunostaining using a monoclonal antibody to human MCP-1 (5D3-F7). At 8 h after injury, the predominant cell type staining positive for MCP-1 was the monocyte/macrophage. Staining was also observed in occasional scattered neutrophils, but MCP-1 protein could not be detected in smooth muscle cells or on extracellular matrix within the sensitivity constraints posed by our methodology. Our results are consistent with invading monocyte/macrophages having a major input into the production of this chemokine in the arterial wall following injury. The fact that MCP-1 expression accompanied monocyte/macrophage presence in damaged artery, rather than preceding it, is suggestive that continued MCP-1 expression is required for functions other than chemoattraction.
Asunto(s)
Arterias/lesiones , Arterias/metabolismo , Quimiocina CCL2/genética , Macrófagos/fisiología , Monocitos/fisiología , Animales , Cateterismo/efectos adversos , Quimiocina CCL2/biosíntesis , Quimiocina CCL2/inmunología , Endotelio Vascular/lesiones , Endotelio Vascular/fisiología , Regulación de la Expresión Génica , Humanos , Hiperplasia , Arteria Ilíaca/lesiones , Arteria Ilíaca/patología , Arteria Ilíaca/fisiopatología , Interleucina-8/genética , Masculino , Inhibidor 1 de Activador Plasminogénico/genética , ARN Mensajero/biosíntesis , Porcinos , Factores de Tiempo , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
The process of tissue invasion by the yeast Candida albicans after subcutaneous inoculation into the footpad of mice has been examined by light and electron microscopy. The blastospores germinated within 2 hr after injection. Hyphal and, to a lesser extent, pseudohyphal elements were observed penetrating into the adjacent striated muscle fibers and the endothelial cells of capillaries and destroying the collagen bundles of the interstitium. Electron microscopy showed evidence of structural tissue degradation associated with the mycelial elements, particularly at the apical tip. This was presumably due to fungal enzymatic activity. By 3 hr after infection, polymorphonuclear leucocytes had engulfed both the extracellular and intracellular mycelia, and often there was structural evidence of mycelial degradation. The results provide morphological evidence that initial tissue damage following subcutaneous infection is caused by the mycelial growth form.
Asunto(s)
Candida albicans/patogenicidad , Candidiasis/microbiología , Fibras Musculares Esqueléticas/microbiología , Músculo Esquelético/microbiología , Animales , Candida albicans/citología , Candida albicans/crecimiento & desarrollo , Candidiasis/patología , Endocitosis , Femenino , Ratones , Ratones Endogámicos DBA , Fibras Musculares Esqueléticas/patología , Fibras Musculares Esqueléticas/ultraestructura , Músculo Esquelético/patología , Neutrófilos/microbiología , Neutrófilos/ultraestructura , Sarcolema/ultraestructuraRESUMEN
Using novel methodology, this study describes the kinetics of rod outer segment (ROS) phagocytosis and digestion by human retinal pigment epithelial (RPE) cells in vitro and examines the effect of certain lysosomal enzyme inhibitors on ROS digestion in these cells. Human RPE cells displayed saturation of phagocytosis with respect to both ROS concentration and time. While surface-binding and ingestion phases of ROS phagocytosis saturated after 24-36 h, the rate of ROS digestion reached a maximal level within 24 h. Increasing the concentration of zinc in the culture medium from 1.9 to 100 microM had no significant effect on ROS digestion. The effects of swainsonine (an alpha-mannosidase inhibitor), pepstatin (an aspartic protease inhibitor), and leupeptin (a cysteine protease inhibitor) were also examined. At 6 h, ROS digestion was reduced 27.3 +/- 15.3% by swainsonine, 69.4 +/- 20.9% by pepstatin, and 77.0 +/- 14.4% by leupeptin. The effect of these inhibitors declined with increasing time. This study is the first to demonstrate the functional importance of cysteine and aspartic proteases in the digestion of ROS by RPE cells in vitro.
Asunto(s)
Lisosomas/enzimología , Fagocitosis , Epitelio Pigmentado Ocular/metabolismo , Segmento Externo de la Célula en Bastón/metabolismo , Animales , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Bovinos , Membrana Celular/metabolismo , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Humanos , Cinética , Leupeptinas/farmacología , Lisosomas/efectos de los fármacos , Lisosomas/ultraestructura , Manosidasas/antagonistas & inhibidores , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Pepstatinas/farmacología , Epitelio Pigmentado Ocular/citología , Epitelio Pigmentado Ocular/ultraestructura , Swainsonina/farmacología , Zinc/farmacología , alfa-ManosidasaRESUMEN
The results from this investigation suggest that chemotactic factor(s) from damaged myofibers attract polymorphonuclear leukocytes (PML) and macrophages to the site of injury, while exudate macrophages but not PML induce a strong positive chemotactic response in myogenic cells. The AB and BB isoforms of platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta), basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF) (all of these are secreted by macrophages) were also shown to be chemoattractants for muscle precursor cells (MPC). The AA isoform of PDGF did not appear to have any such chemotactic effect on MPC. Macrophages were also shown, under tissue culture conditions, to stimulate the proliferation of MPC. This mitogenic effect was similarly demonstrated for the BB isoform of PDGF, bFGF, and TGF-beta but not for the AA or AB isoforms of PDGF nor for LIF. The results indicate that macrophages play a pivotal role in the orchestration of muscle fiber reconstitution.
