RESUMO
Myoblast fusion has been studied in cultures of chick embryonic muscle utilizing ultrastructural techniques. The multinucleated muscle cells (myotubes) are generated by the fusion of two plasma membranes from adjacent cells, apparently by forming a single bilayer that is particle-free in freeze-fracture replicas. This single bilayer subsequently collapses, and cytoplasmic continuity is established between the cells. The fusion between the two plasma membranes appears to take place primarily within particle-free domains (probably phospholipid enriched), and cytoplasmic unilamellar, particle-free vesicles are occasionally associated with these regions. These vesicles structurally resemble phospholipid vesicles (liposomes). They are present in normal myoblasts, but they are absent in certain fusion-arrested myoblast popluations, such as those treated with either 5-bromo-deoxyuridine (BUdR), cycloheximide (CHX), or pospholipase C (PLC). The unilamellar, particle-free vesicles are present in close proximity to the plasma membranes, and physical contact is observed frequently between the vesicle membrane and the plasma membrane. The regions of vesicle membrane-plasma membrane interaction are characteristically free of intramembrane particles. A model for myoblast fusion is presented that is based onan interpretation of these observations. This model suggests that the cytoplasmic vesicles initiate the generation of particle-depleted membrane domains, both being essential components in the fusion process.
Assuntos
Fusão Celular , Membrana Celular/ultraestrutura , Músculos/ultraestrutura , Animais , Embrião de Galinha , Técnicas de Cultura , Junções Intercelulares/ultraestrutura , Microscopia Eletrônica de Varredura , Modelos Biológicos , Músculos/embriologiaRESUMO
Cell-to-cell communication was characterized in prefusion chick embryo myoblast cultures, and it was determined that the prefusion myoblasts can interact via gap junctions, ionic coupling, and metabolic coupling. The biological relevance of this communication was supported by the detection of gap junctions between myoblasts in embryonic muscle. Communication was also examined in fusion-arrested cultures to determine its potential relationship to fusion competency. In cultures that were fusion arrested by treatment with either 1.8 mM ethyleneglycolbis-(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA), 3.3 X 10(-6) M 5-bromodeoxyuridine (BUdR), or 1 microgram/ml cycloheximide (CHX), both gap junctions and ionic coupling were present. Therefore, it is possible to conclude that cell communication is not a sufficient property by itself, to generate fusion between myob-asts. The potential role of communication in myogenesis is discusssed with respect to these observations.
Assuntos
Comunicação Celular , Junções Intercelulares/fisiologia , Músculos/citologia , Diferenciação Celular , Fusão Celular , Células Cultivadas , Eletrofisiologia , Junções Intercelulares/ultraestrutura , Uridina/metabolismoRESUMO
The nicotinic acetylcholine receptor from electrogenic tissue of Torpedo californica was solubilized by tryptic digestion of membrane fragments obtained from autolysed tissue, without use of detergent. The water-soluble acetylcholine receptor was purified by affinity chromatography on a cobra-toxin-Sepharose resin. The purified receptor bound 4000-6000 pmol per mg protein of alpha-[125I]bungarotoxin, and toxin-binding was specifically inhibited by cholinergic ligands. Gel filtration revealed a single molecular species of Stokes radius 125 +/- 10 A and on sucrose gradient centrifugation one major peak was observed of 20-22 S. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and beta-mercaptoethanol revealed two major polypeptides of mol. wt. 30 000 and 48 000.
