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BACKGROUND: In the spring of 2020 during the first wave of the pandemic an above-average number of residents and staff in nursing homes fell ill and accounted for the highest number of incidences. Due to the pandemic, managers in nursing homes had to make new decisions on a daily basis as well as interpret and integrate decisions made by higher level authorities. AIM OF THE STUDY: The aim was to describe the decisions that had to be made by the managers of nursing homes in dealing with the COVID-19 pandemic and related consequences. MATERIAL AND METHODS: A qualitative multicentre cross-sectional design was chosen. Data collection was conducted with semi-structured telephone interviews. The recorded audio data were transcribed, analyzed using the framework analysis method and reflected in peer debriefings. RESULTS: A total of 78 interviews were conducted in 43 nursing homes and 3 main themes with 10 subthemes emerged: decisions about social participation, decisions on quarantine and isolation and staff adjustments. DISCUSSION: Clearer information and directives for the implementation of measures are needed, e.g. through standardized guidelines nationwide. Additionally, public health departments should play a stronger and more responsible role in a pandemic situation. The consequences of their decisions were hardly foreseeable for the managers and were marked by uncertainty. Responsibilities for and consequences of pandemic-related decisions should be further evaluated to empower managers in times of crises.
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COVID-19 , Pandemias , COVID-19/epidemiología , Estudios Transversales , Humanos , Cuidados a Largo Plazo , SARS-CoV-2RESUMEN
Most of the world's crops depend on pollinators, so declines in both managed and wild bees raise concerns about food security. However, the degree to which insect pollination is actually limiting current crop production is poorly understood, as is the role of wild species (as opposed to managed honeybees) in pollinating crops, particularly in intensive production areas. We established a nationwide study to assess the extent of pollinator limitation in seven crops at 131 locations situated across major crop-producing areas of the USA. We found that five out of seven crops showed evidence of pollinator limitation. Wild bees and honeybees provided comparable amounts of pollination for most crops, even in agriculturally intensive regions. We estimated the nationwide annual production value of wild pollinators to the seven crops we studied at over $1.5 billion; the value of wild bee pollination of all pollinator-dependent crops would be much greater. Our findings show that pollinator declines could translate directly into decreased yields or production for most of the crops studied, and that wild species contribute substantially to pollination of most study crops in major crop-producing regions.
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Agricultura , Productos Agrícolas , Polinización , Animales , Abejas , Abastecimiento de Alimentos , Estados UnidosRESUMEN
We tested the gravitational 1/r^{2} law using a stationary torsion-balance detector and a rotating attractor containing test bodies with both 18-fold and 120-fold azimuthal symmetries that simultaneously tests the 1/r^{2} law at two different length scales. We took data at detector-attractor separations between 52 µm and 3.0 mm. Newtonian gravity gave an excellent fit to our data, limiting with 95% confidence any gravitational-strength Yukawa interactions to ranges <38.6 µm.
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BACKGROUND: Endomyocardial biopsy (EMB) is considered the gold standard for diagnosing myocardial involvement in most inflammatory conditions, including systemic lupus erythematosus (SLE). However, EMBs are rarely performed, and most of the myocardial histopathology reports in SLE consist of postmortem data. We therefore sought to describe the histopathologic findings of contemporary EMBs in SLE performed in clinical practice. METHODS: A retrospective review of histopathology reports from SLE patients who underwent EMB from 1994 to 2017 was performed. A total of 41 SLE patients had cardiac pathology reports. Of these, 11 histopathology reports were EMBs, and the remaining were valvular specimens. RESULTS: A total of 11 SLE EMBs were reviewed. It was found that 45% of the patients had hypertension, 27% had coronary artery disease, 9% had hyperlipidemia, and 36% had end-stage renal disease. None had diabetes or smoked. The mean left ventricular ejection fraction was 37%. On histopathology, 10 had mild interstitial fibrosis, 9 had myocyte hypertrophy, 3 had organized blood clots, and 3 had a mild infiltration of lymphocytes and macrophages without clear evidence of myocarditis. None had vasculitis, endocarditis, ischemia, amyloid deposition, or lamellar or curvilinear inclusions. CONCLUSION: EMBs are rarely performed in SLE. In this case series, nonspecific interstitial fibrosis and myocyte hypertrophy were the most common findings, suggesting EMBs have limited value in the diagnosis of cardiac involvement in SLE and rather rule out competing conditions. These data support the need for diagnostic methods with high sensitivity and specificity for SLE heart disease.