Asunto(s)
Quimiotaxis/fisiología , Macrófagos/fisiología , Músculos/fisiología , Regeneración/fisiología , Animales , Bromodesoxiuridina , División Celular/efectos de los fármacos , División Celular/fisiología , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Inmunohistoquímica , Isomerismo , Linfocinas/farmacología , Macrófagos/citología , Macrófagos/ultraestructura , Ratones , Ratones Endogámicos , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Músculos/citología , Músculos/ultraestructura , Neutrófilos/citología , Neutrófilos/fisiología , Neutrófilos/ultraestructura , Factor de Crecimiento Derivado de Plaquetas/farmacología , Células Madre/citología , Células Madre/fisiología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
In regenerating skeletal muscle, sarcoplasmic extensions containing variable numbers of nuclei, widely referred to as 'buds' or 'stumps', are formed at the ends of damaged myofibres. In this paper we investigated whether the nuclei seen in the buds results from fusion of myogenic cells or from migration of myonuclei to the sealed ends of damaged myofibres in murine muscle regenerating after crush injury. The fusion of mononuclear and multinucleate myogenic cells to the buds was demonstrated by transmission electron microscopy. In order to elucidate the frequency and kinetics of cytoplasmic continuity between myotubes and sealed myofibres, we labelled the damaged myofibres with carbocyanine dye DiI (which inserts into the lipid bilayer and travels down continuous membranes) and the samples were then examined by confocal scanning microscopy. This technique showed that there was little fusion between myotubes and myofibres during the first 6 days after crush injury, but significant fusion had occurred by the tenth day especially at the newly sealed region of the damaged myofibre. A scheme for the repair of damaged skeletal muscle is presented.
Asunto(s)
Músculos/fisiología , Animales , Carbocianinas , Membrana Celular/ultraestructura , Histocitoquímica , Membrana Dobles de Lípidos , Ratones , Microscopía Electrónica , Músculos/citología , Músculos/ultraestructura , Regeneración/fisiología , Sarcolema/ultraestructuraRESUMEN
To investigate the role of proliferating local and emigrating circulatory leucocytes in skeletal muscle regeneration in mice, their bone marrow was ablated with whole body irradiation and compared with the effects of local irradiation. The results indicate that (1) the sealing of damaged myofibres is a function of local cells and is not dependent on the presence of infiltrating leucocytes; (2) the formation of sarcoplasmic projections at the ends of damaged myofibres is dependent on leucocyte infiltration; (3) nuclei in the sarcoplasmic projections are probably derived from fusion of muscle precursor cells; (4) most muscle precursor cells in vivo replicate at least once before fusion; and (5) both replication and fusion of muscle precursors can occur in the absence of infiltrating leucocytes. These results are discussed with respect to the interaction of various cell populations during regeneration of skeletal muscle, and are of clinical significance to pathological changes seen in many myopathies.
Asunto(s)
Músculos/fisiología , Regeneración/fisiología , Animales , Citoplasma/fisiología , Citoplasma/ultraestructura , Femenino , Leucocitos/fisiología , Ratones , Ratones Endogámicos , Microscopía Electrónica , Músculos/citología , Músculos/lesiones , Músculos/efectos de la radiación , Músculos/ultraestructura , Enfermedades Musculares/patología , Retículo Sarcoplasmático/fisiología , Irradiación Corporal TotalRESUMEN
In this report we demonstrate for the first time that differentiating myogenic cells, geographically located between the plasmalemma and external lamina of myofibres in the satellite cell position, can fuse directly with the plasmalemma of undamaged segments of mature myofibres.
Asunto(s)
Fusión de Membrana , Músculos/fisiología , Miofibrillas/fisiología , Regeneración/fisiología , Animales , Citoplasma/fisiología , Citoplasma/ultraestructura , Femenino , Ratones , Ratones Endogámicos , Músculos/ultraestructura , Miofibrillas/ultraestructuraRESUMEN
During short term culture of murine resident peritoneal macrophages, increasing the temperature from 37 to 39 degrees C resulted in an increased activity of several surface receptors (FcR and receptor for gluteraldehyde-fixed sheep red blood cells), enhanced phagocytosis of yeast particles, improved spreading, and an accelerated reduction of nitroblue tetrazolium. At 41 degrees C, however, significant reduction of several functional properties (endocytosis of colloidal gold and horseradish peroxidase, phagocytosis of yeast particles) and a decrease in the reduction of nitroblue tetrazolium, the incorporation of tritiated uridine, and Fc and C3b surface receptor activity were observed. In addition morphological evidence of apoptosis, observed in a small number of cells cultured at 39 degrees C and in the majority of macrophages maintained at 41 degrees C, was confirmed by DNA electrophoresis. The data indicates that a reduction of several functional activities of macrophages occurs at 41 degrees C and apoptosis may largely account for these effects.
