RESUMO
BACKGROUND: RGM medium is an agar-based, selective culture medium designed for the isolation of nontuberculous mycobacteria (NTM) from the sputum of patients with cystic fibrosis (CF). We evaluated RGM medium for the detection of NTM in patients with CF (405 samples), bronchiectasis (323 samples) and other lung diseases necessitating lung transplantation (274 samples). METHODS: In total, 1002 respiratory samples from 676 patients were included in the study. Direct culture on RGM medium, with incubation at two temperatures (30 °C and 37 °C), was compared with conventional culture of decontaminated samples for acid-fast bacilli (AFB) using both a solid medium (Löwenstein-Jensen medium) and a liquid medium (the Mycobacterial Growth Indicator Tube; MGIT). RESULTS: For all three patient groups, significantly more isolates of NTM were recovered using RGM medium incubated at 30 °C than by any other method (sensitivity: 94.6% vs. 22.4% for conventional AFB culture; P < 0.0001). Significantly more isolates of Mycobacterium abscessus complex were isolated on RGM at 30 °C than by AFB culture (sensitivity: 96.1% vs. 58.8%; P < 0.0001). The recovery of Mycobacterium avium complex was also greater using RGM medium at 30 °C compared to AFB culture (sensitivity: 83% vs. 70.2%), although this difference was not statistically significant and a combination of methods was necessary for optimal recovery (P = 0.21). CONCLUSIONS: In the largest study of RGM medium to date, we reaffirm its utility for isolation of NTM from patients with CF. Furthermore; we show that it also provides an effective tool for culture of respiratory samples from patients with bronchiectasis and other lung diseases.
Assuntos
Bronquiectasia/microbiologia , Fibrose Cística/microbiologia , Doenças Pulmonares Intersticiais/microbiologia , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Micobactérias não Tuberculosas/isolamento & purificação , Doença Pulmonar Obstrutiva Crônica/microbiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Meios de Cultura , Técnicas de Cultura , Feminino , Humanos , Pneumopatias/microbiologia , Transplante de Pulmão , Masculino , Pessoa de Meia-Idade , Mycobacterium abscessus/isolamento & purificação , Complexo Mycobacterium avium/isolamento & purificação , Infecção por Mycobacterium avium-intracellulare/diagnóstico , Sensibilidade e Especificidade , Escarro , Adulto JovemRESUMO
BACKGROUND: Non-Tuberculous Mycobacterial-pulmonary disease (NTM-PD) is increasing in incidence and prevalence. Mycobacterium abscessus (M.abscessus) is a rapid growing multi-resistant NTM associated with severe NTM-PD requiring prolonged antibiotic therapy. Complications of therapy are common but reports on direct complications of active NTM-PD are rare. Vasculitis has been described as a rare complication of NTM-PD, most often in individuals with inherited immune defects. This case is the first to describe an ANCA positive vasculitide (Microscopic Polyangiitis) secondary to M.abscessus pulmonary disease. CASE PRESENTATION: A 70 year old female with bronchiectasis underwent a clinical decline associated with the growth of M.abscessus and was diagnosed with NTM-PD. Before treatment could be initiated she developed small joint arthralgia and a glove and stocking axonal loss sensorimotor neuropathy. Positive Perinuclear Anti-Neutrophil Cytoplasmic Antibodies (P-ANCA) and Myeloperoxidase-ANCA (MPO-ANCA) titres led to a diagnosis of microscopic polyangiitis. Further investigation revealed reduced interferon-gamma production but no other significant immune dysfunction. Dual treatment with immunosuppressive therapy (Corticosteroids/Cyclophosphamide) for vasculitis and antimicrobial therapy for M.abscessus NTM-PD was initiated. Clinical stability was difficult to achieve with reductions in immunosuppression triggering vasculitic flares. One flare led to retinal vein occlusion with impending visual loss requiring escalation in immunosuppression to Rituximab infusions. An increase in immunosuppression led to a deterioration in NTM-PD necessitating alterations to antibiotic regimes. Adverse effects including alopecia and Achilles tendonitis have further limited antibiotic choices resulting in a strategy of pulsed intra-venous therapy to stabilise NTM-PD. CONCLUSIONS: This is the first reported case of an ANCA positive vasculitis secondary to M.abscessus pulmonary disease. This rare but important complication had a significant impact on the patient adding to the complexity of an already significant disease and treatment burden. The potential role of reduced interferon-gamma production in this case highlights the importance of investigating immune function in those with mycobacterial infection and the intricate relationship between mycobacterial infection and immune dysfunction. Immune dysfunction caused by genetic defects or immunosuppressive therapy is a known risk factor for NTM-PD. Balancing immunosuppressive therapy with prolonged antimicrobial treatment is challenging and likely to become more common as the number of individuals being treated with biologics and immunosuppressive agents increases.