RESUMEN
Immature spinal cord, unlike adult, has an ability to repair itself following injury. Evidence for regeneration, structural repair and development of substantially normal locomotor behaviour comes from studies of marsupials due to their immaturity at birth. We have compared morphological, cellular and molecular changes in spinal cords transected at postnatal day (P)7 or P14, from 3 h to 2 weeks post-injury, in South American opossums (Monodelphis domestica). A bridge between severed ends of cords was apparent 5 days post-injury in P7 cords, compared to 2 weeks in P14. The volume of neurofilament (axonal) material in the bridge 2 weeks after injury was 30% of control in P7- but < 10% in P14-injured cords. Granulocytes accumulated at the site of injury earlier (3 h) in P7 than in P14 (24 h)-injured animals. Monocytes accumulated 24 h post-injury and accumulation was greater in P14 cords. Accumulation of GFAP-positive astrocytes at the lesion occurred earlier in P14-injured cords. Neurites and growth cones were identified ultrastructurally in contact with astrocytes forming the bridge. Results using mouse inflammatory gene arrays showed differences in levels of expression of many TGF, TNF, cytokine, chemokine and interleukin gene families. Most of the genes identified were up-regulated to a greater extent following injury at P7. Some changes were validated and quantified by RT-PCR. Overall, the results suggest that at least some of the greater ability to recover from spinal cord transection at P7 compared to P14 in opossums is due to differences in inflammatory cellular and molecular responses.
Asunto(s)
Monodelphis/fisiología , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Médula Espinal , Factores de Edad , Animales , Animales Recién Nacidos , Conducta Animal , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Granulocitos/patología , Granulocitos/ultraestructura , Microscopía Electrónica de Transmisión , Regeneración Nerviosa , Neuroglía/patología , Neuroglía/ultraestructura , Neuronas/patología , Neuronas/ultraestructura , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Médula Espinal/crecimiento & desarrollo , Médula Espinal/metabolismo , Médula Espinal/patología , Factores de TiempoRESUMEN
The presence of progressive white matter atrophy following traumatic brain injury (TBI) has been reported in humans as well as in animal models. However, a quantitative analysis of progressive alterations in myelinated axons and other cellular responses to trauma has not been conducted. This study examined quantitative differences in myelinated axons from several white and gray matter structures between non-traumatized and traumatized areas at several time points up to 1 year. We hypothesize that axonal numbers decrease over time within the structures analyzed, based on our previous work demonstrating shrinkage of tissue in these vulnerable areas. Intubated, anesthetized male Sprague-Dawley rats were subjected to moderate (1.8-2.2 atm) parasagittal fluid-percussion brain injury, and perfused at various intervals after surgery. Sections from the fimbria, external capsule, thalamus and cerebral cortex from the ipsilateral hemisphere of traumatized and sham-operated animals were prepared and. estimated total numbers of myelinated axons were determined by systematic random sampling. Electron micrographs were obtained for ultrastructural analysis. A significant (P<0.05) reduction in the number of myelinated axons in the traumatized hemisphere compared to control in all structures was observed. In addition, thalamic and cortical axonal counts decreased significantly (P<0.05) over time. Swollen axons and macrophage/microglia infiltration were present as late as 6 months post-TBI in various structures. This study is the first to describe quantitatively chronic axonal changes in vulnerable brains regions after injury. Based on these data, a time-dependent decrease in the number of myelinated axons is seen to occur in vulnerable gray matter regions including the cerebral cortex and thalamus along with distinct morphological changes within white matter tracts after TBI. Although this progressive axonal response to TBI may include Wallerian degeneration, other potential mechanisms underlying this progressive pathological response within the white matter are discussed.
