RESUMO
The neuromuscular junction (NMJ) is the linchpin of nerve-evoked muscle contraction. Broadly considered, the function of the NMJ is to transduce a nerve action potential into a muscle fiber action potential (MFAP). Efficient information transfer requires both cholinergic signaling, responsible for the generation of endplate potentials (EPPs), and excitation, the activation of postsynaptic voltage-gated sodium channels (Nav1.4) to trigger MFAPs. In contrast to the cholinergic apparatus, the signaling pathways that organize Nav1.4 and muscle fiber excitability are poorly characterized. Muscle-specific kinase (MuSK), in addition to its Ig1 domain-dependent role as an agrin-LRP4 receptor, is also a BMP co-receptor that binds BMPs via its Ig3 domain and shapes BMP-induced signaling and transcriptional output. Here we probed the function of the MuSK-BMP pathway at the NMJ using mice lacking the MuSK Ig3 domain ('ΔIg3-MuSK'). Synapses formed normally in ΔIg3-MuSK animals, but the postsynaptic apparatus was fragmented from the first weeks of life. Anatomical denervation was not observed at any age examined. Moreover, spontaneous and nerve-evoked acetylcholine release, AChR density, and endplate currents were comparable to WT. However, trains of nerve-evoked MFAPs in ΔIg3-MuSK muscle were abnormal as revealed by increased jitter and blocking in single fiber electromyography. Further, nerve-evoked compound muscle action potentials (CMAPs), as well as twitch and tetanic muscle torque force production, were also diminished. Finally, Nav1.4 levels were reduced at ΔIg3-MuSK synapses but not at the extrajunctional sarcolemma, indicating that the observed excitability defects are the result of impaired localization of this voltage-gated ion channel at the NMJ. We propose that MuSK plays two distinct roles at the NMJ: as an agrin-LRP4 receptor necessary for establishing and maintaining cholinergic signaling, and as a BMP co-receptor required for maintaining proper Nav1.4 density, nerve-evoked muscle excitability and force production. The MuSK-BMP pathway thus emerges as a target for modulating excitability and functional innervation, which are defective in conditions such as congenital myasthenic syndromes and aging.
RESUMO
By means of spin-polarized electron coincidence spectroscopy we explore the fundamental issue of spin-resolved contributions to the exchange-correlation hole in many-electron systems. We present a joint experimental and theoretical study of correlated electron pair emission from a ferromagnetic Fe(001) surface induced by spin-polarized low-energy electrons. We demonstrate that the contribution to the exchange-correlation hole due to exchange is more extended than the contribution due to the screened Coulomb interaction.
RESUMO
High resolution x-ray lithographic studies of cells from chick embryo hearts dried by the CO2 critical point method have been made with soft x-ray radiation of different wavelengths. A marked difference in the relief replica in polymethyl methacrylate (PMMA) resulting from the differential absorption by the dried cells of carbon K alpha radiation at 4.48 nm and broad band synchrotron radiation (SR) with lambda is greater than 1.5 nm demonstrates the potential usefulness of the technique in making high resolution (approximately or equal to 10 nm) chemical identification of the constitutents which make up the various parts of the cell.
Assuntos
Microanálise por Sonda Eletrônica/métodos , Técnicas Histológicas , Miocárdio/ultraestrutura , Animais , Embrião de Galinha , Microscopia Eletrônica de Varredura , Organoides/ultraestrutura , Fotomicrografia/métodosRESUMO
Improved image quality in soft x-ray contact microscopy can be obtained by examining the resist with transmission rather than scanning electron microscopy. Application of the new technique to air-dried preparations of human blood platelets reveals structures not visible in the same cells with transmission electron microscopy or when the resist is examined by scanning electron microscopy. As seen by the new technique, platelet pseudopods contain a central structure connected to a network in the platelet and dense bodies exhibit a lamellar structure.
