RESUMO
Over the last 30 years there has been considerable interest in the use of functional electrical stimulation (FES) to restore movement to the limbs of paralyzed patients. Spinal cord injury causes a rapid loss in both muscle mass and contractile force. The atrophy is especially severe when the injury involves lower motoneurons because many months after spinal cord injury, atrophy is complicated by fibrosis and fat substitution. In this study we describe the effects of long-term lower motoneuron denervation of human muscle and present the structural results of muscle trained using FES. By means of an antibody for embryonic myosin, we demonstrate that many regenerative events continue to spontaneously occur in human long-term denervated and degenerated muscle (DDM). In addition, using electron microscopy, we describe i) the overall structure of fibers and myofibrils in long-term denervated and degenerated muscle, including the effects of FES, and ii) the structure and localization of calcium release units, or triads; the structures reputed to activate muscle contraction during excitation-contraction coupling (ECC). Both apparatus undergo disarrangement and re-organization following long-term denervation and FES, respectively. The poor excitability of human long-term DDM fibers, which extends to the first periods of FES training, may be explained in terms of the spatial disorder of the ECC apparatus. Its disorganization and re-organization following long-term denervation and FES, respectively, may play a key role in the parallel disarrangement and re-organization of the myofibrils that characterize denervation and FES training. The present structural studies demonstrate that the protocol used during FES training is effective in reverting long-term denervation atrophy and dystrophy. The mean fiber diameter in FES biopsies is 42.2 +/- 14.8 SD (p < 0.0001 vs DDM 14.9 +/- 6.0 SD); the mean percentile of myofiber area of the biopsy is 94.3 +/- 5.7 SD (p < 0.0001 vs DDM 25.7 +/- 23.7 SD); the mean percentile fat area is 2.1 +/- 2.4 SD (p < 0.001 vs DDM 12.8 +/- 12.1 SD); and the mean percentile connective tissue area is 3.6 +/- 4.6 SD (p < 0.001 vs DDM 61.6 +/- 20.1 SD). In DDM biopsies more than 50% of myofibers have diameter smaller than 10 microm, while the FES-trained subjects have more that 50% of myofibers larger than 30 microm. The recovery of muscle mass seems to be the result of both a size increase of the surviving fibers and the regeneration of new myofibers.
Assuntos
Contração Muscular/fisiologia , Denervação Muscular/efeitos adversos , Músculo Esquelético/fisiopatologia , Atrofia Muscular/fisiopatologia , Regeneração/fisiologia , Traumatismos da Medula Espinal/complicações , Potenciais de Ação/fisiologia , Adulto , Sinalização do Cálcio/fisiologia , Tamanho Celular/fisiologia , Terapia por Estimulação Elétrica , Feminino , Humanos , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/patologia , Fibras Musculares Esqueléticas/ultraestrutura , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Atrofia Muscular/patologia , Atrofia Muscular/terapia , Tempo de Reação/fisiologia , Recuperação de Função Fisiológica/fisiologia , Sarcolema/patologia , Sarcolema/ultraestrutura , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
The hypothalamus of the adult ring dove contains acoustic units that respond to species-specific coo vocalization. Loss of nest coo leads to unsuccessful breeding. However, the recovery of nest coo in some doves suggests that these units are capable of self-renewal. We have previously shown that lesioning the hypothalamus generates the addition of new neurons at the lesioned area. In this study, we sought to determine whether lesion-induced new neurons are involved in the recovery of coo-responsive units. We systematically recorded electrical activity in the ventromedial nucleus (VMN) of the hypothalamus, before and after lesion, for varying periods up to 3 months. Recordings were made when the birds were at rest (spontaneous discharge) and when the birds were exposed to acoustic stimulations (evoked discharge). Concurrently, the lesioned area was monitored for changes in cell types by using bromodeoxyuridine (BrdU) to label newly divided cells and NeuN to identify mature neurons. For 1 month after lesion, there was no sign of electrical activity, and only BrdU-labeled cells were present. When the first electrical activity occurred, it displayed abnormal spontaneous bursting patterns. The mature discharge patterns (both spontaneous and evoked) occurred after detection of BrdU+/NeuN+ double-labeled cells 2-3 months postlesion and were similar to those found in intact and sham-lesioned birds. Double-labeled cells bore morphologic characteristics of a neuron and were confirmed with z-stack analysis using confocal laser scanning microscopy. Moreover, double-labeled cells were not stained for glial fibrillary acidic protein (GFAP), suggesting that they were neurons. The number of coo-responsive units was significantly correlated with that of BrdU+/NeuN+ cells. Furthermore, the marker for recording sites revealed that coo-responsive units were colocalized with BrdU+/NeuN+ cells. Taken together, the evidence strongly suggests that lesion-induced addition of new neurons promotes the functional recovery of the adult hypothalamus.