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Cardiopatías/diagnóstico , Lupus Eritematoso Sistémico/complicaciones , Miocardio/patología , Adulto , Biopsia , Ecocardiografía , Femenino , Corazón/fisiopatología , Cardiopatías/fisiopatología , Humanos , Masculino , Persona de Mediana Edad , Miocarditis/diagnóstico , Estudios Retrospectivos , Volumen Sistólico , Función Ventricular Izquierda , Adulto JovenRESUMEN
AIMS: We aim to determine if Pseudomonas fluorescens is a viable biological control for Erwinia tracheiphila within the insect vector, Acalymma vittatum. METHODS AND RESULTS: Pseudomonas fluorescens secreted fluorescein and inhibited growth of E. tracheiphila in disc diffusion assays. To determine if this antagonism was conserved within the insect vector, we performed in vivo assays by orally injecting beetles with bacterial treatments and fluorescent in situ hybridization to determine bacterial presence within the alimentary canal. CONCLUSIONS: Pseudomonas fluorescens inhibited the growth of E. tracheiphila on a nutrient-limiting medium. In situ experiments demonstrated that P. fluorescens is maintained within the alimentary canal of the beetle for at least 4 days, and co-occurred with E. tracheiphila. When beetles were first presented with Pseudomonas and then challenged with E. tracheiphila, E. tracheiphila was not recovered via FISH after 4 days. These data suggest that P. fluorescens has potential as a biological control agent to limit E. tracheiphila within the insect vector. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a novel approach for controlling E. tracheiphila that has the potential to decrease reliance on insecticides, providing a safer environment for pollinators and growers.
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Antibiosis , Escarabajos/microbiología , Erwinia/fisiología , Pseudomonas fluorescens/fisiología , Animales , Erwinia/genética , Tracto Gastrointestinal/microbiología , Hibridación Fluorescente in Situ , Insectos Vectores/microbiología , Pseudomonas fluorescens/genéticaRESUMEN
BACKGROUND: Early childhood interventions positively contribute to health related child development. For these interventions, networks are a necessary prerequisite as they promote interdisciplinary and interprofessional cooperation. This holds especially true for the integration of health system protagonists. METHODS: In a cross-sectional survey local paediatrists, gynaecologists, general practitioners, and psychotherapists were asked about their knowledge, experiences, desires, and reservations regarding cooperation in early childhood intervention networks. RESULTS: 64 out of 1747 (3.7%) eligible clinicians answered the survey. On average they estimated that 10.1% of the families they are treating would benefit from early childhood interventions. Participants rated themselves as competent to offer appropriate early childhood interventions. The youth welfare service was judged as the most important institution for their own professional practice by 84.4%. Additionally to an applicable agenda, a fair group moderation of network meetings was seen as a substantial requirement in order to take part in network meetings. CONCLUSION: Health professionals are important protagonists in early childhood interventions. Clinicians should assess relevant problems in families and offer appropriate support on a regular basis. Alongside clearly defined regional contacts, interprofessional continuing education seems mandatory.
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Protección a la Infancia , Intervención Médica Temprana , Educación no Profesional , Comunicación Interdisciplinaria , Colaboración Intersectorial , Grupo de Atención al Paciente , Médicos , Psicoterapia , Adolescente , Adulto , Niño , Preescolar , Estudios Transversales , Femenino , Alemania , Encuestas de Atención de la Salud , Humanos , Masculino , Persona de Mediana Edad , Evaluación de Necesidades , Relaciones Profesional-Familia , Encuestas y CuestionariosRESUMEN
Fall armyworm, Spodoptera frugiperda (J.E. Smith), is a highly mobile insect pest of a wide range of host crops. However, this pest of tropical origin cannot survive extended periods of freezing temperature but must migrate northward each spring if it is to re-infest cropping areas in temperate regions. The northward limit of the winter-breeding region for North America extends to southern regions of Texas and Florida, but infestations are regularly reported as far north as Québec and Ontario provinces in Canada by the end of summer. Recent genetic analyses have characterized migratory pathways from these winter-breeding regions, but knowledge is lacking on the atmosphere's role in influencing the timing, distance, and direction of migratory flights. The Hybrid Single-Particle Lagrangian Integrated Trajectory (HYSPLIT) model was used to simulate migratory flight of fall armyworm moths from distinct winter-breeding source areas. Model simulations identified regions of dominant immigration from the Florida and Texas source areas and overlapping immigrant populations in the Alabama-Georgia and Pennsylvania-Mid-Atlantic regions. This simulated migratory pattern corroborates a previous migratory map based on the distribution of fall armyworm haplotype profiles. We found a significant regression between the simulated first week of moth immigration and first week of moth capture (for locations which captured ≥ 10 moths), which on average indicated that the model simulated first immigration 2 weeks before first captures in pheromone traps. The results contribute to knowledge of fall armyworm population ecology on a continental scale and will aid in the prediction and interpretation of inter-annual variability of insect migration patterns including those in response to climatic change and adoption rates of transgenic cultivars.
