RESUMO
Two vesicular fractions and one nonvesicular fraction were prepared from crude synaptosomes by differential centrifugation and salting out with ammonium sulfate. Fraction 1 contained a mixture of coated vesicles, material thought to be derived from breakdown of the coats (shell fragments), and plain synaptic vesicles. Fraction 2 contained a mixture of plain synaptic vesicles and flocculent material. Fraction 3 contained flocculent material only. Fractions 1 and 3 were partially purified by passage through a Sephadex column. Fraction 3 contained no shell fragments but contained finer flocculent material which, it is suggested, is composed of unit particles either occurring singly or linked together into chainlike or amorphous aggregates. Each unit particle appears to have four subunits and is here referred to as a tetrasome. Tetrasomes sometimes appear to be attached to the surfaces of the plain synaptic vesicles. Also, it is possible that aggregates of tetrasomes form part of the structure of the presynaptic dense projections.
Assuntos
Encéfalo/citologia , Terminações Nervosas/análise , Vesículas Sinápticas/análise , Animais , Fracionamento Celular , Cromatografia por Troca Iônica , Cobaias , Substâncias Macromoleculares , Microscopia Eletrônica , Modelos Estruturais , Conformação Molecular , Organoides/análise , Coloração e Rotulagem , Frações Subcelulares/análise , Sinaptossomos/análiseRESUMO
Coated vesicles (CVs), plain synaptic vesicles (PSVs), and nonvesicular flocculent material were isolated from synaptosomes and examined with goniometry and high-resolution electron microscopy after either negative staining or various biochemical procedures. The flocculent material (i.e. the presynaptic matrix material except CV shells) is largely composed of particulate or elongated (chainlike) structures; some of this material (here referred to as particle/chain material) is attached to PSVs. The results obtained were: (a) the proteinaceous properties of the CV coat (also referred to as CV shell) and the particle/chain material were demonstrated with chymotrypsin; (b) the CV shell, studied with various negative-staining techniques, differs from the particle/chain material since it has no 3-4-nm globular subunits and reacts differently to alkaline pH; (c) the particle/chain material consists of aggregates of 3-4-nm globular subunits, four of which yield 8-10-nm fine particles; and these particles can be further aggregated into chains 8-10 nm wide and up to 30-60 nm long showing a "hollow" core; (d) vinblastine sulfate induced ringlike or helical crystalloid precipitates closely resembling the vinblastine-induced microtubule crystals reported in the literature, but vinblastine had no effect on either the CV shell material or the particle/chain material.
Assuntos
Encéfalo/ultraestrutura , Glicoproteínas/análise , Vesículas Sinápticas/ultraestrutura , Sinaptossomos/ultraestrutura , Tubulina (Proteína)/análise , Animais , Quimotripsina/farmacologia , Cobaias , Concentração de Íons de Hidrogênio , Microtúbulos , Coloração e Rotulagem , Vesículas Sinápticas/efeitos dos fármacos , Vimblastina/farmacologiaRESUMO
Amplification of the c-myc gene in the human promyelocytic leukemia cell line HL-60 is considered to be one of the major causes of its malignant phenotype. It is also well known since the establishment of the cell line that a culture of HL-60 cells contains a small but fixed percentage of spontaneously differentiated cells. We show that the spontaneous differentiation could be a result of extensive losses of amplified c-myc genes by the findings: (a) the spontaneously differentiated HL-60 cells express Mac-1 (CR3, CD11b/CD18) antigen, irreversibly stop the uptake of [3H]thymidine, and die by apoptosis; (b) these cells, when isolated, and when the copy number of c-myc genes is precisely quantitated, show extensive losses of c-myc genes; and (c) low concentrations of hydroxyurea increase the percentage of spontaneously differentiated cells in which the number of c-myc genes is further decreased. A simple theoretical consideration suggests that an active elimination process(es) must be operating besides the stochastic losses of the extrachromosomally amplified c-myc genes by unequal partition at mitosis.