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/sangue , Bronquiectasia/complicações , Poliangiite Microscópica/diagnóstico , Infecções por Mycobacterium não Tuberculosas/complicações , Mycobacterium abscessus/isolamento & purificação , Idoso , Antibacterianos/uso terapêutico , Feminino , Humanos , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológicoRESUMO
AIM: To assess the effect on radiologist participation in learning from discrepancy meetings (LDMs) in a multisite radiology department by establishing virtual LDMs using OsiriX (Pixmeo). MATERIALS AND METHODS: Sets of anonymised discrepancy cases were added to an OsiriX database available for viewing on iMacs in all radiology reporting rooms. Radiologists were given a 3-week period to review the cases and send their feedback to the LDM convenor. Group learning points and consensus feedback were added to each case before it was moved to a permanent digital LDM library. Participation was recorded and compared with that from the previous 4 years of conventional LDMs. Radiologist feedback comparing the two types of LDM was collected using an anonymous online questionnaire. RESULTS: Numbers of radiologists attending increased significantly from a mean of 12±2.9 for the conventional LDM to 32.7±7 for the virtual LDM (p<0.0001) and the percentage of radiologists achieving the UK standard of participation in at least 50% of LDMs annually rose from an average of 18% to 68%. The number of cases submitted per meeting rose significantly from an average of 11.1±3 for conventional LDMs to 15.9±5.9 for virtual LDMs (p<0.0097). Analysis of 35 returned questionnaires showed that radiologists welcomed being able to review cases at a time and place of their choosing and at their own pace. CONCLUSION: Introduction of virtual LDMs in a multisite radiology department improved radiologist participation in shared learning from radiological discrepancy and increased the number of submitted cases.
Assuntos
Instrução por Computador/métodos , Erros de Diagnóstico/prevenção & controle , Radiologistas/educação , Radiologistas/estatística & dados numéricos , Sistemas de Informação em Radiologia/estatística & dados numéricos , Radiologia/educação , Adulto , Erros de Diagnóstico/estatística & dados numéricos , Feminino , Processos Grupais , Humanos , Disseminação de Informação/métodos , Masculino , Pessoa de Meia-Idade , Variações Dependentes do Observador , Ensino , Reino Unido , Interface Usuário-Computador , Adulto JovemRESUMO
The objective of this study was to determine the disposition of ampicillin in plasma, uterine tissue, lochial fluid, and milk of postpartum dairy cattle. Ampicillin trihydrate was administered by intramuscular (i.m.) injection at a dose of 11 mg/kg of body weight every 24 h (n = 6, total of 3 doses) or every 12 h (n = 6, total of 5 doses) for 3 days. Concentrations of ampicillin were measured in plasma, uterine tissue, lochial fluid, and milk using HPLC with ultraviolet absorption. Quantifiable ampicillin concentrations were found in plasma, milk, and lochial fluid of all cattle within 30 min, 4 h, and 4 h of administration of ampicillin trihydrate, respectively. There was no significant effect of dosing interval (every 12 vs. every 24 h) and no significant interactions between dosing interval and sampling site on the pharmacokinetic variable measured or calculated. Median peak ampicillin concentration at steady-state was significantly higher in lochial fluid (5.27 µg/mL after q 24 h dosing) than other body fluids or tissues and significantly higher in plasma (3.11 µg/mL) compared to milk (0.49 µg/mL) or endometrial tissue (1.55 µg/mL). Ampicillin trihydrate administered once daily by the i.m. route at the label dose of 11 mg/kg of body weight achieves therapeutic concentrations in the milk, lochial fluid, and endometrial tissue of healthy postpartum dairy cattle.