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Migración Animal , Modelos Teóricos , Spodoptera , Animales , Femenino , Masculino , Estaciones del Año , Estados Unidos , Zea maysRESUMEN
OBJECTIVE: CD22 is a surface molecule exclusively expressed on B cells that regulates adhesion and B cell receptor (BCR) signaling as an inhibitory coreceptor of the BCR. Central downstream signaling molecules that are activated upon BCR engagement include spleen tyrosine kinase (Syk) and, subsequently, phospholipase Cγ2 (PLCγ2), which results in calcium (Ca(2+)) mobilization. The humanized anti-CD22 monoclonal antibody epratuzumab is currently being tested in clinical trials. This study was undertaken to determine the potential mechanism by which this drug regulates B cell activation. METHODS: Purified B cells were preincubated with epratuzumab, and the colocalization of CD22 and CD79α, without BCR engagement, was assessed by confocal microscopy. The phosphorylation of Syk (Y348, Y352) and PLCγ2 (Y759) as well as the Ca(2+) flux in the cells were analyzed by flow cytometry upon stimulation of the BCR and/or Toll-like receptor 9 (TLR-9). The influence of CD22 ligation on BCR signaling was assessed by pretreating the cells with epratuzumab or F(ab')(2) fragment of epratuzumab, in comparison with control cells (medium alone or isotype-matched IgG1). RESULTS: Epratuzumab induced colocalization of CD22 and components of the BCR independent of BCR engagement, and also reduced intracellular Ca(2+) mobilization and diminished the phosphorylation of Syk and PLCγ2 after BCR stimulation in vitro. Inhibition of kinase phosphorylation was demonstrated in both CD27- and CD27+ B cells, and this appeared to be independent of Fc receptor signaling. Preactivation of the cells via the stimulation of TLR-9 did not circumvent the inhibitory effect of epratuzumab on BCR signaling. CONCLUSION: These findings are consistent with the concept of targeting CD22 to raise the threshold of BCR activation, which could offer therapeutic benefit in patients with autoimmune diseases.
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Anticuerpos Monoclonales Humanizados/farmacología , Enfermedades Autoinmunes/tratamiento farmacológico , Linfocitos B/efectos de los fármacos , Calcio/metabolismo , Lectina 2 Similar a Ig de Unión al Ácido Siálico/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Adulto , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Linfocitos B/citología , Linfocitos B/metabolismo , Antígenos CD79/inmunología , Antígenos CD79/metabolismo , Relación Dosis-Respuesta Inmunológica , Femenino , Humanos , Fragmentos Fab de Inmunoglobulinas/farmacología , Inmunoglobulina G/farmacología , Masculino , Persona de Mediana Edad , Fosfolipasa C gamma/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/inmunología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/metabolismo , Transducción de Señal/inmunología , Receptor Toll-Like 9/metabolismo , Adulto JovenRESUMEN
We describe a liquid-cryogen free cryostat with ultra-low vibration levels, which allows for continuous operation of a torsion balance at cryogenic temperatures. The apparatus uses a commercially available two-stage pulse-tube cooler and passive vibration isolation. The torsion balance exhibits torque noise levels lower than room temperature thermal noise by a factor of about four in the frequency range of 3-10 mHz, limited by residual seismic motion and by radiative heating of the pendulum body. In addition to lowering thermal noise below room-temperature limits, the low-temperature environment enables novel torsion balance experiments. Currently, the maximum duration of a continuous measurement run is limited by accumulation of cryogenic surface contamination on the optical elements inside the cryostat.