Asunto(s)
Axones/patología , Axones/ultraestructura , Lesiones Encefálicas/patología , Encéfalo/patología , Degeneración Nerviosa/patología , Animales , Atrofia , Masculino , Microscopía Electrónica de Transmisión , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND AND PURPOSE: A reversible model of focal thrombotic stroke was developed in the rat and examined for histological evidence of reperfusion injury after clinically relevant times of recanalization. METHODS: The distal middle cerebral artery of 28 male Sprague-Dawley rats was occluded by 562-nm laser-driven photothrombosis for 0.5, 2, and 3 hours or permanently (each n=7) and was recanalized by 355-nm UV laser irradiation. Occlusive material was examined by transmission electron microscopy. Cortical cerebral blood flow was monitored by laser-Doppler flowmetry. Brain infarcts were examined histologically at 3 days. RESULTS: After occlusion, cortical cerebral blood flow was reduced to 33+/-4% of baseline for all groups and was restored to 82+/-9%, 75+/-3%, and 93+/-7% of baseline for the 0.5-, 2-, and 3-hour groups, respectively, following recanalization after 29+/-8, 38+/-20, and 70+/-33 minutes of UV laser irradiation. The thrombotic occlusion contained compactly aggregated platelets but no fibrin, with length (1.2 to 1.8 mm) proportional to the ischemic period. During recanalization, microchannels containing erythrocytes and scattered leukocytes and bordered by intact disaggregated platelets infiltrated the thrombus. Infarct volumes (mm3) at 3 days were 12+/-3 for the permanent case and 8+/-4, 24+/-3, and 30+/-9 for the 0.5-, 2-, and 3-hour cases, respectively, thus demonstrating reperfusion injury histologically in the latter 2 groups. No hemorrhage was seen. CONCLUSIONS: UV laser-facilitated dissolution of a conventionally refractory platelet thrombus provides a novel and effective method for restoring blood flow without hemorrhagic complications during thrombotic stroke. This was the first observation of histologically confirmed reperfusion injury in such a model.
Asunto(s)
Angioplastia por Láser , Circulación Cerebrovascular , Infarto de la Arteria Cerebral Media/terapia , Daño por Reperfusión , Accidente Cerebrovascular/terapia , Angioplastia por Láser/efectos adversos , Animales , Velocidad del Flujo Sanguíneo , Modelos Animales de Enfermedad , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/fisiopatología , Coagulación con Láser , Flujometría por Láser-Doppler , Masculino , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/etiología , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/fisiopatología , Factores de TiempoAsunto(s)
Proteínas Musculares , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Sinaptofisina/metabolismo , Animales , Dimerización , Microscopía Electrónica , Peso Molecular , Músculo Esquelético/ultraestructura , Pruebas de Precipitina , Unión Proteica , Estructura Cuaternaria de Proteína , Conejos , Sinaptofisina/análogos & derivados , Sinaptofisina/química , Sinaptofisina/aislamiento & purificación , Sinaptofisina/ultraestructuraRESUMEN
Human Schwann cells (SCs) can be isolated and expanded with mitogens using cell culture techniques. These cells have been demonstrated to promote axonal regrowth in both the central and peripheral nervous system. Primary rat SCs can be immortalized with long-term exposure to mitogens. Transplantation of these cells into an autogenic host sciatic nerve results in the formation of tumors. Human cells are, in general, relatively more resistant to malignant transformation, but any potential risk for inducing tumor after transplantation of SCs in humans must be excluded. In this study, the malignant potential of mitogen expanded human SCs injected into the sciatic nerve of immunodeficient mice was investigated. Human SCs were isolated from human peripheral nerves and placed in cell culture, expanded with mitogens (heregulin and forskolin) for many passages (0-6 times), and then injected within the sciatic nerve of Severe Combined Immunodeficient (SCID) rat or mice. As a positive control for tumor formation in this xenograft model, human glioma cells were also injected. The proliferation index (PI) of the human SCs gradually decreased with each passage in cell culture. SC purity remained stable until the 6th passage, and then decreased significantly for older passages, so that the cultures were over-grown with fibroblasts. The incidence for rat or human glioma cells to induce tumors was 100% and 92%, respectively. In contrast, there was no tumor induced by human primary or mitogen expanded SCs. Demyelination, remyelination and formation of connective sheath at the injection site were observed in some cases after injection of the human SCs. Thus, mitogen-expanded human SCs do not produce tumors when transplanted in vivo, which suggests that these cells are safe, and deserve further study towards their use in clinical transplantation.