Assuntos
Ampicilina/farmacocinética , Líquidos Corporais/química , Bovinos/metabolismo , Leite/química , Período Pós-Parto/fisiologia , Útero/metabolismo , Ampicilina/sangue , Animais , Antibacterianos/sangue , Antibacterianos/farmacocinética , Bovinos/sangue , Feminino , Distribuição Tecidual , Útero/químicaRESUMO
Serotonin toxicity results from serotonin excess in the central nervous system from serotonergic drugs. Previous studies suggest an association between T102C polymorphism of the serotonin 2A (5-hydroxytryptamine 2A) receptor gene and serotonergic adverse effects with serotonergic drugs. We aimed to determine whether there is an association between the T102C polymorphism and serotonin toxicity in patients taking serotonergic drug overdoses. Ninety-five patients presenting with serotonergic drug overdoses were examined for serotonin toxicity and had blood collected for DNA analysis. A diagnosis of serotonin toxicity was made in 14 patients (15%) based on the Hunter Serotonin Toxicology Criteria. Four of the 14 patients (29%) with serotonin toxicity had the C/C genotype compared with 20/81 (25%) without serotonin toxicity. There were no differences in age or sex, but the median defined daily dose taken by patients with serotonin toxicity was 27 (14-84) compared with 18 (2-136) in patients without serotonin toxicity (P=0.06). There was no association between serotonin toxicity and the T102C polymorphism in patients taking a serotonergic drug overdose.
Assuntos
Antidepressivos/intoxicação , Polimorfismo Genético , Receptor 5-HT2A de Serotonina/genética , Serotonina/toxicidade , Adulto , Estudos de Coortes , Overdose de Drogas , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Depression is the leading cause of disability worldwide and its effects can be fatal, with over 800,000 people dying by suicide each year. Neuromodulatory treatments such as transcranial magnetic stimulation (TMS) are being used to treat depression. Despite its endorsement by two regulatory bodies: NICE (2016) and the FDA (2008), there are major questions about the treatment efficacy and biological mechanisms of TMS. Ahn et al.'s (2013) justified the use of TMS in a clinical context in an important study indicating that excitatory TMS increases reward responsiveness. A pseudo-replication of this study by Duprat et al., (2016) also found a similar effect of active TMS, but only with the addition of an exploratory covariate to the analyses-trait reward responsiveness. Here we replicate Ahn et al.'s (2013) key study, and to test the reliability of the effects, and their dependency on trait reward responsiveness as described by Duprat et al., (2016). Using excitatory and sham TMS, we tested volunteers using the probabilistic learning task to measure their reward responsiveness both before and after stimulation. We also examined affect (positive, negative) following stimulation. Irrespective of TMS, the task was shown to be sensitive to reward responsiveness. However, we did not show TMS to be effective in increasing reward responsiveness and we did not replicate Ahn et al., (2013) or Duprat et al., (2016)'s key findings for TMS efficacy, where we provide evidence favouring the null. Moreover, exploratory analyses suggested following active stimulation, positive affect was reduced. Given our findings, we question the basic effects, which support the use of TMS for depression, particularly considering potential deleterious effects of reduced positive affect in patients with depression.
Assuntos
Aprendizagem , Estimulação Magnética Transcraniana , Humanos , Reprodutibilidade dos Testes , Resultado do Tratamento , RecompensaRESUMO
Distinguishing diaphragmatic eventration from rupture in the trauma setting can be a considerable challenge. We present a case involving a man suffering from chest pain and with a raised left hemidiaphragm on the chest radiograph after a motor vehicle injury. A review of the literature discusses the use of imaging modalities and subsequent surgical diagnostic procedures in the face of uncertainty.