Assuntos
Deleção de Genes , Genes myc/genética , Leucemia Promielocítica Aguda/genética , Sequência de Bases , Diferenciação Celular/genética , Eletroforese em Gel de Campo Pulsado , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hidroxiureia/farmacologia , Leucemia Promielocítica Aguda/patologia , Testes para Micronúcleos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Células Tumorais CultivadasRESUMO
Hypothesized risk factors for psychostimulant, amphetamine, and cocaine abuse include dopamine (DA) receptor polymorphisms, HIV infection, schizophrenia, drug-induced paranoias, and movement disorders; however, the molecular, cellular, and biochemical mechanisms that predispose to drug sensitivity or drive the development of addiction are incompletely understood. Using the Borna disease rat, an animal model of viral-induced encephalopathy wherein sensitivity to the locomotor and stereotypic behavioral effects of d-amphetamine and cocaine is enhanced (Solbrig et al., 1994, 1998), we identify a specific neurotrophin expression pattern triggered by striatal viral injury that increases tyrosine hydroxylase activity, an early step in DA synthesis, to produce a phenotype of enhanced amphetamine sensitivity. The reactive neurotrophin pattern provides a molecular framework for understanding how CNS viral injury, as well as other CNS adaptations producing similar growth factor activation profiles, may influence psychostimulant sensitivity.
Assuntos
Doença de Borna/metabolismo , Encéfalo/metabolismo , Fatores de Crescimento Neural/biossíntese , Transtornos Relacionados ao Uso de Substâncias/metabolismo , Animais , Western Blotting , Vírus da Doença de Borna/patogenicidade , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Encéfalo/virologia , Química Encefálica , Estimulantes do Sistema Nervoso Central/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/ultraestrutura , Corpo Estriado/virologia , Dextroanfetamina/farmacologia , Suscetibilidade a Doenças/virologia , Relação Dose-Resposta a Droga , Masculino , Atividade Motora/efeitos dos fármacos , Fosforilação , Testes de Precipitina , Ratos , Ratos Endogâmicos Lew , Tirosina 3-Mono-Oxigenase/análise , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Complete spinal transection in adult rats results in poor recovery of hind limb function, whereas significant spontaneous recovery can occur following spinal cord transection in rat neonates. The mechanisms underlying the recovery, however, are poorly understood. Recent studies in rodents suggested that the recovery is not due to axonal regeneration, but rather due to reorganization of the neural circuits in the spinal cord below the injury site, including central pattern generators. Few studies have reported histological evidence for changes in the primary sensory fibers or terminals. Thus, in the present study, we transected spinal cords of rats at thoracic level 8 at postnatal day 5. Four weeks after the injury, biotinylated-dextran amine (BDA), an anterograde tracer, was injected into the dorsal root ganglion of the lumbar spinal cord to examine the localization of sensory fibers and their terminal buttons in the spinal cord. BDA-positive axons in the rat spinal cord following neonatal spinal transection (neo ST) were longer than those in sham-operated or normal rats. The number of terminal buttons was also higher in spinal cords of neo ST rats compared with sham-operated or normal rats. These findings suggest that sensory fibers project more strongly and make more synapses following neo ST to compensate for the lack of supraspinal projections.