Asunto(s)
Transformación Celular Neoplásica , Glioma/patología , Mitógenos/farmacología , Nervios Periféricos/citología , Neoplasias del Sistema Nervioso Periférico/patología , Células de Schwann/citología , Células de Schwann/trasplante , Nervio Ciático , Nervio Ciático/citología , Animales , División Celular , Trasplante de Células , Células Cultivadas , Humanos , Ratones , Ratones SCID , Índice Mitótico , Neoplasias del Sistema Nervioso Periférico/ultraestructura , Ratas , Células de Schwann/efectos de los fármacos , Nervio Ciático/fisiología , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Traumatic injury to the adult human spinal cord most frequently occurs at the mid-to-low cervical segments and produces tetraplegia. To investigate treatments for improving upper extremity function after cervical spinal cord injury (SCI), three behavioral tests were examined for their potential usefulness in evaluating forelimb function in an adult rat model that mimics human low cervical SCI. Testing was conducted pre- and up to 4 weeks post-operation in adult female rats subjected to either contusion injury at the C7 spinal cord segment or sham-surgery. Modified Forelimb Tarlov scales revealed significant proximal and distal forelimb extension dysfunction in lesion rats at l-to-4 weeks post-cervical SCI. The Forelimb Grip Strength Test showed a significant decrease in forelimb grip strength of lesion rats throughout the 4 weeks post-cervical SCI. Significant deficits in reach and pellet retrieval by lesion rats were measured at l-to-4 weeks post-cervical SCI with the conditioned pellet retrieval Staircase Test. The results demonstrate that these qualitative and quantitative forelimb behavioral tests can be used to evaluate forelimb function following low cervical SCI and may be useful to investigate treatments for improving forelimb function in these lesions.
RESUMEN
RN33B, a conditionally-immortalized neuronal cell line, survives and differentiates following grafting into the neocortex and hippocampus of adult and neonatal rat hosts. We have previously shown that these cells assume shapes characteristic of endogenous neurons at the integration site and persist up to 24 weeks post-grafting. In the present study we use electron microscopy and immunohistochemistry to characterize such cells. Differentiated RN33B cells were identical in size to endogenous neurons and their sizes depended on the specific location of integration. RN33B cells in the granule cell layer of the dentate gyrus and CA3 and CA1 pyramidal layers were 9.0, 15.3, and 12.6 microns in diameter, respectively. Grafted RN33B cells received synapses from fibres of host origin. Differentiated cells expressed neuronal markers, but not glial markers. Some differentiated cells expressed glutamate both in vitro and in vivo whereas undifferentiated cells did not. Grafted RN33B cells that differentiated with morphologies similar to CA3 pyramidal neurons and pyramidal cortical neurons expressed Py antigen, a neuronal marker that is differentially expressed in endogenous large pyramidal neurons of the cerebral cortex and large pyramids of hippocampal field CA3. This Py immunoreactivity was region-specific and corresponded to the endogenous pattern of Py immunostaining. Collectively, these data indicate that RN33B cells are capable of region-specific differentiation and have the potential to integrate functionally into the host CNS.
Asunto(s)
Neuronas/ultraestructura , Células Madre/ultraestructura , Animales , Animales Recién Nacidos , Antígenos/análisis , Biomarcadores/química , Línea Celular , Supervivencia Celular/fisiología , Senescencia Celular/fisiología , Ácido Glutámico/análisis , Neuronas/química , Neuronas/trasplante , Terminales Presinápticos/ultraestructura , Células Piramidales/inmunología , Ratas , Ratas Endogámicas Lew , Células Madre/químicaRESUMEN
The dihydropyridine receptors (DHPR) and the ryanodine receptors (RyR) are well-characterized proteins of the triad junctions of skeletal muscle fibers. Recently, a newly discovered 95-kDa protein, triadin, has been purified from rabbit skeletal muscle heavy sarcoplasmic reticulum (SR) vesicles. WE have used indirect immunogold EM to localize triadin to the junctional face of the SR in isolated triads. In addition, we have used indirect immunofluorescence to localize triadin in relation to the DHPR and the RyR in adult and developing rat skeletal muscle. In double immunolabelling experiments of longitudinally oriented adult rat skeletal muscle tissue, triadin-specific and RyR-specific antibodies resulted in a characteristic striated staining pattern. The staining arising from these antibodies completely overlapped when examined by computer analysis of digitized laser scanning confocal microscopy images. A similar result was obtained in double staining experiments using antibodies raised against the DHPR and the RyR suggesting that all three proteins are located in the triads in situ. The developmental expression of the three triad proteins was examined using double labeling of skeletal muscle tissue from several fetal and early postnatal ages. The staining patterns of triadin, RyR, and DHPR antibodies were overlapping throughout development, suggesting that from their earliest appearance the three proteins are components of the triads.