Asunto(s)
Fiebre/fisiopatología , Macrófagos/patología , Macrófagos/fisiología , Cavidad Peritoneal/citología , Animales , Movimiento Celular/fisiología , Células Cultivadas , ADN/metabolismo , Endocitosis/fisiología , Endonucleasas/metabolismo , Macrófagos/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Nitroazul de Tetrazolio/metabolismo , Fagocitosis/fisiología , Receptores de Complemento/fisiología , Receptores de Complemento 3b , Receptores Fc/fisiología , Timidina/metabolismo , Uridina/metabolismoRESUMEN
Fusion of myogenic cells in adult murine skeletal muscle regenerating in vivo was examined at the ultrastructural level. Fusion of myoblast to myoblast, myoblast to myotube, and myotube to myotube was observed by 4 to 5 days after injury. Fusion between myogenic cells (myoblasts or myotubes) lacking a definitive glycocalyx or external lamina (basal lamina) occurred at multiple sites. It was defined by zones of cytoplasmic confluence between apposed cells at sites where contiguous segments of the cell membranes were interrupted while their edges had united resulting in linear continuity; vesicles of varying dimensions were frequent in these areas of fusion. Myoblasts were seen invaginating the surface of myofibres and again vesicles were seen in abundance in such regions. Cilia were often observed at this junctional zone suggesting that they might play a role in fusion. In the one example of probable fusion between a myotube and a myofibre, only a single area of cytoplasmic continuity was apparent.
Asunto(s)
Músculos/fisiología , Regeneración/fisiología , Animales , Fusión Celular , Femenino , Ratones , Ratones Endogámicos , Músculos/ultraestructura , Células Madre/fisiología , Células Madre/ultraestructura , Cicatrización de HeridasRESUMEN
Young adult mice infected with MCMV were shown to develop inflammatory lesions in the peripancreatic and salivary gland adipose tissues. MCMV replication was detected by immunoperoxidase staining and electron microscopy in adipocytes, fibroblasts, endothelial cells and pericytes in brown and white adipose tissues. More infected cells were detected in C3H mice than in BALB/c, BALB.B, BALB.K or C57BL/6 mice. Peripancreatic steatitis consisted of a monocytic infiltrate surrounding focal necrosis of adipocytes, the severity of which was influenced by the route of inoculation, virus dose, and genetic susceptibility to disseminated MCMV-disease. C57BL/6 mice showed the greatest susceptibility with severe coalescing focal inflammation around areas of coagulative necrosis. Salivary gland adipose tissues exhibited lymphocytic steatitis, which was reduced in Nu/Nu mice.
Asunto(s)
Infecciones por Citomegalovirus/complicaciones , Esteatitis/etiología , Tejido Adiposo/ultraestructura , Animales , Infecciones por Citomegalovirus/patología , Ratones , Ratones Endogámicos , Microscopía Electrónica , Páncreas/ultraestructura , Pancreatitis/etiología , Pancreatitis/patología , Glándulas Salivales , Esteatitis/genéticaRESUMEN
The major ultrastructural events in murine skeletal muscle fibers were examined 3 to 24 hr after segmental injury induced by painting with aldehyde fixative. At 3 hr the viable stump of injured myofibers was separated from the necrotic segment by a zone of supercontracted myofibrils. No demarcating membrane was evident at this time, although occasionally collapsed segments of plasmalemma partially covered the viable stump. By 12 hr after injury myonuclei near the viable stump were centrally placed and numerous whorls of membrane material appeared in the vicinity of the Golgi apparatus. At this stage a convoluted, tortuous membrane exhibiting extensive interdigitations has sealed the structurally normal part of the injured fiber. The myoplasm immediately within this demarcating membrane possessed few myofilaments but numerous vesicles and tubules, several of which were continuous with the demarcating membrane; most degraded sarcoplasmic organelles remained external to the demarcating membrane and leukocytes were observed internalizing the debris. It appears that after segmental injury to skeletal muscle fibers, active production of new sarcoplasmic membranes occurs, which contributes to the formation of the part of the plasmalemma that demarcates the viable portion of the muscle fiber from the injured area.
Asunto(s)
Músculos/lesiones , Sarcolema/fisiología , Animales , Femenino , Fijadores , Aparato de Golgi/ultraestructura , Leucocitos/ultraestructura , Ratones , Ratones Endogámicos , Microscopía Electrónica , NecrosisRESUMEN
Mast cells purified from the peritoneal cell population and mast cells derived in culture from bone marrow cells were examined for their sensitivity to murine cytomegalovirus (MCMV) infection in vitro. While up to 70% of mast cells expressed viral antigens, less than 12% of the cells produced infectious virus. Transmission electron microscopy demonstrated nucleocapsids in the nuclei and in association with the cisternal elements of the Golgi apparatus. Some complete virions were found within small cytoplasmic vacuoles. In contrast with previous studies of macrophages and fibroblasts, the susceptibility of mast cells to MCMV infection in vitro was not influenced by the H-2 or non-H-2 genotype of the donor.