Assuntos
Axônios/patologia , Gânglios Espinais/patologia , Células Receptoras Sensoriais/patologia , Traumatismos da Medula Espinal/patologia , Medula Espinal/patologia , Animais , Animais Recém-Nascidos , Axônios/fisiologia , Biotina/análogos & derivados , Dextranos , Modelos Animais de Doenças , Gânglios Espinais/fisiopatologia , Substância Cinzenta/patologia , Substância Cinzenta/fisiopatologia , Vértebras Lombares , Técnicas de Rastreamento Neuroanatômico , Plasticidade Neuronal , Marcadores do Trato Nervoso , Ratos Wistar , Células Receptoras Sensoriais/fisiologia , Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/fisiopatologia , Vértebras TorácicasRESUMO
Spontaneous nerve regeneration beyond the scar frequently occurs in fish after spinal cord lesions, in contrast to mammals. Here we examined the spatiotemporal relationship between the fibrous scar and axonal regeneration in the goldfish. Within 1 week after hemisection of the spinal cord, the open wound was closed by a fibrous scar that was demarcated from the surrounding nervous tissue by the glia limitans, which was immunoreactive for laminin. Within 1 week after hemisection, regenerating axons entered the fibrous scar, and were surrounded by laminin-coated tubular structures continuous with the glia limitans. Regenerating axons that initially entered the fibrous scar were usually accompanied by glial processes. Within 2-3 weeks after hemisection, the tubular structures became enlarged, and the regenerating axons increased in number, fasciculating in the tubules. Glial processes immunoreactive for glial fibrillary acid protein and 5-hydroxytryptamine neurons then entered the tubular structures to associate with the regenerating axons. The tubular structures developed further, creating tunnels that penetrated the fibrous scar, through which the regenerating axons passed. At 6-12 weeks after hemisection, the fibrous scar was smaller and the enlarged tunnels contained many glial processes and several axons. The findings of present study demonstrated that, following spinal lesions in goldfish, regenerating axons enter and pass the scar tissue. The regenerating axons first enter the fibrous scar with glial elements and then grow through laminin-coated tubular structures within the fibrous scar. Invasion by glial processes and neuronal elements into the tubular structures reduces the fibrous scar area and allows for more regenerating axons to pass beyond the fibrous scar.
Assuntos
Axônios/fisiologia , Cicatriz/etiologia , Cicatriz/patologia , Regeneração Nervosa/fisiologia , Traumatismos da Medula Espinal/complicações , Animais , Antígenos/metabolismo , Axônios/ultraestrutura , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Carpa Dourada , Laminina/metabolismo , Microscopia Eletrônica de Transmissão , Atividade Motora/fisiologia , Fibras Nervosas/metabolismo , Fibras Nervosas/patologia , Proteínas do Tecido Nervoso , Proteoglicanas/metabolismo , Piridinas/metabolismo , Recuperação de Função Fisiológica , Rodaminas/metabolismo , Serotonina/metabolismo , Traumatismos da Medula Espinal/patologia , Estatísticas não Paramétricas , Fatores de Tempo , Tubulina (Proteína)/metabolismoRESUMO
The interaction between [131I]-thyroglobulin liposomes and thyroidal lysosomes was used as an in vitro model system for analyzing the relation of colloid droplets to lysosomes in follicular cells. The rates of hydrolysis of [131I]-thyroglobulin in a liposome-lysosome system (Lipo-Lyso system) and a thyroglobulin-lysosome system (TG-Lyso system) were compared. Hydrolysis of thyroglobulin in the Lipo-Lyso system increased hyperbolically and was greater than that in the TG-Lyso system for about 10 h. Liposomal thyroglobulin was not degraded by the 20,000 X g supernatant obtained on disruption of the lysosomal fraction. On varying the pH of the incubation medium, the highest activity was observed under acidic conditions in both systems. Under neutral or weakly alkaline conditions, the Lipo-Lyso system still showed 50% of the maximal hydrolytic activity, while the TG-Lyso system showed no activity. ATP and anaerobic conditions had no effect on either system. Cysteine (5 X 10-2M) and p-chloromercuribenzene sulfonic acid (PCMBS, 10-3 M) had not influence on hydrolysis in the Lipo-Lyso system, but in the TG-Lyso system cysteine greatly increased, and PCMBS significantly reduced the rate of hydrolysis. Dibutyryl cyclic AMP had no effect. Chlorpromazine (Cpz) decreased liposomal thyroglobulin hydrolysis in a concentration-dependent manner. In the TG-Lyso system, concentrations of 10-4M Cpz had no effect. These results strongly suggest that liposomes rapidly fused with lysosomes, providing optimal conditions for hydrolysis of thyroglobulin.