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Objectives: For patients with geriatric frailty, reducing inappropriate medication is an important goal to improve patient safety in primary care. GP-side barriers include knowledge gaps, legal concerns, and lack of communication between the actors involved. The aim was to develop a multi-faceted intervention to facilitate deprescribing and shared prioritisation among frail elderlies with polypharmacy living at home. Methods: Mixed methods study including: 1) scoping review on family conferences, expert panels; 2) group discussions with GPs, mapping of needs and challenges in Primary Care; 3) workshops and expert interviews with GPs, patient advocates, researchers as a basis for a theoretical intervention model; 4) piloting. Results: A major challenge for GPs is to conduct a productive discussion with patients and family cares on deprescribing and drug safety. A guideline for a structured family conference with a medication check and geriatric assessment was developed and proved to be feasible in the pilot study. Conclusion: The intervention developed to facilitate deprescribing and shared prioritisation of drug therapy based on family conferences seems suitable to be tested in a subsequent cRCT. Innovation: Adapting family conferences to primary care for frail patients with polypharmacy.
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As part of an insect resistance management plan to preserve Bt transgenic technology, annual monitoring of target pests is mandated to detect susceptibility changes to Bt toxins. Currently Helicoverpa zea (Boddie) monitoring involves investigating unexpected injury in Bt crop fields and collecting larvae from non-Bt host plants for laboratory diet bioassays to determine mortality responses to diagnostic concentrations of Bt toxins. To date, this monitoring approach has not detected any significant change from the known range of baseline susceptibility to Bt toxins, yet practical field-evolved resistance in H. zea populations and numerous occurrences of unexpected injury occur in Bt crops. In this study, we implemented a network of 73 sentinel sweet corn trials, spanning 16 U.S. states and 4 Canadian provinces, for monitoring changes in H. zea susceptibility to Cry and Vip3A toxins by measuring differences in ear damage and larval infestations between isogenic pairs of non-Bt and Bt hybrids over three years. This approach can monitor susceptibility changes and regional differences in other ear-feeding lepidopteran pests. Temporal changes in the field efficacy of each toxin were evidenced by comparing our current results with earlier published studies, including baseline data for each Bt trait when first commercialized. Changes in amount of ear damage showed significant increases in H. zea resistance to Cry toxins and possibly lower susceptibility to Vip3a. Our findings demonstrate that the sentinel plot approach as an in-field screen can effectively monitor phenotypic resistance and document field-evolved resistance in target pest populations, improving resistance monitoring for Bt crops.
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Bacillus thuringiensis , Mariposas Nocturnas , Animales , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Canadá , Endotoxinas , Proteínas Hemolisinas/genética , Resistencia a los Insecticidas , Control Biológico de Vectores , Plantas Modificadas Genéticamente/genética , Zea mays/genéticaRESUMEN
Wild bees supply sufficient pollination in Cucurbita agroecosystems in certain settings; however, some growers continue to stock fields with managed pollinators due to uncertainties of temporal and spatial variation on pollination services supplied by wild bees. Here, we evaluate wild bee pollination activity in wholesale, commercial pumpkin fields over 3 yr. We identified 37 species of bees foraging in commercial pumpkin fields. Honey bees (Apis mellifera L. [Hymenoptera: Apidae]), squash bees (Eucera (Peponapis) Say, Dorchin [Hymenoptera: Apidae]), and bumble bees (Bombus spp., primarily B. impatiens Cresson [Hymenoptera: Apidae]) were the most active pollinator taxa, responsible for over 95% of all pollination visits. Preference for female flowers decreased as distance from field edge increased for several bee taxa. Visitation rates from one key pollinator was negatively affected by field size. Visitation rates for multiple taxa exhibited a curvilinear response as the growing season progressed and responded positively to increasing floral density. We synthesized existing literature to estimate minimum 'pollination thresholds' per taxa and determined that each of the most active pollinator taxa exceeded these thresholds independently. Under current conditions, renting honey bee hives may be superfluous in this system. These results can aid growers when executing pollination management strategies and further highlights the importance of monitoring and conserving wild pollinator populations.