Assuntos
Diafragma/lesões , Ferimentos não Penetrantes/diagnóstico por imagem , Acidentes de Trânsito , Adulto , Diagnóstico Diferencial , Diafragma/diagnóstico por imagem , Diafragma/cirurgia , Humanos , Masculino , Ruptura , Tomografia Computadorizada por Raios X , Ferimentos não Penetrantes/cirurgiaRESUMO
In this retrospective study, we describe the histopathological findings in seven papillomas and 45 squamous cell carcinomas (SCCs) from psittacine birds, raptors and domestic fowl. The age of affected birds ranged from 3 to 40 years, with median age significantly higher in psittacines (P = 0.014). The majority of tumours were located in the skin (24/52, 46.2%) or uropygial gland (10/52, 19.2%). Thirty of the SCCs (66.7%) were well differentiated and 15 (33.3%) were poorly-differentiated. SCCs exhibited a significantly higher degree of nuclear pleomorphism (P = 0.005) and a greater proportion were ulcerated (P = 0.001) compared with papillomas; however, there was no significant difference in mitotic count (MC) or inflammation score. The expression of cyclo-oxygenase (COX)-2 and E-cadherin was investigated by immunohistochemistry. The COX-2 total score (TS) was significantly higher in SCCs compared with papillomas (P = 0.002), but the difference between COX-2 TS of well- and poorly-differentiated SCCs was not significant. COX-2 labelling was predominantly cytoplasmic, but some tumours had concurrent membranous and/or perinuclear labelling. SCCs with membranous labelling had a significantly higher MC (P = 0.028). A significantly higher proportion of SCCs were negative for E-cadherin compared with papillomas (P = 0.042), but there was no significant difference between well- and poorly-differentiated SCCs. Fourteen papillomas and SCCs from psittacines were also tested by polymerase chain reaction for the presence of Psittacus erithacus papillomavirus 1 and Psittacid herpesvirus 1, but all samples tested negative. We demonstrate for the first time the expression of COX-2 and E-cadherin in avian tissues, and suggest that these markers may be useful in differentiating papillomas from SCCs, particularly when sample size is small.
Assuntos
Doenças das Aves/patologia , Carcinoma de Células Escamosas/veterinária , Papiloma/veterinária , Animais , Biomarcadores Tumorais/análise , Aves , Imuno-Histoquímica , Estudos RetrospectivosRESUMO
Chemotherapy-induced febrile neutropenia is costly in both financial and human terms. The associated costs can be reduced substantially through the development and implementation of national policies and locally agreed protocols for the prevention and management of febrile neutropenia. Patients, the NHS, healthcare professionals and the broader community all stand to benefit from a commitment to effective management of this common and predictable side effect of some chemotherapy regimens for early-stage breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Febre/prevenção & controle , Febre/terapia , Neutropenia/prevenção & controle , Neutropenia/terapia , Antineoplásicos/efeitos adversos , Febre/induzido quimicamente , Humanos , Neutropenia/induzido quimicamente , Medicina Estatal , Reino UnidoRESUMO
More women are living with and surviving breast cancer, because of improvements in breast cancer care. Trastuzumab (Herceptin) has significantly improved outcomes for women with HER2-positive tumours. Concerns about the cardiac effects of trastuzumab (which fundamentally differ from the permanent myocyte loss associated with anthracyclines) led to the development of cardiac guidelines for adjuvant trials, which are used to monitor patient safety in clinical practice. Clinical experience has shown that the trial protocols are not truly applicable to the breast cancer population as a whole, and exclude some women from receiving trastuzumab, even though they might benefit from treatment without long-term adverse cardiac sequelae. Consequently, five oncologists who recruited patients to trastuzumab trials, some cardiologists with whom they work, and a cardiovascular lead general practitioner reviewed the current cardiac guidelines in the light of recent safety data and their experience with adjuvant trastuzumab. The group devised recommendations that promote proactive pharmacological management of cardiac function in trastuzumab-treated patients, and that apply to all patients who are likely to receive standard cytotoxic chemotherapy. Key recommendations include: a monitoring schedule that assesses baseline and on-treatment cardiac function and potentially reduces the overall number of assessments required; intervention strategies with cardiovascular medication to improve cardiac status before, during, and after treatment; simplified rules for starting, interrupting and discontinuing trastuzumab; and a multidisciplinary approach to breast cancer care.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Cardiopatias/prevenção & controle , Monitorização Fisiológica/métodos , Algoritmos , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais Humanizados , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Neoplasias da Mama/fisiopatologia , Feminino , Diretrizes para o Planejamento em Saúde , Coração/fisiopatologia , Cardiopatias/etiologia , Insuficiência Cardíaca/induzido quimicamente , Insuficiência Cardíaca/fisiopatologia , Humanos , Trastuzumab , Reino Unido , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
Microtubule polymer levels in mouse 3T6 fibroblasts and primary cultures of rat hepatocytes can be manipulated by treatment of cells with long term, low doses of colcemid. Such treatment produces a rather uniform population of cells with microtubules of reduced lengths. Using this system, we demonstrate (a) that the rate of tubulin synthesis is sensitive to small changes (10%) in microtubule polymer mass and (b) that the percent of inhibition of synthesis is proportional to the level of soluble tubulin. Experiments with hepatocytes indicate that not only synthesis but the stability of tubulin protein was also regulated to maintain a specific level of tubulin. Treatment of hepatocytes with colcemid or other microtubule-depolymerizing drugs reduced the half-life of tubulin from 50 to 2 h, whereas taxol, which stabilizes microtubules, increased the half-life. To assess the consequences of altering microtubule polymer mass, we have analyzed the effect of controlled depolymerization of microtubules in rat hepatocytes on the processing of endocytosed ligands and found it sensitive to small changes in microtubule polymer levels.