Assuntos
Lipossomos/metabolismo , Lisossomos/metabolismo , Modelos Biológicos , Tireoglobulina/metabolismo , Hormônios Tireóideos/metabolismo , Trifosfato de Adenosina/farmacologia , Anaerobiose , Animais , Bucladesina/farmacologia , Cloromercurobenzoatos/farmacologia , Clorpromazina , Cisteína/farmacologia , Concentração de Íons de Hidrogênio , Hidrólise , Técnicas In Vitro , Ratos , Glândula Tireoide/ultraestrutura , Fatores de TempoRESUMO
Primary sensory trigeminal projections were investigated in the hagfish following application of horseradish peroxidase (HRP) to the sensory branches. In our control preparations we were able to distinguish five sensory ganglia and their respective nerves. HRP application confirmed the almost exclusive relation of each of these nerves to their respective ganglia, with very little overlap. In normal frontal sections of the medulla oblongata, five columns of fibers surrounded by neuronal cell bodies could be clearly distinguished, but the number is probably fortuitous, for there was no one-on-one relationship with the five trigeminal ganglia. From their peripheral connections, we surmised that columns 1 and 3 handle general cutaneous sensation, columns 2, 4, and 5 handle taste sensation, and column 5 handles general mucous cutaneous sensation conveyed by utricular ganglion cells. Dorsally located columns received projections from nerves with dorsal peripheral connections, and more ventrally located columns received projections from nerves with ventral peripheral connections. This relation is the reverse of that seen in other vertebrates.
Assuntos
Peixes/anatomia & histologia , Feiticeiras (Peixe)/anatomia & histologia , Neurônios Aferentes/análise , Nervo Trigêmeo/anatomia & histologia , Vias Aferentes/anatomia & histologia , Vias Aferentes/citologia , Animais , Mapeamento Encefálico , Gânglios/anatomia & histologia , Gânglios/citologia , Peroxidase do Rábano Silvestre , Pele/inervação , Paladar/fisiologia , Nervo Trigêmeo/citologiaRESUMO
The hagfish lateral line system was studied by horseradish peroxidase transganglionic transport. The anterior lateral line nerve innervates the group of lateral line canals situated anteriorly to the eye, and the posterior lateral line nerve innervates the group of canals situated posteriorly to the eye. Although both nerves pass through the muscle fascia at the same point, each runs a different course to the brain. The anterior lateral line nerve runs near the trigeminal nerve and its ganglion is closely attached to the trigeminal ganglion, but both systems are completely independent. The posterior lateral line nerve runs independently of any other cranial nerve and makes a peculiar U-turn at the point of entry to the brain capsule. The anterior lateral line ganglion contains both cutaneous sensory cells (small to large cells) and lateral line sensory cells (small cells); from this ganglion projections run to both the trigeminal sensory nucleus (fine and thick fibers) and medial nucleus of the area acousticolateralis (fine fibers). The posterior lateral line ganglion contains only small lateral line cells that project fine fibers to the medial nucleus of the area acousticolateralis. There are no efferent components in this lateral line system, and its only afferent terminal field is the medial nucleus of the area acousticolateralis.