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Cucurbita , Polinización , Agricultura , Animales , Abejas , Femenino , Flores , Estaciones del AñoRESUMEN
There have been several reports describing paracrystalline arrays in the intermembrane space of mitochondria. On closer inspection these structures appear to be junctions of two adjoining membranes. There are two types. They can be formed between the outer and inner mitochondrial membranes (designated outer-inner membrane junctions) or between two cristal membranes (intercristal membrane junctions). In rat heart, adjoining membranes appeared associated via a central dense midline approximately 30 A wide. In rat kidney, the junction had a ladder-like appearance with electron-dense "bridges" approximately 80 A wide, spaced 130 A apart, connecting the adjoining membranes. We have investigated the conditions which favor the visualization of such structures in mitochondria. Heart mitochondria isolated rapidly from fresh tissue (within 30 min of death) contain membrane junctions in approximately 10-15% of the cross sections. This would indicate that the percentage of membrane junctions in the entire mitochondrion is far greater. Mitochondria isolated from heart tissue which was stored for 1 h at 0 degrees -4 degrees C showed an increased number of membrane junctions, so that 80% of the mitochondrial cross sections show membrane junctions. No membrane junctions are observed in mitochondria in rapidly fixed fresh tissue or in mitochondria isolated from tissue disrupted in fixative. Thus, the visualization of junctions in the intermembrane space of mitochondria appears to be dependent upon the storage of tissue after death. Membrane junctions can also be observed in mitochondria from other stored tissues such as skeletal muscle, kidney, and interstitial cells from large and small intestine. In each case, no such junctions are observed in these tissues when they are fixed immediately after removal from the animal. It would appear that most studies in the literature in which isolated mitochondria from tissues such as heart or kidney were used were carried out on mitochondria which contained membrane junctions. The presence of such structures does not significantly affect normal mitochondrial function in terms of respiratory control and oxidative phosphorylation.
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Mitocondrias , Miocardio/citología , Animales , Sitios de Unión , Bovinos , Glutaral , Riñón/citología , Masculino , Membranas , Microscopía Electrónica , Mitocondrias Musculares , Especificidad de Órganos , Ratas , Factores de Tiempo , UltracentrifugaciónRESUMEN
The fine structure of mitochondria and submitochondrial vesicles depleted of their lipid by extraction with aqueous acetone was studied. Thin sections of mitochondrial membranes depleted of more than 95% of their lipid retained the unit membrane structure. Densitometer tracings of the electron micrographs showed that the unit membrane of extracted mitochondria was, on the average, wider than that of unextracted controls and showed a greater variation in width. The outer membrane was lost in mitochondria from which 80-95% of the lipids was extracted. Inner membrane particles were present on submitochondrial vesicles depleted of up to 85% of their lipids. However, when more than 95% of the lipid was removed, few, if any, particles remained attached to the membranes but many particles were found unattached in the background. When lipid was restored to lipid-deficient preparations, the mitochondrial membranes were found to be devoid of inner membrane particles but were fully active with respect to succinate-cytochrome c reductase activity.
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Lípidos/análisis , Mitocondrias/química , Mitocondrias/ultraestructura , Animales , Bovinos , Membranas Intracelulares/química , Membranas Intracelulares/enzimología , Membranas Intracelulares/ultraestructura , Microscopía Electrónica , Mitocondrias/enzimología , Miocardio/ultraestructura , NADH Deshidrogenasa/metabolismo , Ácido FosfotúngsticoRESUMEN
Fixation of purified sarcoplasmic reticulum (SR) membrane vesicles, using glutaraldehyde supplemented with 1% tannic acid, reveals newly visualized ultrastructure in thin sections. The trilaminar appearance of the membrane is highly asymmetric; the outer electron-opaque layer is appreciably wider (70 A) than the inner layer (20 A). The asymmetry is not referable to lack of penetration of the tannic acid since: (a) SR vesicles made permeable with 1 mM EDTA, pH 8.5, show similar asymmetry; (b) treatment of SR with trypsin results in progressive loss in protein content and decrease in the thickness of the outer layer, until in the limit the trilayer has a symmetric appearance; (c) within the same muscle section, the SR membrane appears highly asymmetric whereas the sarcolemma has a more symmetric appearance; (d) reconstituted SR vesicles have a symmetric appearance with equally broad inner and outer layers (approximately 70 A); the symmetric structure is confirmed by freeze-fracture and negative staining electron microscopy. Heavy and light SR vesicles obtained by isopycnic density sedimentation of purified SR have the same asymmetric appearance of the membrane and seem to differ mainly in that the heavy vesicles contain internal contents consisting largely of Ca++-binding protein. The asymmetry of the SR membrane is referable mainly to the unidirectional alignment of the Ca++ pump protein, the major component (90% of the protein) of the membrane. The asymmetry of the SR membrane can be visualized now for the first time in situ in thin sections of muscle.