Asunto(s)
Canales de Calcio/metabolismo , Proteínas Portadoras , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Envejecimiento/metabolismo , Animales , Animales Recién Nacidos , Canales de Calcio Tipo L , Proteínas de Unión a Calmodulina/metabolismo , Desarrollo Embrionario y Fetal , Feto/metabolismo , Feto/fisiología , Técnica del Anticuerpo Fluorescente , Immunoblotting , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular , Microscopía Inmunoelectrónica , Proteínas del Tejido Nervioso/metabolismo , Conejos , Ratas , Ratas Sprague-Dawley , Canal Liberador de Calcio Receptor de Rianodina , Distribución TisularRESUMEN
Dyads (transverse tubule--junctional sarcoplasmic reticulum complexes) were enriched from rat ventricle microsomes by continuous sucrose gradients. The major vesicle peak at 36% sucrose contained up to 90% of those membranes which possessed dihydropyridine (DHP) binding sites (markers for transverse tubules) and all membranes which possessed ryanodine receptors and the putative junctional foot protein (markers for junctional sarcoplasmic reticulum). In addition, the 36% sucrose peak contained half of the vesicles with muscarine receptors. Vesicles derived from the nonjunctional plasma membrane as defined by a low content of dihydropyridine binding sites per muscarine receptor and from the free sarcoplasmic reticulum as defined by the M(r) 102K Ca2+ ATPase were associated with a diffuse protein band (22-30% sucrose) in the lighter region of the gradient. These organelles were recovered in low yield. Putative dyads were not broken by French press treatment at 8,000 psi and only partially disrupted at 14,000 psi. The monoclonal antibody GE4.90 against skeletal muscle triadin, a protein which links the DHP receptor to the junctional foot protein in skeletal muscle triad junctions, cross-reacted with a protein in rat dyads of the same M(r) as triadin. Western blots of muscle microsomes from preparations which had been treated with 100 mM iodoacetamide throughout the isolation procedure showed that cardiac triadin consisted predominantly of a band of M(r) 95 kD. Higher molecular weight polymers were detectable but low in content, in contrast with the ladder of oligomeric forms in rat psoas muscle microsomes. Cardiac triadin was not dissolved from the microsomes by hypertonic salt or Triton X-100, indicating that it, as well as skeletal muscle triadin, was an integral protein of the junctional SR. The cardiac epitope was localized to the junctional SR by comparison of its distribution with that of organelle markers in both total microsome and in French press disrupted dyad preparations. Immunofluorescence localization of triadin using mAb GE4.90 revealed that intact rat ventricular muscle tissue was stained following a well-defined pattern of bands every sarcomere. This spacing of bands was consistent with the interpretation that triadin was present in the dyadic junctional regions.
Asunto(s)
Proteínas Portadoras , Ventrículos Cardíacos/química , Proteínas Musculares/análisis , Músculo Liso Vascular/química , Animales , Anticuerpos Monoclonales , Western Blotting , Dihidropiridinas/metabolismo , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Epítopos , Técnica del Anticuerpo Fluorescente , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/ultraestructura , Péptidos y Proteínas de Señalización Intracelular , Masculino , Microsomas/química , Microsomas/ultraestructura , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/ultraestructura , Orgánulos/química , Orgánulos/ultraestructura , Ratas , Ratas Sprague-DawleyRESUMEN
The monoclonal antibody, mAb GE 4.90, raised against triadin, a 95 kDa protein of sarcoplasmic reticulum (SR), inhibits the slow phase of Ca2+ release from SR following depolarization of the T-tubule moiety of the triad. The antibody has virtually no effect on the fast phase of depolarization-induced Ca2+ release nor on caffeine-induced Ca2+ release. Since the slow phase of depolarization-induced Ca2+ release is also inhibited by dihydropyridines (DHP), these results suggest that triadin may be involved in the functional coupling between the DHP receptor and the SR Ca2+ channel.