Assuntos
Plaquetas/ultraestrutura , Humanos , Microscopia Eletrônica/instrumentação , Microscopia Eletrônica/métodos , Microscopia Eletrônica de VarreduraRESUMO
Soft x-ray contact microscopy, utilizing single-shot exposures of approximately 60 nanoseconds duration in polymethyl methacrylate, has been realized with a resolution of 300 angstroms. The radiation spectrum is intense in the "window" between 23 and 44 angstroms where water is transparent compared to biological materials, and therefore permits viewing of wet samples.
RESUMO
A 100-nanosecond pulse of long-wavelength x-rays was used to produce high-resolution stop-motion images of living human platelets. Although some aspects of the structure conform to those seen in dehydrated specimens, novel features are apparent. The technique should permit detailed stop-motion examination of the interaction of platelets with their surrounding medium as well as exploration of the phagocytic and secretory activities of a wide variety of other cells.
Assuntos
Plaquetas/ultraestrutura , Microscopia/métodos , Radiação Ionizante , Raios X , Humanos , Microscopia Eletrônica de Varredura , Pseudópodes/ultraestruturaRESUMO
X-ray holography offers the possibility of three-dimensional microscopy with resolution higher than that of the light microscope and with contrast based on x-ray edges. In principle, the method is especially advantageous for biological samples if x-rays in the wavelength region between the carbon and oxygen K edges are used. However, until now the achieved resolution has not exceeded that of the light microscope because of the poor coherence properties of the x-ray sources and the low resolution of the detectors that were available. With a recently developed x-ray source based on an undulator on an electron storage ring, and high resolution x-ray resist, a hologram has been recorded at about 400-angstrom resolution. The experiment utilized x-rays with wavelengths of 24.7 angstroms and required a 1-hour exposure of the pancreatic zymogen granules under study.
Assuntos
Grânulos Citoplasmáticos/ultraestrutura , Precursores Enzimáticos , Holografia/métodos , Animais , Grânulos Citoplasmáticos/enzimologia , Microscopia Eletrônica , Pâncreas/ultraestrutura , Ratos , Raios XRESUMO
X-ray micrographs of biological objects have been obtained with a resolution better than 1000 angstroms by using poly(methyl methacrylate) x-ray resist and carbon Kalpha or synchrotron radiation. Synchrotron radiation allows short exposure times; storage rings especially designed as radiation sources and improved x-ray resists would make exposure times under 1 second possible.
Assuntos
Microscopia/métodos , Raios X , Eucariotos/ultraestrutura , Microscopia/instrumentação , Microscopia Eletrônica de Varredura/métodosRESUMO
Calculations are presented which indicate an extensive suboptical region in the microscopy of biological materials in their natural state which is accessible to ultrasoft x-ray transmission microscopy. Throughout most of the region, radiation dosage levels to the specimen are lower than in electron microscopy.
Assuntos
Microscopia/instrumentação , Radiação Ionizante , Raios X , Transferência de Energia , Microscopia Eletrônica , Proteínas , Doses de Radiação , ÁguaRESUMO
X-ray micrographs of biological materials have been obtained with a resolution better than 100 angstroms by using x-ray resist as the recording medium. A high-resolution scanning electron microscope with a short-focal-length final lens, operating in the "low-loss" mode, is used to make the smallest features in the x-ray replica visible.
Assuntos
Microscopia/instrumentação , Animais , Anuros , Cromossomos/ultraestrutura , Drosophila melanogaster/ultraestrutura , Metacrilatos , Microscopia Eletrônica de Varredura , Epitélio Pigmentado Ocular/ultraestrutura , Rana catesbeiana , Raios XRESUMO
The Upper Wide Angle Viewing System (UWAVS) will be installed on five upper ports of ITER. This paper shows major requirements, gives an overview of the preliminary design with reasons for some design choices, examines self-emitted IR light from UWAVS optics and its effect on accuracy, and shows calculations of signal-to-noise ratios for the two-color temperature output as a function of integration time and divertor temperature. Accurate temperature output requires correction for vacuum window absorption vs. wavelength and for self-emitted IR, which requires good measurement of the temperature of the optical components. The anticipated signal-to-noise ratio using presently available IR cameras is adequate for the required 500 Hz frame rate.