Assuntos
Acústica , Neurônios/fisiologia , Recuperação de Função Fisiológica/fisiologia , Vocalização Animal/fisiologia , Estimulação Acústica/métodos , Potenciais de Ação/fisiologia , Potenciais de Ação/efeitos da radiação , Análise de Variância , Animais , Bromodesoxiuridina/metabolismo , Contagem de Células/métodos , Tamanho Celular/fisiologia , Tamanho Celular/efeitos da radiação , Sobrevivência Celular/fisiologia , Columbidae , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica/métodos , Masculino , Microscopia Confocal/métodos , Neurônios/efeitos da radiação , Fosfopiruvato Hidratase/metabolismo , Fatores de Tempo , Núcleo Hipotalâmico Ventromedial/citologia , Núcleo Hipotalâmico Ventromedial/lesões , Núcleo Hipotalâmico Ventromedial/fisiologiaRESUMO
The study of the dielectric properties of micrometer- or nanometer-scale particles is of particular interest in present-day applications of biomedical engineering. Electrokinetics utilises electrically energised microelectrode structures within microfluidic chambers to noninvasively probe the physiological structure of live cancer cells. A system is described that combines the three complementary techniques of dielectrophoresis (DEP), travelling wave dielectrophoresis (TWD) and electrorotation (ROT) for the first time on a single, integrated chip (3 x 6 mm). The chip employs planar microelectrode arrays fabricated on a silicon substrate to facilitate the synthesis of the various nonuniform electric fields required for the controlled manipulation, measurement and characterization of mammalian cells. A study of the dielectric properties of human malignant cells (Daudi and NCI-H929) was performed to demonstrate the potential and the versatility of the system in providing a fully programmable microsystem.
Assuntos
Linfoma de Burkitt/patologia , Eletroforese/métodos , Mieloma Múltiplo/patologia , Linhagem Celular Tumoral , Tamanho Celular/fisiologia , Eletroforese/instrumentação , Humanos , Microeletrodos , SemicondutoresRESUMO
The amino acid analogue 3-nitrotyrosine (3-NT) is formed in neural cells as a result of the intense stimulation of NMDA glutamate receptors. 3-NT is involved in the pathology of diverse neurodegenerative disorders. The aim of our work is to investigate the sensitivity of cultured neural and glial cells to 3-NT. We report the morphological changes detected on mouse neuroblastoma (C1300) and rat glioma (C6) cell lines cultured in a medium supplemented with different 3-NT concentrations. Western blot displayed a selective incorporation of 3-NT into a single protein that co-migrated with tubulin. Both cell lines showed morphological changes, nuclear suffering, decreased viability and growth inhibition (starting from 90 and 360 microM for C1300 and C6, respectively). Such effects were dose-dependent, though glioma cells showed severe alterations at higher 3-NT concentrations. Our results point out a higher 3-NT sensitivity in the neural cells studied in comparison with those of glial origin. The dramatic toxicity of 3-NT in neural cells suggests further investigations focused on the biochemical mechanisms at the roots of neurodegenerative diseases.
Assuntos
Doenças Neurodegenerativas/metabolismo , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Tirosina/análogos & derivados , Tirosina/toxicidade , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/patologia , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Glioma , Camundongos , Neuroblastoma , Doenças Neurodegenerativas/fisiopatologia , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/metabolismo , Neurônios/patologia , Ratos , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Tirosina/metabolismoRESUMO
It was previously shown that tyrosine hydroxylase (TH) immunoreactivity in the terminals of the lateral efferents of the cochlea is decreased by acoustic trauma and that sound preconditioning counteracted this decrease [Hear Res 174 (2002) 124]. Here we identify those neurons in the lateral olivocochlear system (LOC) in the brainstem that regulates the peripheral expression of TH in the cochlea. By employing retrograde tracing techniques, dextran-labeled neurons were found predominantly in the ipsilateral LOC system including lateral superior olive (LSO), and the surrounding periolivary regions (dorsal periolivary nucleus [DPO], dorsolateral periolivary nucleus [DLPO], lateral nucleus of trapezoid body [LNTB]). Employing immunocytochemistry, it was found that a control group had 35% of the ipsilateral LOC neurons positively stained with TH. Of the total population of TH neurons, 77% were double-stained (TH and dextran) in the LOC system. Acoustic trauma decreased the number of TH positive neurons in the LSO and the surrounding DLPO, and caused a reduction of TH fiber immunolabeling in these regions. Changes were not found in the DPO or the LNTB after acoustic trauma. Sound conditioning protected against the decrease of TH immunolabeling by acoustic trauma and increased the fiber staining for TH in the LSO and DLPO, but not in the DPO or the LNTB. These results provide evidence that TH positive neurons are present in the LOC system in the guinea-pig. It is now demonstrated that protection against acoustic trauma by sound conditioning has a central component that is governed by TH in the LSO and the surrounding periolivary DLPO region.