Asunto(s)
Calcio/metabolismo , Proteínas Portadoras , Proteínas Musculares/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Anticuerpos Monoclonales , Cinética , Proteínas Musculares/antagonistas & inhibidores , ConejosRESUMEN
A monoclonal antibody, GE 4.90, has been produced following immunization of mice with the 95-kDa protein (triadin) of terminal cisternae of rabbit fast skeletal muscle isolated in nondenaturing detergent. The antibody binds to a protein of Mr95K in Western blots of microsomal vesicles electrophoresed in the presence of mercaptoethanol. The greatest intensity of the immunoblot reaction is to enriched terminal cisternae vesicles while little binding is seen to longitudinal reticulum and transverse tubules. The content of antigen in different microsomal subfractions has been estimated by immunoassay: terminal cisternae/triads contain 5.6 micrograms/mg of protein while heavy terminal cisternae contain 32 micrograms/mg. The molar content of triadin in vesicles is approximately the same as that of the ryanodine receptor. When Western blots of gels of terminal cisternae are run in nonreducing conditions, little protein of Mr95K is visible. A number of bands, however, forming a ladder of higher molecular weight are discerned, indicating that the 95-kDa protein forms a disulfide-linked homopolymer. A biotinylated aromatic disulfide reagent (biotin-HPDP) labels the 95-kDa protein, the junctional foot protein, and the Mr 106K protein described by others as a Ca(2+)-release channel (SG 106). This latter protein migrates in gel electrophoresis under nonreducing conditions at a molecular weight different from that of the 95-kDa protein. We did not detect any alteration of binding of the 95-kDa protein to the dihydropyridine receptor or junctional foot protein dependent on the state of oxidation of cysteine residues of either triadin or receptor protein used as the overlay probe.
Asunto(s)
Proteínas Portadoras , Proteínas Musculares/metabolismo , Receptores Colinérgicos/metabolismo , Receptores Nicotínicos/metabolismo , Retículo Sarcoplasmático/química , Animales , Anticuerpos Monoclonales , Western Blotting , Canales de Calcio , Cisteína/metabolismo , Disulfuros , Sustancias Macromoleculares , Microsomas/metabolismo , Peso Molecular , Proteínas Musculares/análisis , Proteínas Musculares/aislamiento & purificación , Músculos , Conejos , Canal Liberador de Calcio Receptor de Rianodina , Retículo Sarcoplasmático/metabolismoRESUMEN
It has been previously recognized that a number of protocols may cause breakage of the triad junction and separation of the constituent organelles of skeletal muscle. We now describe a fraction of triad junctions which is refractory to the known protocols for disruption. Triads were passed through a French press and the dissociated organelles were separated on a sucrose density gradient, which was assayed for PN200-110, ouabain and ryanodine binding. Ryanodine binding showed a single peak at the density of heavy terminal cisternae. On the other hand, the PN200-110 and ouabain, which are external membrane ligands, bound in two peaks: one at the free transverse tubule region and the other at the light terminal cisternae. Similarly, a two peak pattern of PN200-110 and ouabain binding was observed when triad junctions were broken by the Ca2(+)-dependent protease, calpain, which selectively hydrolyzes the junctional foot protein. The light terminal cisternae vesicles were subjected to three different procedures of junctional breakage: French press, hypertonic salt treatment, and protease digestion using calpain or trypsin. The treated membranes were then centrifuged on density gradients. Only extensive trypsin digestion caused a partial shift of ouabain activity into the free transverse tubule region. These observations suggest that the triads are a composite mixture of breakage susceptible, "weak," and breakage resistant, "strong," triads. Scatchard analysis of PN200-110 suggests that the transverse tubules of strong triads contain a relatively high number of dihydropyridine receptors compared to those of weak triads. Thin section electron microscopic images of the strong triads comparable to those of intact muscle are presented.