RESUMO
A young man with chronic auditory hallucinations was treated according to the principle that increasing external auditory stimulation decreases the likelihood of auditory hallucinations. Listening to a radio through stereo headphones in conditions of low auditory stimulation eliminated the patient's hallucinations.
Assuntos
Estimulação Acústica/métodos , Percepção Auditiva , Alucinações/terapia , Rádio/instrumentação , Adulto , Feminino , Humanos , Masculino , Transtornos Mentais/psicologiaRESUMO
We have characterized the early biosynthetic forms of the histidine-rich protein (HisRP), a major, granule-bound protein (Mr 58 000) of the avian malarial parasite Plasmodium lophurae. We have translated poly(A)-containing, size-selected parasite mRNA in the wheat germ cell-free system in the presence of [3H]histidine. HisRP was synthesized as a larger precursor (Mr 63 000). When dog pancreas microsomal membranes were present in the cell-free system during translation, a still larger form of HisRP (Mr 66 000) was detected. This larger form was segregated into the dog pancreas microsomal vesicles and was core glycosylated. Presumably, it corresponds to an intermediate form located in the parasite rough endoplasmic reticulum (RER). The difference in the Mr of approx. 8 000 between this RER associated 'pro' form and the granule-bound, mature form of HisRP suggests that proteolytic processing occurs upon transport from the RER to the granule. Segregation and core glycosylation were strictly coupled to translation and were not observed upon posttranslational addition of microsomal membranes. Thus, the early events in the biosynthesis of HisRP are similar to those established for secretory and lysosomal proteins.
Assuntos
Plasmodium/metabolismo , Biossíntese de Proteínas , Proteínas , Animais , Cães , Retículo Endoplasmático/metabolismo , Técnicas In Vitro , Microssomos/metabolismo , Peso Molecular , Pâncreas/metabolismo , Precursores de Proteínas/biossínteseRESUMO
Recombinant cDNA clones representing the carboxy-terminal portion of the histidine-rich protein of Plasmodium lophurae and the 3' untranslated region of the mRNA have been sequenced. Histidine accounts for 78% of the predicted amino acid sequence. The DNA and protein sequences in this region differ significantly from published sequences deduced from cloned genomic DNA of P. lophurae. Sequence data from two independent cDNA clones, comparison of restriction endonuclease sites present in genomic DNA, genomic and cDNA clones, gene titrations, S1 nuclease digestion of cDNA-mRNA hybrids and comparison of predicted and published data for the amino acid composition of the histidine-rich protein all suggest that P. lophurae contains one histidine-rich protein gene and that the sequence of the 3' coding region of this gene has been correctly deduced from the cDNA clones.