Assuntos
Receptores Colinérgicos/química , Receptores Colinérgicos/isolamento & purificação , Animais , Biofísica/métodos , Bungarotoxinas/metabolismo , Membrana Celular/metabolismo , Centrifugação com Gradiente de Concentração , Cromatografia de Afinidade , Cromatografia em Gel , Detergentes/farmacologia , Eletroforese em Gel de Poliacrilamida , Concentração Inibidora 50 , Mercaptoetanol/farmacologia , Peptídeos/química , Receptores Colinérgicos/metabolismo , Sefarose/farmacologia , Sacarose/farmacologia , Torpedo , Água/químicaRESUMO
Reduced in vitro T cell mitogen-induced transformation, low proportion of T cells and increased proportion of non-T cells were found in blood mononuclear cells of patients with severe burns 3-12 days after the injury. High spontaneous proliferation of non-T cells was observed and could be related mainly to the B cell fraction. Monocytes mediated suppression of mitogen-stimulated T cell proliferation. We further studied the role of monocytes in the enhanced suppressor activity of Con A-activated T cells and found that in this assay system, the patient's T cells mediated suppression in collaboration with monocytes. In vitro, increased suppressor function was probably the result of in vivo stimulation of inhibitory activity ascribed to both monocytes and T cells of patients. Addition of indomethacin to cell cultures markedly reduced suppression of lymphocyte proliferation. Less significant reduction was noted when the patient's T cells were activated in vitro by Con A. Adjuvant treatment of burn patients with indomethacin may play a role in alleviating suppression of immune response in these patients.
Assuntos
Queimaduras/imunologia , Comunicação Celular , Tolerância Imunológica , Monócitos/imunologia , Linfócitos T Reguladores/imunologia , Adolescente , Adulto , Idoso , Concanavalina A/farmacologia , Feminino , Humanos , Contagem de Leucócitos , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Monócitos/fisiologia , Linfócitos T/imunologia , Linfócitos T Reguladores/fisiologiaRESUMO
The present study was performed to identify at which stages during spinal cord histogenesis the extracellular proteolysis (i.e. the plasmin-generating system) is expressed by the developing tissue. We report that the embryonic chick spinal cord in the course of its histogenesis produces the protease, plasminogen activator. Specific activity of plasminogen activator in the developing embryo was monitored daily, in whole cords (from day 4 until hatching) and in the ventral and dorsal halves of the cord (6-17 days) and was biochemically determined by the fibrinolytic assay. The enzyme-specific activity of the developing cord was found to be very low at early embryonic stages when neuroblasts proliferate and it is markedly enhanced (14-fold at its maximal level) at later stages when the neurons are postmitotic and only the glial cells proliferate. The pattern of plasminogen activator specific activities does not remain at a constant level: it appears to be developmentally regulated, since it is decreased and enhanced during 3 temporal stages of the spinal cord histogenesis. These coincide with the stages when a transient increase in cell proliferation, presumably of glia, was monitored as determined daily by the spinal cord DNA content (microgram DNA/mg protein). Furthermore, on days 7-9 of embryonic age, the two halves of the cord differ in their plasminogen activator specific activities; activity levels of the ventral halves were found to be higher than their dorsal counterparts with significant difference on day 7. These correspond with the well-established differentiation gradient in the ventral-to-dorsal axis of the cord.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Ativadores de Plasminogênio/metabolismo , Medula Espinal/embriologia , Animais , Divisão Celular , Sobrevivência Celular , Embrião de Galinha , Curare/farmacologia , DNA/análise , Espaço Extracelular/enzimologia , Neurônios Motores , Medula Espinal/enzimologiaRESUMO
Previous studies suggest that motor recovery does not occur after spinal cord injury because reactive glia abort the natural repair processes. A permanent wound gap is left in the cord and the brain-cord circuitry consequently remains broken. Single-dose x-irradiation destroys reactive glia at the damage site in transected adult rat spinal cord. The wound then heals naturally, and a partially functional brain-cord circuitry is reconstructed. Timing is crucial; cell ablation is beneficial only within the third week after injury. Data presented here point to the possibility of translating these observations into a clinical therapy for preventing the paralysis following spinal cord injury in the human. The lesion site (at low thoracic level) in severed adult rat spinal cord was treated daily, over the third week postinjury, with protocols of fractionated radiation similar to those for treating human spinal cord tumors. This resulted, as with the single-dose protocol, in wound healing and restoration of some hindquarter motor function; in addition, the beneficial outcome was augmented. Of the restored hindlimb motor functions, weight-support and posture in stance was the only obvious one. Recovery of this motor function was partial to substantial and its incidence was 100% instead of about 50% obtained with the single-dose treatment. None of the hindlimbs, however, regained frequent stepping or any weight-bearing locomotion. These data indicate that the therapeutic outcome may be further augmented by tuning the radiation parameters within the critical time-window after injury. These data also indicate that dose-fractionation is an effective strategy and better than the single-dose treatment for targeting of reactive cells that abort the natural repair, suggesting that radiation therapy could be developed into a therapeutic procedure for repairing injured spinal cord.