Asunto(s)
Músculos/ultraestructura , Animales , Bloqueadores de los Canales de Calcio/metabolismo , Canales de Calcio , Fraccionamiento Celular/métodos , Técnicas In Vitro , Isradipino , Cinética , Microscopía Electrónica , Microsomas/metabolismo , Microsomas/ultraestructura , Músculos/metabolismo , Orgánulos/metabolismo , Orgánulos/ultraestructura , Oxadiazoles/metabolismo , Conejos , Receptores Colinérgicos/metabolismo , Receptores Nicotínicos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/ultraestructura , TripsinaRESUMEN
Polyclonal antibodies have been developed against the junctional feet or spanning protein from skeletal muscle triads. These probes in combination with immunogold labels have been used to localize the spanning protein by electron microscope of isolated vesicles from terminal cisternae/triads. The spanning protein antibodies specifically bind to the electron dense junctional feet. In vesicles permeabilized by hypotonic treatment or by saponin, some gold particles may be seen on the luminal side of the vesicle. Trypsin treatment of vesicles causes complete loss of the 300 K spanning protein from SDS gels while dot blots show that some but not all the antigenic activity is lost. This treatment is associated with the loss of the electron dense projections from the membrane surface and is coincident with the loss of immunogold staining when antibody is added to the intact vesicles. On the other hand, in experiments in which the luminal portions of the isolated vesicles have been made accessible to the polyclonal antibodies by sectioning lightly fixed vesicles before immunogold tagging, extensive gold labelling was found to occur in trypsin treated vesicles which have lost detectable projections from the cytoplasmic side of the membrane. These data support the view that the spanning protein projects from the sarcoplasmic reticulum towards the transverse tubules but further suggest that spanning protein extends into and probably through the sarcoplasmic reticulum membrane in accord with the proposition that it is a Ca2+ channel.
Asunto(s)
Proteínas Musculares/análisis , Músculos/análisis , Organoides/ultraestructura , Animales , Membrana Celular/ultraestructura , Inmunohistoquímica , Microscopía Electrónica , Músculos/citología , Músculos/ultraestructura , ConejosAsunto(s)
Membranas Intracelulares/ultraestructura , Microsomas/ultraestructura , Microtúbulos/ultraestructura , Músculos/ultraestructura , Adenosina Trifosfatasas/metabolismo , Animales , Calcio/metabolismo , Fraccionamiento Celular/métodos , Técnica de Fractura por Congelación , Membranas Intracelulares/metabolismo , Microscopía Electrónica , Microsomas/metabolismo , Microtúbulos/metabolismo , Peso Molecular , Músculos/metabolismo , Proteínas/aislamiento & purificación , ConejosRESUMEN
A monoclonal antibody has been developed against the putative junctional protein or spanning protein (SP) from skeletal muscle triads. By immuno-affinity chromatography, we have purified this protein. The native protein has a molecular mass of 630-800 kD, as determined by gel filtration and rate zonal centrifugation. Within the limits of the methods used, the basic unit of the SP appears to be a dimer. In electron micrographs, it is shown to exhibit a circular profile with a diameter of approximately 100 A. In thin section analysis, the protein is frequently observed as parallel tracks of electron-dense particles bordering a translucent core. We suggest that the basic unit of the junctional structure is a dimer of 300-kD subunits and that four such entities constitute the intact SP. The purified protein has been used to develop polyclonal antibodies. By immunoelectron microscopy using immunogold probes, the SP has been localized to the junctional gap of the triad. By attaching the SP to an affinity resin, three proteins have been identified as forming associations with the SP. The Mrs of the proteins are 150, 62, and 38 kD; the 62-kD protein is calsequestrin.