Assuntos
Encéfalo/anatomia & histologia , Peixes/anatomia & histologia , Feiticeiras (Peixe)/anatomia & histologia , Nervos Periféricos/anatomia & histologia , Vias Aferentes/anatomia & histologia , Animais , Peroxidase do Rábano Silvestre , Nervos Periféricos/citologiaRESUMO
Immunoreactivity for substance P and cholecystokinin-8 was examined in the nerve fibers in the central autonomic nucleus, a cell column for sympathetic preganglionic neurons, in the filefish Stephanolepis cirrhifer. Substance P-immunoreactive fibers were distributed throughout the entire rostrocaudal extent, but were more abundant in the caudal part of the column, where substance P-immunoreactive varicosities sometimes made contacts with the sympathetic preganglionic neurons. Cholecystokinin-8-immunoreactive fibers were found almost entirely in the rostral part of the column, where a dense network of varicosities was in close apposition to a considerable number of the sympathetic preganglionic neurons. Double labeling immunohistochemistry showed that substance P fibers and cholecystokin-8 fibers were entirely different, and distinct from serotonin-immunoreactive fibers. By using immunoelectron microscopy, synaptic specialization was sometimes observed between the dendrites of preganglionic neurons and varicosities immunoreactive for substance P and cholecystokinin-8. Substance P- and cholecystokinin-8 fibers were seen from the descending trigeminal tract, through the dorsolateral funiculus and the ventral portion of the dorsal horn, to the central autonomic nucleus. After colchicine treatment, substance P-immunoreactive perikarya were found in the cranial and spinal sensory ganglia. These results suggest that the sympathetic preganglionic neurons of the filefish receive innervation by substance P fibers and cholecystokinin fibers, and that the former might be of primary sensory origin. Topographical distribution of cholecystokinin-8-immunoreactive terminals in the central autonomic nucleus along the rostrocaudal extent might underlie the differential regulation of sympathetic activity via a distinct population of sympathetic preganglionic neurons.
Assuntos
Fibras Autônomas Pré-Ganglionares/metabolismo , Colecistocinina/metabolismo , Peixes/metabolismo , Terminações Pré-Sinápticas/metabolismo , Medula Espinal/metabolismo , Substância P/metabolismo , Sistema Nervoso Simpático/metabolismo , Animais , Fibras Autônomas Pré-Ganglionares/citologia , Peixes/anatomia & histologia , Gânglios Sensitivos/citologia , Gânglios Sensitivos/metabolismo , Bulbo/citologia , Bulbo/metabolismo , Vias Neurais/citologia , Vias Neurais/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Sincalida/metabolismo , Medula Espinal/citologia , Sistema Nervoso Simpático/citologiaRESUMO
The four kinds of oligopeptides specific in amino acid sequence to a rat dopamine transporter (DAT), peptide-1-peptide-4, were chemically synthethized. An attempt to produce antipeptide antibodies against these oligopeptides was made with an in vitro immunization method. Two monoclonal antibodies, MAbs H-1a and H-1b, were produced against one of the oligopeptides, peptide-1. Western blot analysis confirmed that the two antibodies recognized an approximately 85,000 Da protein in a synaptosomal fraction prepared from the rat striatum but none in the fraction from the cerebellum. The specificity of the antibody to DAT was also confirmed by an antibody absorption test using two synthetic oligopeptides, one of which is specific only to DAT. These results have confirmed the specificity of the present antibody to DAT. The expression and subcellular localization of DAT were immunohistochemically examined with MAbs H-1a and H-1b in PC12 cells treated with nerve growth factor (NGF). The antibody labeled the surface of PC12 cells. When the cells were treated with NGF, the expression of DAT was significantly emphasized, first in the area mainly including the Golgi apparatus and rough endoplasmic reticulum and then on the surface of growth cones from the beginning of neurite outgrowth. DAT was detected by Western blot analysis in a microsomal fraction prepared from PC12 cells. The activity of DAT in the PC12 cells was pharmacologically confirmed by the uptake of [3H]-dopamine and blockade by uptake inhibitors. The NGF treatment doubled the dopamine uptake activity. GBR12909, a specific inhibitor of DAT, blocked the [3H]-dopamine at a concentration of 10(-7) M. The expression of DAT and norepinephrine transporter (NET) mRNA in the PC12 cells was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). DAT mRNA significantly increased in the NGF-treated cells after 7 days of incubation, whereas NET mRNA markedly decreased.