Assuntos
Genes , Plasmodium/genética , Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , DNA/análise , Enzimas de Restrição do DNA , DNA Recombinante , Endonucleases/metabolismo , Minicomputadores , Hibridização de Ácido Nucleico , Proteínas/análise , RNA Mensageiro/análise , Sequências Repetitivas de Ácido Nucleico , Endonucleases Específicas para DNA e RNA de Cadeia SimplesRESUMO
PURPOSE: Keratoconus is characterized by thinning and scarring of the central region of the cornea. Previous research showed that, in corneas obtained from patients with keratoconus, lysosomal enzyme activities are elevated, whereas levels of protease inhibitors such as alpha1-proteinase inhibitor are reduced. This study was undertaken to examine further the expression of a spectrum of proteolytic enzymes and protease inhibitors. METHODS: Corneal buttons were collected from patients with keratoconus, healthy subjects, and patients with other corneal diseases. Immunohistochemical staining was performed on paraffin sections. Enzymatic assays and western blot analysis were carried out for cathepsins B and G. In addition, an in situ zymography procedure was used to examine the gelatin- and casein-digesting activities in corneas with keratoconus. RESULTS: An enhanced staining was found with antibodies to cathepsins B and G. Enzymatic assays and western blotting confirmed that the levels of these two enzymes were elevated in corneas with keratoconus. No alteration was noted with any of the matrix metalloproteinase (MMP) family members and other enzymes and inhibitors examined, although in situ zymography did indicate an increase in net gelatin- and casein-digesting activities in corneas with keratoconus. These activities were mostly abolished by inhibitors for serine and cysteine proteinases, but not by those for MMPs and aspartic proteinases. CONCLUSIONS: Levels of cathepsins B and G are increased in corneas with keratoconus. These enzymes may contribute to the heightened in situ gelatin- and casein-digesting activities, leading to abnormalities in keratoconus.
Assuntos
Córnea/enzimologia , Endopeptidases/metabolismo , Ceratocone/enzimologia , Inibidores de Proteases/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Criança , Pré-Escolar , Córnea/patologia , Doenças da Córnea/enzimologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Técnicas Imunoenzimáticas , Ceratocone/patologia , Pessoa de Meia-IdadeRESUMO
PURPOSE: To compare the levels of alpha 2-macroglobulin, one of the major proteinase inhibitors, in corneas with keratoconus to those in normal human corneas and corneas with other diseases. METHODS: An immunoperoxidase technique was used to visualize the presence of alpha 2-macroglobulin in the corneas. Western blot analysis was performed, and the levels of this inhibitor in extracts of keratoconus and normal human corneas were subsequently analyzed by a dot blot assay. RESULTS: alpha 2-Macroglobulin was demonstrated immunohistochemically in the epithelium, stroma, and endothelium of all corneal sections. Compared with normal human control specimens, the staining intensity in the epithelium of keratoconus corneas was markedly reduced. The majority of scarred and other diseased corneas exhibited normal staining intensity for alpha 2-macroglobulin. Dot blot assays showed that the alpha 2-macroglobulin levels in the epithelial and stromal extracts of keratoconus corneas were lower than those found in normal human control counterparts. CONCLUSION: Keratoconus corneas contained a reduced level of alpha 2-macroglobulin. This result lends further support to the hypothesis that degradation processes may be aberrant in keratoconus.
Assuntos
Córnea/metabolismo , Ceratocone/metabolismo , alfa-Macroglobulinas/análise , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Criança , Pré-Escolar , Doenças da Córnea/metabolismo , Humanos , Técnicas Imunoenzimáticas , Lactente , Pessoa de Meia-IdadeRESUMO
PURPOSE: Keratoconus is characterized by thinning and scarring of the central region of the cornea. The authors have shown, in corneas obtained from patients with keratoconus, that lysosomal enzyme activities are elevated, whereas levels of protease inhibitors such as alpha 1-proteinase inhibitor (alpha 1-PI) are reduced. This study was undertaken to examine further the gene expression of cathepsin G, acid phosphatase, and alpha 1-PI in keratoconus corneas. METHODS: Corneal buttons were collected from patients with keratoconus, normal subjects, and patients with other corneal diseases. In situ hybridization was performed on paraffin sections using a tritium-labeled probe for cathepsin G or alpha 1-PI. Competitive polymerase chain reaction (PCR) was used to determine the messenger RNA (mRNA) levels for lysosomal acid phosphatase and alpha 1-PI in epithelial and stromal cells of keratoconus corneas. RESULTS: Silver grains, indicative of positive in situ hybridization products, were observed in all three cell types of normal corneas for both DNA probes. Compared with normal and other diseased controls, the labeling was enhanced for cathepsin G but was diminished for alpha 1-PI in the epithelium of keratoconus corneas. Competitive PCR showed that the mRNA level for acid phosphatase was higher and that the mRNA level for alpha 1-PI was lower in keratoconus corneas. CONCLUSIONS: These results indicate that the mRNA level for degradative enzymes in increased and that for alpha 1-PI it is reduced in keratoconus corneas. This study provides the first evidence that the altered expression of multiple enzymes and inhibitors in keratoconus occurs at the gene level. Furthermore, it implicates a possible role of coordinated transcriptional regulation of gene expressions in keratoconus.