Assuntos
Vias Eferentes/enzimologia , Núcleo Olivar/enzimologia , Órgão Espiral/enzimologia , Ponte/enzimologia , Tirosina 3-Mono-Oxigenase/metabolismo , Estimulação Acústica , Animais , Catecolaminas/biossíntese , Tamanho Celular/fisiologia , Dextranos , Vias Eferentes/citologia , Cobaias , Perda Auditiva Provocada por Ruído/fisiopatologia , Perda Auditiva Provocada por Ruído/prevenção & controle , Imuno-Histoquímica , Neurônios/citologia , Neurônios/enzimologia , Núcleo Olivar/citologia , Órgão Espiral/citologia , Órgão Espiral/lesões , Ponte/citologiaRESUMO
The synthetic compound NO-1886 is a lipoprotein lipase activator that has been proven to be highly effective in lowering plasma triglycerides and elevating high-density lipoprotein cholesterol. Recently, we found that NO-1886 also had a plasma glucose-reducing action in high-fat/high-sucrose diet-induced diabetic rabbits. In the current study, we investigated the effects of NO-1886 on the morphology of adipocytes, plasma levels of tumor necrosis factor-alpha (TNF-alpha) and free fatty acids (FFA) in miniature pigs fed a high-fat/high-sucrose diet. Our results showed that feeding a high-fat/high-sucrose diet to miniature pigs increased the size of adipocytes, and the plasma levels of TNF-alpha, FFA, and glucose. This diet also induced insulin resistance and impaired the acute insulin response to glucose loading. Supplementing 1% NO-1886 to the high-fat/high-sucrose diet inhibited adipocyte enlargement, and suppressed plasma levels of TNF-alpha, FFA, and glucose. The decrease in plasma TNF-alpha and FFA was simultaneous with the decrease in plasma glucose. We also found an increased whole body glucose clearance and an increased acute insulin response to intravenous glucose loading by NO-1886 supplementation. These data suggest that NO-1886 improves the glucose metabolism in high-fat/high-sucrose diet-induced diabetic minipigs by decreasing fat deposit, and suppressing plasma TNF-alpha and FFA levels. Therefore, NO-1886 is potentially beneficial for the treatment of insulin-resistant syndrome.
Assuntos
Adipócitos/efeitos dos fármacos , Benzamidas/farmacologia , Gorduras na Dieta/administração & dosagem , Sacarose Alimentar/administração & dosagem , Ácidos Graxos não Esterificados/antagonistas & inibidores , Glucose/metabolismo , Compostos Organofosforados/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Ácidos Graxos não Esterificados/sangue , Feminino , Inibidores do Crescimento/farmacologia , Hipolipemiantes/farmacologia , Masculino , Suínos , Porco Miniatura , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Electrical stimulation training is known to alter skeletal muscle characteristics after a spinal cord injury, but the effect of load on optimizing the training protocol has not been fully investigated. This study investigated two electrical-stimulation training regimes with different loads on intramuscular parameters of the paralyzed lower limbs. Six paraplegic individuals with a spinal cord injury underwent electrical stimulation training (45 min daily for 3 days per week for 10 weeks). One leg was trained statically with load, and the contralateral leg was trained dynamically with minimal load. Isometric force assessed with 35-HZ stimuli increased significantly in both legs from baseline, with the static-trained leg also being significantly higher than the dynamic-trained leg. The vastus lateralis muscle of the statically trained leg showed a significant increase in type I fibers, fiber cross-sectional area, capillary-to-fiber ratio, and citrate synthase activity when compared to both baseline and the dynamically trained leg. Relative oxygenation of the vastus lateralis muscle as determined by near infrared spectroscopy was also significantly greater after static training. This study indicates that the load that is applied to paralyzed muscle during an electrical stimulation training program is an important factor in determining the amount of muscle adaptation that can be achieved.