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Fijadores , Taninos Hidrolizables , Músculos/ultraestructura , Retículo Sarcoplasmático/ultraestructura , Taninos , Animales , Técnica de Fractura por Congelación , Modelos Moleculares , Conejos , Proteína G de Unión al Calcio S100/análisis , Retículo Sarcoplasmático/análisis , Coloración y Etiquetado , Fracciones SubcelularesRESUMEN
Zonal centrifugation has been used to isolate a fraction from bovine liver which appears to be derived from the Golgi apparatus. Morphologically, the fraction consists mainly of sacs and tubular elements. Spherical inclusions, probably lipoproteins, are occasionally seen in negative stains of this material. The preparation is biochemically unique. UDP-galactose:N-acetyl glucosamine, galactosyl transferase activity is concentrated about 40-fold in this fraction compared to the homogenate. Rotenone- or antimycin-insensitive DPNH- or TPNH- cytochrome c reductase activities are 60-80% of the level of activities found in microsomes. Purified organelles from bovine liver such as plasma membranes, rough microsomes, mitochondria and nuclei have negligible levels of galactosyl transferase. Some activity is present in smooth microsomes but at a level compatible with the possible presence of Golgi membranes in this fraction. The Golgi fraction does not contain appreciable amounts of enzymes such as ATPase, 5'-nucleotidase, glycosidase, glucose-6-phosphatase, acid phosphatase, or succinate-cytochrome c reductase. Similar fractions isolated from bovine epididymis also have very high levels of galactosyl transferase. The fraction is heavily osmicated when incubated for long periods of time at elevated temperatures, a characteristic property of Golgi membranes.
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Aparato de Golgi/análisis , Hígado/citología , Fosfatasa Ácida/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Antimicina A , Isótopos de Carbono , Bovinos , Membrana Celular/enzimología , Núcleo Celular/enzimología , Centrifugación Zonal , Cromatografía por Intercambio Iónico , Citocromos , Epidídimo/citología , Galactosa , Glucosidasas/metabolismo , Aparato de Golgi/enzimología , Hexosaminas , Calor , Concentración de Iones de Hidrógeno , Lipoproteínas/análisis , Masculino , Microscopía Electrónica , Microsomas/enzimología , Mitocondrias Hepáticas/enzimología , Nucleotidasas/metabolismo , Oxidorreductasas/análisis , Oxidorreductasas/antagonistas & inhibidores , Rotenona , Transferasas/análisis , Nucleótidos de UraciloRESUMEN
A procedure has been devised for isolation of triads (t-tubule/sarcoplasmic reticulum (SR) junctional complexes) from rabbit skeletal muscle. The procedure consists of preparation of a heavy microsomal fraction followed by two sequential 90-min sucrose gradient centrifugations to enrich the triads. A pyrophosphate/phosphate/magnesium buffer system was introduced to decrease aggregation in order to achieve effective separation. The preparation time is 12 h. Some differences between purified triads isolated by two variants of this method are noted. The purity of the triad fractions has been estimated by particle counting to be in the vicinity of 50%. There is good retention of morphology and Ca++-loading activity and enrichment in Na+,K+-ATPase and adenylate cyclase. The triads are practically devoid of contractile elements, mitochondria, and free plasmalemma, and low in content of light SR. The method for obtaining enriched triads is reproducible, and sufficient yields are obtained for structural, biochemical, and functional characterization.