Assuntos
Fosfatase Ácida/genética , Catepsinas/genética , Córnea/metabolismo , Regulação Enzimológica da Expressão Gênica , Ceratocone/metabolismo , RNA Mensageiro/metabolismo , Serina Endopeptidases/genética , Inibidores de Serina Proteinase/genética , alfa 1-Antitripsina/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Catepsina G , Criança , Pré-Escolar , Primers do DNA/química , Sondas de DNA , Eletroforese em Gel de Ágar , Humanos , Hibridização In Situ , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
Three male patients are described in whom anorgasmia developed during treatment with fluoxetine for depression. During attempts to treat the anorgasmia with cyproheptadine, all three patients suffered a relapse of depressive symptoms. The possible mechanism of this effect is discussed in relation to serotonergic systems.
Assuntos
Ciproeptadina/farmacologia , Transtorno Depressivo/tratamento farmacológico , Fluoxetina/efeitos adversos , Disfunções Sexuais Psicogênicas/induzido quimicamente , Adulto , Ciproeptadina/efeitos adversos , Ciproeptadina/uso terapêutico , Transtorno Depressivo/psicologia , Interações Medicamentosas , Fluoxetina/antagonistas & inibidores , Fluoxetina/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Orgasmo/efeitos dos fármacos , Receptores de Serotonina/efeitos dos fármacos , Recidiva , Disfunções Sexuais Psicogênicas/tratamento farmacológicoRESUMO
We present three cases of acne rosacea keratitis that developed in childhood. All three children were prepubescent and demonstrated characteristic dermatologic findings involving the nose, cheeks, and/or chin. The patients had complained of ocular irritation for at least 6 months, and in one case symptoms were reported by the family to have occurred intermittently since age 4 years. All three children showed evidence of meibomian gland inflammation; two patients demonstrated bilateral keratitis, the third had only unilateral involvement. In each case, oral tetracycline hydrochloride or doxycycline hyclate was necessary to relieve symptoms. Rosacea keratitis should be considered in the differential diagnosis of chronic keratoconjunctivitis during childhood.
Assuntos
Ceratite/diagnóstico , Rosácea/diagnóstico , Administração Oral , Administração Tópica , Antibacterianos/administração & dosagem , Antibacterianos/uso terapêutico , Criança , Diagnóstico Diferencial , Feminino , Temperatura Alta/uso terapêutico , Humanos , Ceratite/complicações , Ceratite/terapia , Masculino , Oftalmoscopia , Recidiva , Rosácea/complicações , Rosácea/terapia , Acuidade VisualRESUMO
All three types of lattice corneal dystrophy are inherited and localized, and they largely involve linear corneal amyloid deposits. We encountered two white families with lattice corneal dystrophy which closely resembled type III. Four generations of one family and three of another family exhibited lattice corneal dystrophy. Because both families are from Caccamo, Sicily, Italy, we believe it is likely that both are from a single mutation. Thick, ropy lattice lines were seen to traverse the corneas almost from limbus to limbus and were easily detected with direct illumination. Histopathologic examination revealed accumulations of varying sized amyloid deposits in the stroma and ribbons of amyloid between the stroma and Bowman's layer typical of lattice corneal dystrophy type III. We have named the disease in this family lattice corneal dystrophy type IIIA, because of three differences from lattice corneal dystrophy type III: the presence of corneal erosions, the occurrence in whites, and the autosomal dominant inheritance pattern.