Assuntos
Fígado/metabolismo , Tubulina (Proteína)/biossíntese , Animais , Células Cultivadas , Demecolcina/farmacologia , Endocitose , Fibroblastos/citologia , Fibroblastos/metabolismo , Imunofluorescência , Homeostase , Cinética , Fígado/citologia , Fígado/efeitos dos fármacos , Substâncias Macromoleculares , Masculino , Camundongos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Ratos , Ratos EndogâmicosRESUMO
Stereological analysis of hepatic fine structure in Fischer 344 male rats at 1, 6, 10, 16, 20, 25, and 30 mo of age revealed differences in the amounts and distributions of hepatocellular organelles as a function of sublobular location or animal age. Between 1 and 16 mo of age, both the centrolobular and periportal hepatocytes increased in volume by 65 and 35%, respectively. Subsequently, the cell volumes declined until the hepatocytes of 30-mo-old rats approached the size of those found in the youngest animals. Regardless of animal age, the centrolobular cells were consistently larger than the corresponding periportal hepatocytes. The cytoplasmic and ground substance compartments reflected similar changes in their volumes, although there was no significant alteration in the nuclear volume. The volumes of the mitochondrial and microbody compartments increased and decreased concomitant with the changes in average hepatocyte size. Both lobular zones in the 30-mo-old rats contained significantly smaller relative volumes of mitochondria than similar parenchyma in 16-mo-old animals. The volume density of the dense bodies (lysosomes) increased markedly in both lobular zones between 1 and 30 mo of age, confirming reports of an age-dependent increase in this organelle. The surface area of the endoplasmic reticulum in the centrolobular and periportal hepatocytes reached its maximum level in the 10-mo-old rats and subsequently declined to amounts which approximated those measured in the 1-mo-old animals. This age-related loss of intracellular membrane is attributable to a significant reduction in the surface area of the smooth-surfaced endoplasmic reticulum (SER) in animals beyond 16 mo of age. The amount of rough-surfaced endoplasmic reticulum (RER) in the periportal parenchymal cells was unaffected by aging, but the centrolobular hepatocytes of 30-mo-old animals contained 90% more RER than similar cells in the youngest rats. The centrolobular parenchyma contained more SER and the portal zones more RER throughout the age span studied. These quantitative data suggest that (a) certain hepatic fine structural parameters undergo marked changes as a function of animal age, (b) there exists a gradient in hepatocellular fine structure across the entire liver lobule, and (c) there are remarkable similarities in hepatocyte ultrastructure between very young and senescent animals, including cell size and the amount of SER.