Assuntos
Terapia por Estimulação Elétrica/normas , Músculo Esquelético/fisiopatologia , Paraplegia/terapia , Traumatismos da Medula Espinal/fisiopatologia , Adulto , Capilares/citologia , Capilares/fisiologia , Tamanho Celular/fisiologia , Citrato (si)-Sintase/metabolismo , Tolerância ao Exercício/fisiologia , Humanos , Contração Isométrica/fisiologia , Pessoa de Meia-Idade , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/citologia , Neovascularização Fisiológica/fisiologia , Consumo de Oxigênio/fisiologia , Paraplegia/etiologia , Paraplegia/fisiopatologia , Resultado do Tratamento , Suporte de Carga/fisiologiaRESUMO
Choline, a compound present in many foods, has recently been classified as an essential nutrient for humans. Studies with animal models indicate that the availability of choline during the prenatal period influences neural and cognitive development. Specifically, prenatal choline supplementation has been shown to enhance working memory and hippocampal long-term potentiation (LTP) in adult offspring. However, the cellular mechanisms underlying these effects remain unclear. Here we report that choline supplementation, during a 6-day gestational period, results in greater excitatory responsiveness, reduced slow afterhyperpolarizations (sAHPs), enhanced afterdepolarizing potentials (ADPs), larger somata, and greater basal dendritic arborization among hippocampal CA1 pyramidal cells studied postnatally in juvenile rats (20-25 days of age). These data indicate that dietary supplementation with a single nutrient, choline, during a brief, critical period of prenatal development, alters the structure and function of hippocampal pyramidal cells.
Assuntos
Colina/farmacologia , Suplementos Nutricionais , Hipocampo/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal , Potenciais de Ação/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Contagem de Células/métodos , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Impedância Elétrica , Estimulação Elétrica/métodos , Feminino , Hipocampo/citologia , Hipocampo/fisiologia , Técnicas In Vitro , Masculino , Técnicas de Patch-Clamp/métodos , Gravidez , Células Piramidais/citologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/efeitos da radiação , Ratos , Ratos Sprague-DawleyRESUMO
Previous reports have demonstrated that extracts of the plant Uncaria tomentosa inhibit tumor cell proliferation and inflammatory responses. We have confirmed that C-Med 100, a hot water extract of this plant, inhibits tumor cell proliferation albeit with variable efficiency. We extend these findings by showing that this extract also inhibits proliferation of normal mouse T and B lymphocytes and that the inhibition is not caused by toxicity or by induction of apoptosis. Further, the extract did not interfere with IL-2 production nor IL-2 receptor signaling. Since there was no discrete cell cycle block in C-Med 100-treated cells, we propose that retarded cell cycle progression caused the inhibition of proliferation. Collectively, these data suggested interference with a common pathway controlling cell growth and cell cycle progression. Indeed, we provide direct evidence that C-Med 100 inhibits nuclear factor kappa B (NF-kappa B) activity and propose that this at least partially causes the inhibition of proliferation.
Assuntos
Unha-de-Gato/química , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , NF-kappa B/antagonistas & inibidores , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Adulto , Animais , Linfócitos B/efeitos dos fármacos , Unha-de-Gato/citologia , Divisão Celular/fisiologia , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Genes Reporter/fisiologia , Humanos , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Mitógenos/antagonistas & inibidores , Mitógenos/farmacologia , NF-kappa B/biossíntese , NF-kappa B/genética , Extratos Vegetais/química , Extratos Vegetais/uso terapêutico , Plantas Medicinais/química , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Células Tumorais Cultivadas , UncariaRESUMO
The influence of direct electric stimulation on nerve regeneration was studied in a model of crush injury of the sciatic nerve of rats. Forty-three rats were used and distributed in four groups according to the procedure: (1) intact nerve, inactive circuit; (2) crush injury, inactive circuit; (3) intact nerve, active circuit; (4) crush injury, active circuit. The low intensity continuous current circuit (1 microA) was implanted in the lumbar region, the anode being fixed to the muscles proximally and the cathode below the nerve distally to the lesion site. The Sciatic Functional Index (SFI) was evaluated at weekly intervals for 3 weeks, the sciatic nerve being resected on the 21st day for histologic and morphometric studies. The SFI progressively improved and the average fiber nerve density recovered to a nearly normal value in Group 2 and increased in Group 4 compared with the control groups (1 and 3), but this was accompanied by a decreased average fiber nerve diameter. Both number and diameter of inter and intra-fascicular blood vessels increased in the stimulated nerves. We conclude that low intensity direct electric stimulation enhances nerve regeneration following a controlled nerve crush injury and increases blood supply by increasing number and diameter of vasa nervorum.