Assuntos
Proteínas de Transporte/metabolismo , Dopamina/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Neuritos/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Antagonistas de Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Imuno-Histoquímica , Dados de Sequência Molecular , Fatores de Crescimento Neural/metabolismo , Células PC12 , Peptídeos/síntese química , Peptídeos/imunologia , Reação em Cadeia da Polimerase , RatosRESUMO
The potential of purple corn color (PCC), a natural anthocyanin, to modify colorectal carcinogenesis was investigated in male F344/DuCrj rats, initially treated with 1,2-dimethylhydrazine (DMH), receiving 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the diet. After DMH initiation, PCC was given at a dietary level of 5.0% in combination with 0.02% PhIP until week 36. No PCC-treatment-related changes in clinical signs, body weight and food consumption were found. Incidences and multiplicities of colorectal adenomas and carcinomas in rats initiated with DMH were clearly increased by PhIP. In contrast, lesion development was suppressed by PCC administration. Furthermore, in the non-DMH initiation groups, induction of aberrant crypt foci by PhIP tended to be decreased by the PCC supplementation. The results thus demonstrate that while PhIP clearly exerts promoting effects on DMH-induced colorectal carcinogenesis, these can be reduced by 5.0% PCC in the diet, under the present experimental conditions.
Assuntos
Adenocarcinoma/prevenção & controle , Adenoma/prevenção & controle , Antocianinas/farmacologia , Anticarcinógenos/farmacologia , Neoplasias Colorretais/prevenção & controle , Glucosídeos/farmacologia , Zea mays/química , 1,2-Dimetilidrazina/administração & dosagem , Adenocarcinoma/induzido quimicamente , Adenocarcinoma Mucinoso/induzido quimicamente , Adenocarcinoma Mucinoso/prevenção & controle , Adenoma/induzido quimicamente , Administração Oral , Animais , Antocianinas/administração & dosagem , Antocianinas/química , Anticarcinógenos/administração & dosagem , Peso Corporal/efeitos dos fármacos , Carcinógenos/administração & dosagem , Cocarcinogênese , Doenças do Colo/induzido quimicamente , Doenças do Colo/prevenção & controle , Neoplasias Colorretais/induzido quimicamente , Esquema de Medicação , Ensaios de Seleção de Medicamentos Antitumorais , Glucosídeos/administração & dosagem , Glucosídeos/química , Hiperplasia , Imidazóis/administração & dosagem , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Neoplasias do Jejuno/induzido quimicamente , Neoplasias do Jejuno/prevenção & controle , Masculino , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/prevenção & controle , Neoplasias da Próstata/induzido quimicamente , Neoplasias da Próstata/prevenção & controle , Ratos , Ratos Endogâmicos F344 , Glândulas Seminais/efeitos dos fármacos , Glândulas Seminais/patologiaRESUMO
RATIONALE AND OBJECTIVES: Hepatocellular carcinomas (HCCs) usually consist of components of different histologic grade. Using surgically resected specimens, the authors obtained high-resolution magnetic resonance (MR) images and studied the relationship between histologic grade of HCCs and MR signal intensity. METHOD: In vitro MRI was performed on 15 small (less than 20-mm diameter) HCCs and 2 patients with adenomatous hyperplasia (AH) within 1 hour of surgical resection. In these 17 lesions, 24 macroscopic nodular components corresponding to MR images were recognized pathologically. The difference in MR signal intensity was examined by using tumor/phantom (T/P) signal-intensity ratio. In addition, the correlations between signal intensity on MRI and histologic criteria for grading HCC were studied. RESULTS: On T2-weighted images, grade II HCCs had significantly greater T/P values than grade I HCCs (P < .01). There is the correlation (r = .88, P < .001) between nucleocytoplasmic (N/C) ratio and signal intensity of small HCCs and AHs on T2-weighted images. CONCLUSION: The N/C ratio is an important factor relating the signal intensity to the histologic grade of these lesions on T2-weighted images.