Asunto(s)
Proteínas Musculares/aislamiento & purificación , Músculos/análisis , Animales , Anticuerpos Monoclonales/inmunología , Cromatografía de Afinidad , Cromatografía en Gel , Microscopía Electrónica , Proteínas Musculares/inmunología , Músculos/ultraestructura , ConejosRESUMEN
Junctional sarcoplasmic reticulum (SR) has been identified in microsomes from canine ventricular muscle by the presence of calsequestrin and ryanodine-sensitive Ca2+ release channels. These properties, however, are not common to cardiac cells from all species. Seiler et al (1) have recently described a high Mr polypeptide in canine junctional SR similar to the spanning protein subunits of skeletal muscle triads. We now report the existence of a polypeptide with the same mobility in SR from rabbit ventricular muscle and show that those cardiac membranes can associate with transverse (T-) tubules from rabbit skeletal muscle in K cacodylate medium. We propose that this polypeptide and the reaction with T-tubules be considered as criteria for the identification of cardiac junctional SR.
Asunto(s)
Miocardio/ultraestructura , Retículo Sarcoplasmático/ultraestructura , Animales , Calcio/metabolismo , Calsecuestrina/análisis , Electroforesis en Gel de Poliacrilamida , Canales Iónicos/análisis , Microscopía Electrónica , Peso Molecular , Conejos , Rianodina/análisisRESUMEN
The junction of isolated triads can be mechanically broken by passage through a French press and subsequently reformed by incubation of the isolated organelles with certain salts of weak acids (e.g., K cacodylate, K propionate, and K butyrate). In contrast, other salts (e.g., KCl, K phosphate, and K benzoate) are ineffective in promoting triad formation. An endogenous factor obtained from a muscle homogenate acts in the same manner as these artificial compounds. When rabbit skeletal muscle is homogenized in a KCl solution and centrifuged to remove large cellular components and membrane fractions, an endogenous factor is extracted into the high speed supernatant which promotes the reformation of mechanically broken triads. A three-stage purification of this factor has been achieved using: ammonium sulfate fractionation, adsorption chromatography, and molecular sieve chromatography. SDS-PAGE showed that the protein was purified to homogeneity and had a subunit Mr of 34,000 daltons. This protein has the following characteristics: it exists in 0.1 M KCl as a polymeric substance with an estimated Mr = 123,000 on molecular sieve chromatography and a Mr = 155,000 on sedimentation equilibrium; it promotes the formation of triadic vesicles from isolated organelles in a low ionic strength medium; Both this protein and cacodylate share the property of specifically catalyzing the association and aggregation of junctional proteins which had previously been dissolved by neutral detergent and salt; it appears to be identical to an extrinsic constituent of terminal cisternae, which has been described as a protein of Mr = 34K. It is not clear, however, whether this protein is a necessary and integral component of the junctional feet or whether it exerts predominantly a catalytic role in the formation of the triad junction.
Asunto(s)
Proteínas Musculares/aislamiento & purificación , Músculos/ultraestructura , Organoides/ultraestructura , Animales , Fraccionamiento Celular/métodos , Microscopía Electrónica , Microsomas/ultraestructura , Peso Molecular , Proteínas Musculares/fisiología , Músculos/fisiología , Conejos , Sales (Química)RESUMEN
Treatment of both transverse tubules and terminal cisternae with a combination of Triton X-100 and hypertonic K cacodylate causes dissolution of nonjunctional proteins and selective retention of membrane fragments which are capable of junction formation. Treatment of vesicles with Triton X-100 and either KCl or K gluconate causes complete dissolution of all components. Therefore K cacodylate exerts a specific preservative action on the junctional material. The membrane fragment from treatment of transverse tubules with Triton X-100 + cacodylate contains a protein of Mr = 80,000 in SDS gel electrophoresis as the predominant protein while lipid composition is enriched in cholesterol. The membrane fragment retains in electron microscopy the trilaminar appearance of the intact vesicles. Freeze fracture of transverse tubule fragments reveals a high density of low-profile, intercalated particles, which frequently form strings or occasional small arrays. The fragments from Triton X-100 plus cacodylate treatment of terminal cisternae include the protein of Mr = 80,000 as well as the spanning protein of the triad, calsequestrin, and some minor proteins. The fragments are almost devoid of lipid and display an amorphous morphology suggesting membrane disruption. The ability of the transverse tubular fragment, which contains predominantly the Mr = 80,000 protein, to form junctions with terminal cisternae fragments suggests that it plays a role in anchoring the membrane to the junctional processes of the triad. The junctional proteins may be solubilized in a combination of nonionic detergent and hypertonic NaCl. Subsequent molecular sieve chromatography gives an enriched preparation of the spanning protein. This protein has subunits of Mr = 300,000, 270,000 and 140,000 and migrates in the gel as a protein of Mr = 1.2 X 10(6) indicating a polymeric structure.