Assuntos
Terapia por Estimulação Elétrica/métodos , Fibras Nervosas Mielinizadas/fisiologia , Regeneração Nervosa/fisiologia , Traumatismos dos Nervos Periféricos , Neuropatia Ciática/terapia , Animais , Vasos Sanguíneos/fisiologia , Vasos Sanguíneos/ultraestrutura , Contagem de Células , Tamanho Celular/fisiologia , Modelos Animais de Doenças , Terapia por Estimulação Elétrica/instrumentação , Eletrodos Implantados/tendências , Masculino , Microscopia Eletrônica , Neovascularização Fisiológica/fisiologia , Fibras Nervosas Mielinizadas/ultraestrutura , Nervos Periféricos/irrigação sanguínea , Nervos Periféricos/ultraestrutura , Ratos , Ratos Wistar , Recuperação de Função Fisiológica/fisiologia , Neuropatia Ciática/patologia , Neuropatia Ciática/fisiopatologia , Resultado do TratamentoRESUMO
Stellate cells within the ventral cochlear nucleus (VCN) are a diverse cell group that have been classified according to their size and morphology. Some of these stellate cell types constitute major projection neurones into the brainstem and directly into the inferior colliculus, while others are implicated in more local processing. It is still not clear whether a specific physiological profile is uniquely associated with each distinct type of stellate cell. To investigate such associations, we have analysed 23 units with a battery of physiological stimuli in vivo and then examined their shape and outputs following juxtacellular labelling with biocytin. Five physiologically identified groups of cells were filled. These formed two major response classes: onset cells and chopper cells. The two classes could be separated purely on morphological grounds. The onset cells had large somata, large symmetrical dendritic trees and profuse axonal branches that were restricted to the cochlear nucleus on one (On-L) or both sides (On-C) of the brainstem. The chopper cells had smaller, asymmetric, dendritic trees, which were either planar or marginal, had smaller somata and an output axon that left via the trapezoid body. We have confirmed profuse projections into the dorsal cochlear nucleus from all onset cells, and more focal projections from some members of all three groups of chopper cells.
Assuntos
Vias Auditivas/citologia , Percepção Auditiva/fisiologia , Axônios/ultraestrutura , Núcleo Coclear/citologia , Dendritos/ultraestrutura , Lisina/análogos & derivados , Estimulação Acústica , Potenciais de Ação/fisiologia , Animais , Vias Auditivas/fisiologia , Axônios/fisiologia , Tamanho Celular/fisiologia , Núcleo Coclear/fisiologia , Dendritos/fisiologia , Feminino , Cobaias , Masculino , Transmissão Sináptica/fisiologiaRESUMO
The culture conditions of a multiphase fermentation involving morphologically complex mycelia were simulated in order to investigate the influence of mycelial morphology (Trichoderma harzianum) on castor oil and air dispersion. Measurements of oil drops and air bubbles were obtained using an image analysis system coupled to a mixing tank. Complex interactions of the phases involved could be clearly observed. The Sauter diameter and the size distributions of drops and bubbles were affected by the morphological type of biomass (pellets or dispersed mycelia) added to the system. Larger oil drop sizes were obtained with dispersed mycelia than with pellets, as a result of the high apparent viscosity of the broth, which caused a drop in the power drawn, reducing oil drop break-up. Unexpectedly, bubble sizes observed with dispersed mycelia were smaller than with pellets, a phenomenon which can be explained by the segregation occurring at high biomass concentrations with the dispersed mycelia. Very complex oil drops were produced, containing air bubbles and a high number of structures likely consisting of small water droplets. Bubble location was influenced by biomass morphology. The percentage (in volume) of oil-trapped bubbles increased (from 32 to 80%) as dispersed mycelia concentration increased. A practically constant (32%) percentage of oil-trapped bubbles was observed with pelleted morphology at all biomass concentrations. The results evidenced the high complexity of phases interactions and the importance of mycelial morphology in such processes.