Assuntos
Fracionamento da Dose de Radiação , Recuperação de Função Fisiológica/efeitos da radiação , Traumatismos da Medula Espinal/radioterapia , Animais , Feminino , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/fisiologia , Regeneração/fisiologia , Regeneração/efeitos da radiação , Medula Espinal/fisiologia , Medula Espinal/efeitos da radiação , Vértebras Torácicas , Cicatrização/fisiologia , Cicatrização/efeitos da radiaçãoRESUMO
In the present study, Aloe vera gel (AVG) was applied to experimental second-degree burns in guinea pigs, and its effects on epithelialization, wound contraction, newly formed granulation tissue, and regeneration of hair follicles was compared with that effected by 1% silver sulfadiazine cream (AgSD). Epithelialization (%mean +/- SEM) on postburn day 8, 16, and 24 of the AVG-treated wounds was 38.72% +/- 2.71%, 60.34% +/- 3.28%, and 92.46% +/- 2.26%, respectively, while that of the AgSD-treated burns was 53.35% +/- 2.65%, 94.84% +/- 2.65%, and 100%, respectively (P less than .001). Contraction of the AVG-wounds was significantly higher than that of the AgSD-treated burns during 24 days of the study (P less than .001). The thickness of the newly formed granulation tissue was higher in the AVG-treated wounds (P less than .001), while the hair follicles count was significantly lower (P less than .001) compared with the AgSD-treated burns. It is concluded that this preparation of Aloe vera gel hindered the healing process of the present burn wound model when compared with 1% silver sulfadiazine cream.
Assuntos
Queimaduras/fisiopatologia , Extratos Vegetais/farmacologia , Cicatrização/efeitos dos fármacos , Administração Cutânea , Animais , Feminino , Géis , Tecido de Granulação/efeitos dos fármacos , Cobaias , Cabelo/efeitos dos fármacos , Microcirculação/efeitos dos fármacos , Distribuição Aleatória , Sulfadiazina de Prata/farmacologia , Pele/irrigação sanguíneaRESUMO
We present a case of microlaryngoscopy in a patient with an unexpectedly difficult airway. The airway was managed by using an oesophageal-tracheal Combitube (Kendall-Sheridan, Argyle, NY) (ETC) and a fibre-optic bronchoscope (Pentax-Japan-5 mm).
Assuntos
Complicações Intraoperatórias , Doenças da Laringe/cirurgia , Laringoscopia/métodos , Prega Vocal/cirurgia , Idoso , Broncoscopia/métodos , Feminino , Tecnologia de Fibra Óptica , Humanos , Intubação Intratraqueal/instrumentaçãoAssuntos
Acetilcolinesterase/isolamento & purificação , Órgão Elétrico/enzimologia , Acetilcolinesterase/análise , Aminoácidos/análise , Sulfato de Amônio , Anilidas , Animais , Sítios de Ligação , Centrifugação com Gradiente de Concentração , Inibidores da Colinesterase , Cromatografia de Afinidade , Cromatografia em Gel , Cyprinidae , Eletroforese , Eletroforese Descontínua , Isoenzimas , Concentração Osmolar , Polissacarídeos , Compostos de Amônio Quaternário , TripsinaRESUMO
In vitro conditions were defined under which Schwann cells, from a population of dissociated embryonic chicken spinal cord cells, migrate along the growing neuronal fibers and wrap bundles as well as individual axons, in a pattern similar to that found in a developing peripheral nervous system in vivo. The migration of Schwann cells and their wrapping of nerve fibers was found to be a function of plasmin activity in the growth medium. It was determined that at least one cell type among the spinal cord cells is producing plasminogen activator, the enzyme that activates the plasminogen that is a constituent of any serum. It is concluded that, to achieve wrapping of neurons by Schwann cells in culture, it is essential to have an active plasmin-generating system in the medium. It is hypothesized that the Schwann cell produces plasminogen activator. The possible role of both the Schwann cell and the plasminogen possible role of both the Schwann cell and the plasminogen activator in the formation of the neuromuscular junction is discussed.