Assuntos
Fígado/ultraestrutura , Envelhecimento , Animais , Contagem de Células , Retículo Endoplasmático/ultraestrutura , Fígado/anatomia & histologia , Masculino , Microssomos Hepáticos/ultraestrutura , Mitocôndrias Hepáticas/ultraestrutura , Organoides/ultraestrutura , Ratos , Ratos Endogâmicos F344RESUMO
Hepatic parenchymal cells from adult rats, established in vitro as a monolayer, have been evaluated by electron microscopy. Within 24 h after the initial seeding, the incubated cells were polygonal and in close apposition with three to six neighboring cells. The ultrastructure of the monolayer cells was examined at this time and after 3 and 10 days of incubation. With the exception of a few enlarged mitochondria, organelles in both the 1- and 3-day monolayer cells were indistinguishable quantitatively and morphologically from those found in the intact liver. After 10 days of incubation, however, the rough-surfaced endoplasmic reticulum (RER) had become dilated and vesiculated. In all cells studied, portions of RER were found in a close spatial relationship to mitochondria. From its frequency, this association appeared to be more than fortuitous, and the organelle complex may represent a functional unit necessary for new membrane formation, as suggested previously. The Golgi complexes of 1- and 3-day cells contained very low density lipoprotein-sized particles, which suggests that the monolayer cells synthesize lipoproteins. These electron microscope observations demonstrate that adult hepatic parenchymal cells in monolayer retain for several days the subcellular structural elements characteristic of normally functioning hepatocytes.
Assuntos
Fígado/citologia , Animais , Membrana Celular , Núcleo Celular , Células Cultivadas , Retículo Endoplasmático , Complexo de Golgi , Técnicas In Vitro , Corpos de Inclusão , Regeneração Hepática , Masculino , Microscopia Eletrônica , Microtúbulos , Mitocôndrias Hepáticas , Perfusão , RatosRESUMO
Asialoglycoproteins are taken up by the rat liver for degradation; rat polymeric IgA is taken up via a separate receptor, secretory component (SC), for quantitative delivery to bile. There is negligible uptake of these ligands by the converse receptor, and only a low level of missorting of ligands to opposite destinations. The two pathways are not cross-inhibitable and operate independently (Schiff, J.M., M. M. Fisher, and B. J. Underdown, 1984, J. Cell Biol., 98:79-89). We report here that when human IgA is presented as a ligand in the rat, it is processed using elements of both pathways. To study this in detail, different IgA fractions were prepared using two radiolabeling methods that provide separate probes for degradation or re-secretion. Behavior of intravenously injected human polymeric IgA in the rat depended on its binding properties. If deprived of SC binding activity by affinity adsorption or by reduction and alkylation, greater than 80% of human IgA was degraded in hepatic lysosomes; radioactive catabolites were released into bile by a leupeptin-inhibitable process. If prevented from binding to the asialoglycoprotein receptor by competition or by treatment with galactose oxidase, human IgA was cleared and transported to bile directly via SC, but its uptake was about fivefold slower than rat IgA. Untreated human IgA was taken up rapidly by the asialoglycoprotein receptor, but depended on SC binding to get to bile: the proportion secreted correlated 1:1 with SC binding activity determined in vitro, and the IgA was released into bile with SC still attached. These results demonstrate that human IgA is normally heterovalent: it is first captured from blood by the asialoglycoprotein receptor, but escapes the usual fate of asialoglycoproteins by switching to SC during transport. Since the biliary transit times of native human and rat IgA are the same, it is probable that the receptor switching event occurs en route. This implies that the two receptors briefly share a common intracellular compartment.
Assuntos
Imunoglobulina A/metabolismo , Fragmentos de Imunoglobulinas/metabolismo , Fígado/metabolismo , Receptores Fc , Receptores Imunológicos/metabolismo , Componente Secretório/metabolismo , Animais , Receptor de Asialoglicoproteína , Bile/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Compartimento Celular , Galactose Oxidase/metabolismo , Humanos , Leupeptinas/farmacologia , Ligantes/metabolismo , Lisossomos/metabolismo , Masculino , Ligação Proteica , Ratos , Ratos Endogâmicos , Especificidade da Espécie , Fatores de TempoRESUMO
By scanning and transmission electron microscopy we have shown that insulin rapidly reversed changes in surface membrane conformation and polysome profile induced by the transfer of actively growing Balb/c 3T3 fibroblasts from a serum-containing to a serum-free medium. Morphometric analysis of polysome profiles revealed a 94% aggregation of total f ribosomes during logarithmic growth. This figure fell to 78% after 18 h of serum starvation. The number of f ribosomes per unit area of cytoplasm also fell. 1 h of insulin treatment restored aggregation to 92% and increased the number of f ribosomes per unit area of cytoplasm by 22%. Scanning electron microscopy of logarithmically growing cells revealed an abundance of surface microvilli, whereas serum starvation promoted a smooth surface with few microvilli. After 1 h of insulin treatment, microvilli reappeared with a distribution and subcellular organization characteristic of exponential growth. This study shows the combined and rapid effect of insulin on the regulation of polysome formation and the promotion of a specific surface membrane conformation in cultured cells. The observations are consistent with the knowledge that insulin, acting on the surface membrane, can influence such parameters as membrane transport, and the rates of protein and RNA synthesis.