Assuntos
Ar , Óleo de Rícino/química , Técnicas de Cultura de Células/métodos , Micélio/citologia , Micélio/fisiologia , Reologia/métodos , Trichoderma/citologia , Trichoderma/fisiologia , Reatores Biológicos , Óleo de Rícino/metabolismo , Divisão Celular , Tamanho Celular/fisiologia , Coloides/química , Meios de Cultura/química , Fermentação/fisiologia , Gases/química , Micélio/química , Tamanho da Partícula , Propriedades de Superfície , Trichoderma/químicaRESUMO
Mitogen-activated protein kinases (MAP kinases) play a key role in the regulation of cell survival and death. Effects of Treponema denticola ATCC 35405 on ERK, p38 and JNK MAP kinases, and cell behavior was studied using non-keratinizing periodontal ligament epithelial cells (PLE) in vitro. Compared to Chinese hamster ovary cells, human cervix adenocarcinoma cells, human osteosacroma cells and human gingival fibroblasts, PLE cells were much more resistant to T. denticola-induced reduction in cell viability, assayed by tetrazolium and crystal violet assays. A low dose of 5 x 10(7) T. denticola cells/ml increased DNA synthesis ([3H]thymidine uptake) in PLE cells but at higher concentrations DNA synthesis was decreased. TUNEL staining analysis showed that about 50% of epithelial cells in onolayers died through apoptosis when exposed to a high dose of 10(11) T. denticola/ml for 24 h. Morphological light and electron microscopic analysis supported the idea that both apoptotic and necrotic cell death took place. Rounding, membrane damage, fragmentation and detachment were observed in selective cells of both mono- and multilayered PLE cultures challenged with T. denticola. Western blot analysis using MAP kinase phosphospecific antibodies showed that T. denticola strongly but transiently activated ERK1 and ERK2, signals mediating cell proliferation, and JNK and p38, kinases mediating apoptosis. While a specific inhibitor of the ERK MAP kinase pathway prevented the T. denticola stimulation of cell proliferation, inhibitor of p38 increased the cell numbers in T. denticola-treated cultures. The results suggest that T. denticola activates epithelial cell MAP kinase signal pathways controlling cell proliferation and cell survival. In addition, T. denticola exerts cytotoxic effects that appear to predominate at higher bacterial concentrations.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno , Sistema de Sinalização das MAP Quinases/fisiologia , Ligamento Periodontal/fisiologia , Treponema/fisiologia , Adenocarcinoma/patologia , Animais , Apoptose/fisiologia , Western Blotting , Células CHO , Adesão Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular , Membrana Celular/ultraestrutura , Tamanho Celular/fisiologia , Sobrevivência Celular/fisiologia , Cricetinae , DNA/biossíntese , Células Epiteliais/patologia , Células Epiteliais/fisiologia , Fibroblastos/fisiologia , Gengiva/citologia , Humanos , Marcação In Situ das Extremidades Cortadas , MAP Quinase Quinase 4 , Microscopia Eletrônica , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Osteossarcoma/patologia , Ligamento Periodontal/citologia , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The subthalamic nucleus participates in the control of movement and is considered a surgical target in the treatment of parkinsonian symptoms. Using the rat brain in vitro slice technique we show that sustained high-frequency (>100 Hz) electrical stimulation (i.e., 'tetanic stimulation') of the nucleus, as used in humans to treat Parkinson's disease, silenced subthalamic neurons. Two main cell types were identified. 'Tonic cells' (68%) showed delayed inward rectification, fired continuously, switched to bursting and stopped firing when strongly depolarized with injected current. Tetanic stimulation of the nucleus induced a steady depolarization (approximately 18 mV) that triggered action potentials at a high rate followed by bursts and finally (approximately 25 s) totally silenced tonic cells. The control tonic activity was recovered rapidly (<10 s) after ending stimulation. 'Phasic cells' (25%) discharged a single initial brief burst of action potentials both when depolarized by prolonged current injection and tetanic stimulation and did not show inward rectification. An infrequent cell type called 'phasic-tonic' (7%) showed a mixed discharge. We suggest that the silencing effect of tetanic stimulation is not a frequency-dependent presynaptic depression and could result from the gradual inactivation of Na+-mediated action potentials. These findings suggest that the remission of parkinsonian symptoms by treatment with high-frequency electrical stimulation of the subthalamic nucleus in humans may primarily reside on its capacity to suppress the action potential activity of subthalamic neurons.
Assuntos
Potenciais de Ação/fisiologia , Inibição Neural/fisiologia , Neurônios/fisiologia , Doença de Parkinson/fisiopatologia , Núcleo Subtalâmico/fisiologia , Transmissão Sináptica/fisiologia , Animais , Tamanho Celular/fisiologia , Dendritos/fisiologia , Dendritos/ultraestrutura , Estimulação Elétrica , Terapia por Estimulação Elétrica , Neurônios/citologia , Doença de Parkinson/terapia , Ratos , Canais de Sódio/fisiologia , Núcleo Subtalâmico/citologia , Sinapses/fisiologiaRESUMO
Volume changes were studied in Beta vulgaris storage root vacuoles, using video microscopy, when exposed to hypotonic conditions. The osmotic gradient was either step-applied or progressively imposed in perfusion experiments. Preincubation at low pH (6.6) or with HgCl2 strongly reduced the vacuoles' water permeability, measured in step experiments. Furthermore, the volumetric response depended on the rate with which the aniso-osmotic condition was established. In perfusion experiments a "plateau value" (osmotic equilibrium or steady-state volume value) was observed, which was significantly lower than the theoretically expected one. Furthermore, if vacuoles were preincubated in presence of HgCl2 or at low pH and then the hypo-osmotic challenge was applied in perfusion experiments, a still lower "plateau value" was observed. This reduction was concentration-dependent and completely reversible. In these conditions, when HgCl2 concentration was 300 mM or medium pH was 6.6, the volume change was abolished. In other experiments, when urea iso-osmotically replaced mannitol, a reversible, pH-dependent volumetric response was observed. These results can be interpreted accepting that 1) mercury-sensitive water channels, present in the studied structure, were blocked by low pH during the hypo-osmotic challenge; 2) modification of water permeability prevents excessive swelling during the osmotic shock; 3) the effectiveness of this last mechanism depended on the osmotic challenge rate; and 4) additionally, urea reflection coefficients were also modified by reduced medium pH.