Assuntos
Fibrinolisina/metabolismo , Neurônios/ultraestrutura , Células de Schwann/fisiologia , Animais , Movimento Celular , Células Cultivadas , Embrião de Galinha , Meios de Cultura , Espaço Extracelular/enzimologia , Fibrinólise , Junção Neuromuscular/ultraestrutura , Neurônios/fisiologia , Ativadores de Plasminogênio/metabolismo , Medula Espinal/citologiaRESUMO
The role of astroglia in nervous system histogenesis/regeneration (morphogenesis) was studied as a function of cell age. The effect of inoculated astroglia at different cell maturation stages on axonal growth was examined in a peripheral nerve regenerating model system. This model system consists of rat sciatic nerve stumps that regenerate through an empty silicone chamber (Lundborg et al.: Journal of Neuropathology and Experimental Neurology 41:412-422, 1982). Rat astroglial cell populations grown in cell culture were suspended either in a liquid (physiological solution) or in a solid (isotonic collagen gel) medium and inoculated within the silicone chamber at the time of surgery. Immature and mature cell populations, at ages corresponding to 9-69 postnatal days (P9-P69), were inoculated, and their effect on neural growth was analyzed by histological, immunocytochemical, and ultrastructural techniques, 2-6 weeks later. Astroglial cells differentially modulated the process of nerve regeneration, an effect that is a function of the cells' developmental stage. P19 astroglia and older cells inhibited the regeneration process, encapsulating the axons in a glia-limitans-like structure. Immature astrocytes (P9) did not seem to interfere with the regeneration process; nerve outgrowth in their presence resembled and was comparable to the ones obtained in the presence of inoculated Schwann cells. The differential effects of the developing astroglia were not significantly changed by the inoculation media, e.g., liquid or solid, and were already pronounced 2 weeks after neural transection. It is suggested by the results of the study that the role and function of astroglia in nervous system morphogenesis are changing with cell differentiation. Adult astrocytes seem to downregulate axonal growth; presumably, their function is to confine the neurites within designated structural and functional boundaries.
Assuntos
Astrócitos/citologia , Modelos Neurológicos , Regeneração Nervosa , Nervos Periféricos/citologia , Animais , Astrócitos/metabolismo , Astrócitos/ultraestrutura , Diferenciação Celular , Células Cultivadas , Microscopia Eletrônica , Nervos Periféricos/fisiologia , Nervos Periféricos/ultraestrutura , Ratos , Ratos Endogâmicos , Nervo IsquiáticoRESUMO
The role of the plasmin-generating system, a serum component, in the development of dissociated embryonic chick spinal cord cells in culture was studied. Studies were performed in a defined system where the cells were maintained in a serum-free medium. Under these conditions the cells produce plasminogen activator. It was found that plasminogen, when added to the chemically defined culture medium at concentrations of 0.2-0.75 microgram/ml, stimulates [3H]thymidine uptake (as expressed per total DNA) in a dose-response manner. This mitogenic effect is abolished by the protease inhibitors leupeptin and aprotinin. Trypsin, but not chymotrypsin, can produce similar effects. It is concluded that plasmin, which is produced as a result of the activation of plasminogen, is a component that serves as a proliferation factor in developing spinal cords in culture.