Assuntos
Carcinoma Hepatocelular/diagnóstico , Neoplasias Hepáticas/diagnóstico , Fígado/patologia , Imageamento por Ressonância Magnética , Idoso , Carcinoma Hepatocelular/patologia , Feminino , Humanos , Hiperplasia/diagnóstico , Hiperplasia/patologia , Técnicas In Vitro , Neoplasias Hepáticas/patologia , Imageamento por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-IdadeRESUMO
Coated vesicles isolated from bovine brain contained a protein kinase(s) which phosphorylated phosvitin and an endogenous protein with a molecular weight (Mr) of 48,000. A clathrin light chain (Mr 33,000), a constituent of the coat structure of the coated vesicles, was also phosphorylated when histone was added to the incubation medium. The clathrin light chain was phosphorylated with GTP as well as ATP as the phosphoryl donor. The phosphorylation reaction was inhibited by heparin. An additional 1.35 mol of PO4/mol was incorporated into the clathrin light chain which had contained approximately 1.5 mol of PO4/mol when the coated vesicles were incubated with ATP, Mg2+, and histone. Phosphoamino acid determination revealed the presence of 32P-phosphorylated threonine and serine in phosvitin, threonine in the endogenous protein (Mr 48,000) and serine in the clathrin light chain (Mr 33,000).
Assuntos
Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Endossomos/metabolismo , Histonas/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Química Encefálica , Bovinos , Guanosina Trifosfato/metabolismo , Heparina/farmacologia , FosforilaçãoRESUMO
Coated vesicles prepared from bovine brain contained cyclic nucleotides- and Ca2+-calmodulin-independent protein kinases which in the presence of Mg2+ catalyzed the phosphorylation of an endogenous 48,000 Mr protein of coated vesicles (C-48), phosvitin and troponin T. Phosvitin was phosphorylated either in the presence of ATP or GTP. The phosphorylation of C-48, on the other hand, was specific for ATP. Heparin inhibited the phosphorylation of phosvitin but not that of C-48. Mn2+ inhibited the phosphorylation of phosvitin, while Mn2+ substituted for Mg2+ in the phosphorylation of C-48. When the coated vesicles were prepared in the presence of NaF, C-48 contained 2.5-2.8 mol of phosphate/mol. On incubation with Mg2+ and ATP, C-48 incorporated 1.2-1.6 mol of phosphate/mol. With C-48 as a substrate, the value of its apparent Km for ATP was 6 microM. With phosvitin as a substrate, the value of its apparent Km was 20 microM. The phosphorylated amino acid residues in the phosvitin were identified as serine and threonine. Phosphothreonine was detected in C-48. These results suggest that brain coated vesicles possess two different classes of protein kinase, a casein kinase II and C-48 kinase.
Assuntos
Encéfalo/enzimologia , Invaginações Revestidas da Membrana Celular/metabolismo , Endossomos/metabolismo , Proteínas Quinases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Bovinos , Guanosina Trifosfato/metabolismo , Heparina/farmacologia , Cinética , Manganês/farmacologia , Proteínas de Membrana/metabolismo , Fosvitina/metabolismo , Sais/farmacologia , Especificidade por Substrato , Troponina/metabolismo , Troponina TRESUMO
Proximal gastric vagotomy has been widely used as a surgical treatment for peptic ulcer disease. However, it is technically complex and time-consuming. Moreover, it may cause circulatory problems in the gastric mucosa. We have reported a new method of blood flow-preserving vagotomy with a carbon dioxide laser (CO2 laser vagotomy) developed in our laboratory. To assess its efficacy, we used cysteamine-induced ulcer and measured gastric mucosal blood flow in rats. The incidence of cysteamine-induced ulcer formation was reduced significantly in the group that underwent CO2 laser vagotomy compared with a group treated with proximal gastric vagotomy. Gastric mucosal blood flow was significantly better in the CO2 laser vagotomy group. Long-term follow-up of acid reduction was evaluated in dogs by the pentagastrin-stimulation test. Acid reduction in dogs was satisfactory during the 12 months of this study. CO2 laser vagotomy is a new, easy, time-saving, and circulatory-preserving technique for peptic ulcer disease.