Assuntos
Beta vulgaris/metabolismo , Permeabilidade da Membrana Celular/fisiologia , Citoplasma/química , Água/metabolismo , Beta vulgaris/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Concentração de Íons de Hidrogênio , Manitol/metabolismo , Cloreto de Mercúrio/farmacologia , Concentração Osmolar , Pressão Osmótica , Raízes de Plantas , Ureia/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismoRESUMO
Tylosema esculentum is a long-lived perennial species endemic to arid areas of southern Africa. Its potential as a crop species has long been recognized as a result of the high oil and protein content of its seeds. The reproductive biology and breeding systems of the species were investigated in wild and experimental populations growing in Botswana. Field observations confirmed that the species is heterostylous with the pistil and anthers exhibiting reciprocal heights in the two morphs, although pollen size and sculpturing do not vary. The wet, nonpapillate stigma characteristic of the species is the first to be reported in the Caesalpinioideae. In vivo and in vitro diallel crossing experiments demonstrated that a diallelic self-incompatability system exists in T. esculentum. The major site of pollen tube inhibition in the intramorph crosses was found to be in the style. This is the first report of functional heterostyly in the Fabaceae and of a confirmed self-incompatibility system in the Caesalpinioideae. Three separate lines of evidence, the monitoring of fruit development in open-pollinated plants, fruit set in diallel crossing experiments, and observations made in wild populations, demonstrated that fruit set and, by implication, seed set, are very low in this species. Floral abscission was a major limitation to the production of mature pods but there were also significant losses at other developmental stages of fruit production. The results suggest that low seed set may be an adaptation of the species to an environment in which rainfall is scarce.
Assuntos
Fabaceae/anatomia & histologia , Caules de Planta/anatomia & histologia , Alelos , Botsuana , Cruzamento/métodos , Tamanho Celular/fisiologia , Cruzamentos Genéticos , Fabaceae/genética , Fabaceae/fisiologia , Frutas/genética , Frutas/crescimento & desenvolvimento , Epiderme Vegetal/anatomia & histologia , Caules de Planta/genética , Caules de Planta/fisiologia , Pólen/anatomia & histologia , Reprodução/fisiologiaRESUMO
The study was conducted to identify the self-incompatibility mechanism in Eucalyptus globulus ssp. globulus. Controlled self- and cross-pollinations were conducted on individual flowers from three mature trees that had self-incompatibility levels of 76, 99.6 and 100%. Flowers were harvested at 4, 6 and 8 weeks after pollination. Embryology was investigated by bright field microscopy on material harvested at 4 and 6 weeks after pollination. Fertilization had taken place at 4 weeks after pollination with zygotes and free nuclear endosperm visible. There was a greater proportion of healthy, fertilized ovules in the cross- compared with the self-pollination treatment, and approx. half the ovules examined from both pollen treatments were not fertilized or were degenerating. By 6 weeks after pollination a few zygotes were starting to divide. The number of healthy, fertilized ovules was still greater in the cross-pollination treatment, but the number of healthy fertilized ovules was lower in both treatments compared with 4 weeks after pollination, and many ovules were degenerating. Fertilized ovules were significantly larger than non-fertilized or degenerating ovules and this difference was detectable by eye at 6 and 8 weeks after pollination. The mechanism of self-incompatibility appears to have both late pre- and post-zygotic components.
Assuntos
Eucalyptus/fisiologia , Caules de Planta/fisiologia , Pólen/fisiologia , Sementes/crescimento & desenvolvimento , Algoritmos , Tamanho Celular/fisiologia , Eucalyptus/embriologia , Fertilidade/fisiologia , Fatores de TempoRESUMO
This study presents an analysis of the distribution and organization pattern of axons originating from the substantia nigra pars reticulata and projecting to the thalamus in monkeys. Biotin dextran amine was iontophoretically injected into different parts of the substantia nigra pars reticulata of monkeys (African green monkeys and macaques). In whatever part of the substantia nigra the injection was made, numerous axonal endings were found to be distributed within different thalamic regions: the ventral anterior nucleus and mainly its magnocellular part, the most ventromedial part of the ventral lateral nucleus, and the mediodorsal and parafascicular nuclei. Moreover, the nigrothalamic projection appeared to be topographically organized. Ten anterogradely labeled axons were reconstructed from serial sections. The axons terminated in three to six terminal fields per axon located in a relatively small portion of only one thalamic region. These terminal fields were variable in size and comprised 4-43 very thin, varicose branches. They consisted either of different axonal branches of the same axon or of different axons and covered 10-31 thalamic cell bodies. These findings demonstrate that the overall morphological organization of individual nigral axons is complex and allows single axons to influence thalamic neurons via a combination of divergent, convergent, and amplification processes.