Assuntos
Fibrinolisina/fisiologia , Mitógenos , Neuroglia/fisiologia , Medula Espinal/embriologia , Animais , Divisão Celular , Células Cultivadas , Embrião de Galinha , DNA/biossíntese , Proteínas do Tecido Nervoso/metabolismo , Ativadores de Plasminogênio , Medula Espinal/metabolismo , Timidina/metabolismoRESUMO
The role of the serum proteolytic system plasminogen/plasminogen activator as a biochemical tool used by the glia or neurons, or both, for maintaining their temporary and flexible cellular interactions during histogenesis of the nervous system is under study. The present report identifies a glia cell type, the Schwann cell, as one of the cellular components of the nervous system that uses extracellular proteolysis at the time of the tissue construction. Purified dividing mouse Schwann cells in culture produce extracellular plasminogen activator. The levels of extracellular plasminogen-activator activity, as measured by the biochemical fibrinolytic assay, were directly related to the proliferation rates of the Schwann cells. The cellular plasminogen-activator specific activity at the maximal rate of cell proliferation was 3-4 times higher than that of the cells at low rate of mitosis. It is concluded that plasminogen-activator activity is expressed predominantly by the proliferating Schwann cell populations, suggesting that the extracellular proteolysis is used by the tissue at those stages when the cells divide.
Assuntos
Ativadores de Plasminogênio/metabolismo , Células de Schwann/enzimologia , Animais , Diferenciação Celular , Divisão Celular , Meios de Cultura , Espaço Extracelular/enzimologia , Camundongos , Células de Schwann/citologiaRESUMO
Mechanical injury to the adult mammalian spinal cord results in permanent morphological disintegration including severance/laceration of brain-cord axons at the lesion site. We report here that some of the structural consequences of injury can be averted by altering the cellular components of the lesion site with x-irradiation. We observed that localized irradiation of the unilaterally transected adult rat spinal cord when delivered during a defined time-window (third week) postinjury prevented cavitation, enabled establishment of structural integrity, and resulted in regrowth of severed corticospinal axons through the lesion site and into the distal stump. In addition, we examined the natural course of degeneration and cavitation at the site of lesion with time after injury, noting that through the third week postinjury recovery processes are in progress and only at the fourth week do the destructive processes take over. Our data suggest that the adult mammalian spinal cord has innate mechanisms required for recovery from injury and that timed intervention in certain cellular events by x-irradiation prevents the onset of degeneration and thus enables structural regenerative processes to proceed unhindered. We postulate that a radiation-sensitive subgroup of cells triggers the delayed degenerative processes. The identity of these intrusive cells and the mechanisms for triggering tissue degeneration are still unknown.
Assuntos
Degeneração Neural/efeitos da radiação , Regeneração Nervosa/efeitos da radiação , Traumatismos da Medula Espinal/radioterapia , Animais , Feminino , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Cicatrização , Raios XRESUMO
Mechanical injury to the adult mammalian spinal cord results in permanent loss of structural integrity at the lesion site and of the brain-controlled function distal to the lesion. Some of these consequences were permanently averted by altering the cellular constituents at the lesion site with x-irradiation delivered within a critical time window after injury. We have reported in a separate article that x-irradiation of sectioned adult rat spinal cord resulted in restitution of structural continuity and regrowth of severed corticospinal axons across and deep into the distal stump. Here, we report that after x-ray therapy of the lesion site severed corticospinal axons of transected adult rat spinal cord recover electrophysiologic control of activity of hindlimb muscles innervated by motoneurons distal to the lesion. The degree of recovery of control of muscle activity was directly related to the degree of restitution of structural integrity. This restitution of electrophysiologic function implies that the regenerating corticospinal axons reestablish connectivity with neurons within the target field in the distal stump. Our data suggest that recovery of structural continuity is a sufficient condition for the axotomized corticospinal neurons to regain some of their disrupted function in cord regions distal to the lesion site.