Assuntos
Terapia a Laser , Vagotomia Gástrica Proximal , Animais , Dióxido de Carbono , Cisteamina , Cães , Úlcera Duodenal/induzido quimicamente , Úlcera Duodenal/cirurgia , Mucosa Gástrica/irrigação sanguínea , Masculino , Pentagastrina , Ratos , Ratos Endogâmicos , Fluxo Sanguíneo RegionalRESUMO
A fraction of synaptic junctional complex (SJC) was prepared from rat synaptosomes and served as antigen material to produce monoclonal antibodies (Mab) for examining the component proteins of the SJC. An antibody, Mab SJ-8, was obtained, which recognized a protein with a molecular weight of 82,000 Da in the SJC preparation by immunoblot analysis. The immunohistochemical localization of the 82 kDa protein was studied with the rat cerebellum. Mab SJ-8 labeled the peripheral areas of the Purkinje and granule cells. Small punctate areas were also stained in the molecular layer with SJ-8. Intracellular localization of the protein was examined with rat brain synaptosomes. Immunoelectron microscopy demonstrated that Mab SJ-8 strongly labeled the postsynaptic density (PSD) and also a fibrous network spreading out of it. However, the antibody did not label the pre- or post-synaptic membrane or the cleft material.
Assuntos
Citoesqueleto/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sinapses/metabolismo , Animais , Anticorpos Monoclonais , Eletroforese em Gel de Poliacrilamida , Imuno-Histoquímica , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Imunoeletrônica , Ratos , Ratos Wistar , Frações Subcelulares/química , Frações Subcelulares/metabolismoRESUMO
After horseradish peroxidase was applied to the main trunk of the mouse vagal nerve, anterogradely labeled cells in the vagal ganglia and fibers in the solitary complex, and retrogradely labeled cells in the dorsal motor nucleus and the ambiguous nucleus were observed. Most of the cells in the nodose ganglion were labeled, but only a few cells in the jugular ganglion were labeled. Heavily labeled nerve terminals and fibers were found in 3 areas in the solitary nucleus: i.e., the lateral half of the medial nucleus, the ventrolateral nucleus, and the commissural nucleus. There was only weak labeling in the dorsolateral nucleus, ventral nucleus, and intermediate nucleus. Substance P immunoreactive neurons in the vagal ganglia were found in the jugular ganglion and the dorsal part of the nodose ganglion, but not in the ventral part of the nodose ganglion. Substance P immunoreactivity in the solitary nucleus was moderate in the commissural nucleus and the intermediate nucleus, but was lacking or very weak in the lateral half of the medial nucleus, ventral nucleus, dorsolateral nucleus, and ventrolateral nucleus. We conclude that most substance P containing fibers in the main trunk of the vagal nerve project centrally to the commissural nucleus and peripherally to some of the thoracic viscera.
Assuntos
Substância P/metabolismo , Nervo Vago/metabolismo , Animais , Peroxidase do Rábano Silvestre , Bulbo/citologia , Bulbo/metabolismo , Camundongos , Vias Neurais/anatomia & histologia , Gânglio Nodoso/citologia , Gânglio Nodoso/metabolismo , Nervo Vago/citologiaRESUMO
Crotaline snakes, which have infrared-sensitive pit organs, provide a good model for linking neuron morphology with sensory modality. In the trigeminal ganglion of the habu, Trimeresurus flavoviridis, cells positive for calcitonin gene-related peptide-like (CGRP) immunoreactivity were found to be of two types, darkly stained and lightly stained. They were pseudo-unipolar, having an axon divided into stem, peripheral branch, and central branch, all of which were 1 micron or less in diameter. Other, CGRP-negative cells in the ganglion were also pseudo-unipolar, but much larger. In configuration, some of the positive cells were similar to the neurons with A-delta fibers, and others to the neurons with C fibers that have been reported by other workers. On the basis of their distribution and density, and physiological studies by other workers, the CGRP-positive cells were judged to be not part of the infrared-receptive system, but to be involved in the transmission of nociception in small fibers.