Asunto(s)
Membranas Intracelulares/ultraestructura , Proteínas de la Membrana/aislamiento & purificación , Proteínas Musculares/aislamiento & purificación , Músculos/ultraestructura , Animales , Ácido Cacodílico , Colesterol/aislamiento & purificación , Detergentes , Electroforesis en Gel de Poliacrilamida , Técnica de Fractura por Congelación , Lípidos de la Membrana/aislamiento & purificación , Microscopía Electrónica , Modelos Moleculares , Peso Molecular , Octoxinol , Fosfolípidos/aislamiento & purificación , Polietilenglicoles , ConejosRESUMEN
Prolongation of rat pancreatic islet allograft survival by a prior 7-day period of tissue culture was demonstrated, confirming previous reports by others. We then sought to identity those cells in islets capable of stimulating allograft rejection (Ia antigen-bearing cells) and to determine whether such cells and/or their Ia antigens might be reduced by tissue culture. Freshly isolated and 7-day-cultured Wistar-Furth rat islets were incubated with a mouse anti-rat Ia nonpolymorphic monoclonal antibody, then with peroxidase-conjugated goat anti-mouse antibody, and processed for electron microscopy. Peroxidase (Ia)-positive lymphocytes, macrophages, and capillary endothelial cells were identified in fresh but not in cultured islets. A radioligand assay, using 125I-protein A, revealed a 45% decrease in binding of Ia antibody to cultured compared with fresh islet cells. We conclude that Ia antigen-bearing lymphocytes, macrophages, and capillary endothelial cells in rat islets are reduced by tissue culture and that this may account, at least in part, for the decreased immunogenicity of cultured islet allografts.
Asunto(s)
Antígenos de Histocompatibilidad Clase II/análisis , Islotes Pancreáticos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Técnicas de Cultivo , Femenino , Supervivencia de Injerto , Técnicas para Inmunoenzimas , Islotes Pancreáticos/ultraestructura , Trasplante de Islotes Pancreáticos , Masculino , Microscopía Electrónica , Ensayo de Unión Radioligante , Ratas , Ratas Endogámicas Lew , Ratas Endogámicas WF , Factores de TiempoRESUMEN
Tannic acid mordanting during fixation of isolated vesicles from skeletal muscle enhanced the resolution of the images. Isolated triadic junctions displayed two characteristic features not previously described: (a) a clear gap separated terminal cisternae from transverse tubules; (b) this gap was bridged by a separating array of structures which resembled the "feet" of intact muscle. When the triad was broken in a French press and subsequently reassembled by joining the two organelles, a similar gap was seen but the structure of the feet was less well defined. When the membrane of the triad was extracted by Triton X-100, the junctional region was retained and a similar gap between the two organelles could be discerned. The terminal cisternae characteristically displayed a thickening of the cytoplasmic leaflet of the membrane in select areas in which electron-dense material was apposed on the luminal leaflet. This thickened membrane was not observed in longitudinal reticulum or in terminal cisternae regions distal to the electron-dense matter. This thickened leaflet was not invariably associated with the junction, and some junctional regions did not display discernible thickening of the membrane. When the triad was treated with KCl, the electron-dense aggregate was dispersed and the thickened leaflet of the terminal cisternae dissipated, whereas the triadic junctional region with its feet remained unchanged. KCl treatment caused dissolution of three proteins of Mr = 77,000, 43,000, and 38,000. Treatment of Triton-resistant vesicles with KCl caused the loss of electron-dense aggregate but did not otherwise influence the appearance of the junction. A good degree of correlation both qualitatively and in quantitative parameters between the isolated vesicles and the intact muscle was observed.