Assuntos
Neurônios Motores/fisiologia , Regeneração Nervosa , Traumatismos da Medula Espinal/radioterapia , Animais , Eletromiografia , Feminino , Membro Posterior/inervação , Movimento/fisiologia , Músculos/inervação , Ratos , Ratos Sprague-Dawley , Medula Espinal/anatomia & histologia , Fatores de Tempo , Raios XRESUMO
Plasminogen activator (PA) is a key enzyme in control of the cascade of extracellular proteolytic activities, proteases that degrade the extracellular components. Mammalian cells produce two molecular forms of PA, the urokinase type (u-PA) and the tissue type (t-PA); the u-PA type enzyme regulates cell migration/invasion and related tissue plasticity events. Thus, these plasticity properties of cells are defined by their PAs' biochemical profiles. The capacity of the differentiating glial cells of the central nervous system (CNS) to express and regulate the two types of PA activities has been examined as a function of cell age in culture. Results of the study suggest that only the immature astrocyte is endowed with these plasticity properties. Differentiating heterogeneous rat glial cells in culture express PA activity. Astroglia were identified as the primary source for the glial PA activity, as no PA activity was detected in the purified oligodendroglia. Cellular PA activity levels of differentiating rat and mouse astroglia are developmentally regulated. The specific activity of PA reached its highest level in rat astroglia at a cell age corresponding to 20-32 postnatal days (P20-P32) and in mouse astroglia at P8-P14; thereafter, this declined (three- to fourfold decrease) within 2 weeks to a low value. At comparable ages (P0-P35), the magnitudes of the PA specific activities of the differentiating rat astroglia and of the developing cerebrum, the tissue from which these cells were purified, were similar. Differentiating rat astroglia produce u-PA and t-PA, the cellular content of both is developmentally regulated, and the u-PA form is only found in the immature cells. u-PA is the predominant form in the immature astrocyte until age P13. Both forms are found in cells at ages P14-P30, and at later stages u-PA disappears while the t-PA type persists as the sole form. After 3 more weeks neither of the PA types was detected. Astroglia express also PA inhibitory activity; the rat astroglial PA inhibitor (PAI) seemed to be identical to PAI-1, one of the known types of PAIs. Stimulation of astroglial proliferation by their subculturing in contrast to Schwann cells did not lead to an increase; rather, beyond a certain cell age (P13) it resulted in a threefold irreversible decline in the PA specific activity of the daughter cells. It has been established that various biochemical properties of CNS mature glia appear on schedule with cell age in culture, thus defining "mature"glia in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Astrócitos/fisiologia , Plasticidade Neuronal , Ativadores de Plasminogênio/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Conformação Molecular , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Oligodendroglia/fisiologia , Ativadores de Plasminogênio/química , Inibidores de Proteases/farmacologia , Ratos , Ratos EndogâmicosRESUMO
We examined the potential of x-irradiation, at clinical dose levels, to manipulate the cellular constituents and thereby change the consequences of transection injury to adult mammalian central nervous tissue (rat olfactory bulb). Irradiation resulted in reduction or elimination of reactive astrocytes at the site of incision provided that it was delivered within a defined time window postinjury. Under conditions optimal for the elimination of gliosis (15-18 days postinjury), irradiation of severed olfactory bulbs averted some of the degenerative consequences of lesion. We observed that irradiation was accompanied by prevention of tissue degeneration around the site of lesion, structural healing with maintenance of the typical cell lamination, and rescue of some axotomized mitral cells (principal bulb neurons). Thus radiation resulted in partial preservation of normal tissue morphology. It is postulated that intrusive cell populations are generated in response to injury and reactive astrocytes are one such group. Our results suggest that selective elimination of these cells by irradiation enabled some of the regenerative processes that are necessary for full recovery to maintain their courses. The cellular targets of these cells, their modes of intervention in recovery, and the potential role of irradiation as a therapeutic modality for injured central nervous system are discussed.