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Fraccionamiento Celular/métodos , Membranas Intracelulares/ultraestructura , Microtúbulos/ultraestructura , Músculos/ultraestructura , Retículo Sarcoplasmático/ultraestructura , Adenilil Ciclasas/metabolismo , Animales , Tampones (Química) , Calcio/metabolismo , Centrifugación por Gradiente de Densidad , Difosfatos , Magnesio , Músculos/metabolismo , Fosfatos , Conejos , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
We have developed a procedure to isolate, from skeletal muscle, enriched terminal cisternae of sarcoplasmic reticulum (SR), which retain morphologically intact junctional "feet" structures similar to those observed in situ. The fraction is largely devoid of transverse tubule, plasma membrane, mitochondria, triads (transverse tubules junctionally associated with terminal cisternae), and longitudinal cisternae, as shown by thin-section electron microscopy of representative samples. The terminal cisternae vesicles have distinctive morphological characteristics that differ from the isolated longitudinal cisternae (light SR) obtained from the same gradient. The terminal cisternae consist of two distinct types of membranes, i.e., the junctional face membrane and the Ca2+ pump protein-containing membrane, whereas the longitudinal cisternae contain only the Ca2+ pump protein-containing membrane. The junctional face membrane of the terminal cisternae contains feet structures that extend approximately 12 nm from the membrane surface and can be clearly visualized in thin section through using tannic acid enhancement, by negative staining and by freeze-fracture electron microscopy. Sections of the terminal cisternae, cut tangential to and intersecting the plane of the junctional face, reveal a checkerboardlike lattice of alternating, square-shaped feet structures and spaces each 20 nm square. Structures characteristic of the Ca2+ pump protein are not observed between the feet at the junctional face membrane, either in thin section or by negative staining, even though the Ca2+ pump protein is observed in the nonjunctional membrane on the remainder of the same vesicle. Likewise, freeze-fracture replicas reveal regions of the P face containing ropelike strands instead of the high density of the 7-8-nm particles referable to the Ca2+ pump protein. The intravesicular content of the terminal cisternae, mostly Ca2+-binding protein (calsequestrin), is organized in the form of strands, sometimes appearing paracrystalline, and attached to the inner face of the membrane in the vicinity of the junctional feet. The terminal cisternae preparation is distinct from previously described heavy SR fractions in that it contains the highest percentage of junctional face membrane with morphologically well-preserved junctional feet structures.
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Músculos/ultraestructura , Retículo Sarcoplasmático/ultraestructura , Animales , ATPasa de Ca(2+) y Mg(2+) , ATPasas Transportadoras de Calcio/análisis , Fraccionamiento Celular , Centrifugación Zonal/métodos , Técnica de Fractura por Congelación , Microscopía Electrónica , Fosfolípidos/análisis , Conejos , Retículo Sarcoplasmático/enzimologíaRESUMEN
This study is concerned with the characterization of the morphology of the calcium release channel of sarcoplasmic reticulum (SR) from fast-twitch skeletal muscle, which is involved in excitation-contraction coupling. We have previously purified the ryanodine receptor and found it to be equivalent to the feet structures, which are involved, in situ, in the junctional association of transverse tubules with terminal cisternae of SR. The receptor is an oligomer of a single high molecular weight polypeptide and when incorporated into phospholipid bilayers, has channel conductance which is characteristic of calcium release in terminal cisternae of SR. The purified channel can be observed by electron microscopy using different methods of sample preparation, with complementary views being observed by negative staining, double staining, thin section and rotary shadowing electron microscopy. Three views can be observed and interpreted: (a) a square face which, in situ, is junctionally associated with the transverse tubule or junctional face membrane; (b) a rectangle equivalent to the side view; and (c) a diamond shape equivalent to the side view, of which the base portion appears to be equivalent to the transmembrane segment. Negative staining reveals detailed substructure of the channel. A computer averaged view of the receptor displays fourfold symmetry and ultrastructural detail. The dense central mass is divided into four domains with a 2-nm hole in the center, and is enclosed within an outer frame which has a pinwheel appearance. Double staining shows substructure of the square face in the form of parallel linear arrays (six/face). The features of the isolated receptor can be correlated with the structure observed in terminal cisternae vesicles. Sections tangential to the junctional face membrane reveal that the feet structures (23-nm squares) overlap so as to enclose smaller square spaces of approximately 14 nm/side. We suggest that this is equivalent to the transverse tubule face and that the terminal cisternae face is smaller (approximately 17 nm/face) and has larger alternating spaces as a consequence of the tapered sides of the foot structures. Image reconstruction analysis appears to be feasible and should provide the three-dimensional structure of the channel.