Assuntos
Biotina/análogos & derivados , Chlorocebus aethiops/anatomia & histologia , Macaca fascicularis/anatomia & histologia , Vias Neurais/citologia , Terminações Pré-Sinápticas/ultraestrutura , Substância Negra/citologia , Tálamo/citologia , Animais , Tamanho Celular/fisiologia , Chlorocebus aethiops/fisiologia , Dextranos , Corantes Fluorescentes , Macaca fascicularis/fisiologia , Masculino , Vias Neurais/fisiologia , Terminações Pré-Sinápticas/fisiologia , Substância Negra/fisiologia , Tálamo/fisiologiaRESUMO
The purpose of this study was to examine postnatal development of connectional specificity of corticospinal terminals. We labeled a small population of primary motor cortex neurons with the anterograde tracer biotinylated dextran amine. We reconstructed individual corticospinal segmental axon terminals in the spinal gray matter in cats of varying postnatal ages and adults. We found that at days 25 and 35 the segmental termination field of reconstructed axons was large, estimated to cover more than half of the contralateral gray matter. Branches and varicosities were sparse and had a relatively uniform distribution. When we examined the terminal fields of multiple axons, reconstructed over the same set of spinal sections (120-200 microm), we found that there was extensive overlap. By day 55, the morphology and termination fields had changed remarkably. There were many short branches, organized into discrete clusters, and varicosities were preferentially located within these clusters. The termination field of individual axons was substantially reduced compared with that of younger animals, and there was minimal overlap between the terminals of neighboring corticospinal neurons. In adults, a further reduction was seen in the spatial extent of terminals, branching, and varicosity density. Termination overlap was not substantially different from that in PD 55 animals. Development of spatially restricted clusters of short terminal branches and dense axonal varicosities occurred just prior to development of the motor map in primary motor cortex and may be necessary for ensuring that the corticospinal system can exert a dominant influence on skilled limb movement control in maturity.
Assuntos
Biotina/análogos & derivados , Padronização Corporal/fisiologia , Gatos/crescimento & desenvolvimento , Diferenciação Celular/fisiologia , Córtex Motor/crescimento & desenvolvimento , Terminações Pré-Sinápticas/ultraestrutura , Células Piramidais/citologia , Tratos Piramidais/crescimento & desenvolvimento , Medula Espinal/crescimento & desenvolvimento , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos , Gatos/anatomia & histologia , Gatos/fisiologia , Tamanho Celular/fisiologia , Dextranos , Feminino , Corantes Fluorescentes , Cones de Crescimento/fisiologia , Cones de Crescimento/ultraestrutura , Córtex Motor/citologia , Córtex Motor/fisiologia , Terminações Pré-Sinápticas/fisiologia , Células Piramidais/fisiologia , Tratos Piramidais/citologia , Tratos Piramidais/fisiologia , Medula Espinal/citologia , Medula Espinal/fisiologiaRESUMO
Each stage of the striate cortical circuit extracts novel information about the visual environment. We asked if this analytic process reflected laminar variations in synaptic physiology by making whole-cell recording with dye-filled electrodes from the cat's visual cortex and thalamus; the stimuli were flashed spots. Thalamic afferents terminate in layer 4, which contains two types of cell, simple and complex, distinguished by the spatial structure of the receptive field. Previously, we had found that the postsynaptic and spike responses of simple cells reliably followed the time course of flash-evoked thalamic activity. Here we report that complex cells in layer 4 (or cells intermediate between simple and complex) similarly reprised thalamic activity (response/trial, 99 +/- 1.9 %; response duration 159 +/- 57 ms; latency 25 +/- 4 ms; average +/- standard deviation; n = 7). Thus, all cells in layer 4 share a common synaptic physiology that allows secure integration of thalamic input. By contrast, at the second cortical stage (layer 2+3), where layer 4 directs its output, postsynaptic responses did not track simple patterns of antecedent activity. Typical responses to the static stimulus were intermittent and brief (response/trial, 31 +/- 40 %; response duration 72 +/- 60 ms, latency 39 +/- 7 ms; n = 11). Only richer stimuli like those including motion evoked reliable responses. All told, the second level of cortical processing differs markedly from the first. At that later stage, ascending information seems strongly gated by connections between cortical neurons. Inputs must be combined in newly specified patterns to influence intracortical stages of processing.