Assuntos
Membrana Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Insulina/farmacologia , Polirribossomos/efeitos dos fármacos , Animais , Sangue , Linhagem Celular , Meios de Cultura , Citoplasma , Retículo Endoplasmático/efeitos dos fármacos , Fibroblastos , Complexo de Golgi/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , MicrotúbulosRESUMO
The specific localization and the characterization of the parathyroid hormone (PTH) receptor in bone have been studied using 18-d embryonic chick calvariae and biologically active, electrolytically labeled [125I] bovine PTH(1-34). Binding was initiated by adding [125I]-bPTH(1-34) to bisected calvariae at 30 degrees C. Steady state binding was achieved at 90 min at which time 10 mg drg wt of calvaria specifically bound 17% of the added [125I]bPTH(1-34). Nonspecific binding in the presence of 244 nM unlabeled bPTH(1-34) was less than 2%. Insulin, glucagon, and calcitonin (1 microgram/ml) did not compete for PTH binding sites. Half-maximal inhibition of binding was achieved at concentrations of unlabeled bPTH(1-34) or bPTH(1-84) of about 10 nM. The range of concentration (2-100 nM) over which bPTH(1-34) and bPTH(1-84) stimulated cyclic 3'5'adenosine monophosphate (cAMP) production was similar to that which inhibited the binding of [125I]bPTH(1-34). Light microscope autoradiograms showed that grains were concentrated over cells (osteoblasts and progenitor cells) at the external surface of the calvariae and in trabeculae. In the presence of excess unlabeled PTH, labeling of control autoradiograms was reduced to near background levels. No labeling of osteocytes or osteoclasts was observed. At the electron microscopic level, grains were localized primarily over cell membranes. A quantitative analysis of grain distribution suggested that cellular internalization of PTH occurred.
Assuntos
Osso e Ossos/embriologia , Receptores de Superfície Celular/metabolismo , Animais , Osso e Ossos/metabolismo , Osso e Ossos/ultraestrutura , Membrana Celular/metabolismo , Embrião de Galinha , AMP Cíclico/metabolismo , Cinética , Microscopia Eletrônica , Hormônio Paratireóideo/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de Hormônios Paratireóideos , Relação Estrutura-AtividadeRESUMO
Epidermal growth factor (EGF), circulating in the blood, is taken up by rat liver hepatocytes by means of specific and saturable receptor-mediated endocytosis. These experiments were undertaken to determine (a) the transport pathway(s) of EGF taken up by rat liver and (b) the effects of lysosomal inhibition on its transport. 125I-EGF was injected into rat portal veins, and bile samples were collected and analyzed for both total and immunoprecipitable radioactivity. In addition, the livers were examined by electron microscopic autoradiography. Some animals received injections of chloroquine before surgery, to disrupt lysosomal function. The results indicate that most of the EGF taken up by the hepatocytes is transported to lysosomes and degraded. However, a small but significant percentage of endocytosed EGF is transported by a pathway independent of the lysosomal system, resulting in secretion of intact EGF: (a) Both degraded and immunoprecipitable EGF are secreted into bile. (b) Immunoprecipitable radioactivity peaks at 20 min after EGF injection, whereas degradation-associated radioactivity does not peak until 40 min postinjection. (c) EGF isolated from bile is specifically taken up by isolated hepatocytes in monolayer culture, indicating that it is still recognizable by the EGF receptor. (d) When the lysosomal system is inhibited with chloroquine, secretion of degraded EGF is significantly inhibited, whereas the amount of intact EGF secreted into bile is unchanged. The utilization by liver of a dual transport process for EGF represents an unusual system of intracellular ligand processing, whose physiological significance has yet to be determined.
Assuntos
Endocitose , Fator de Crescimento Epidérmico/metabolismo , Fígado/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Autorradiografia , Bile/metabolismo , Cloroquina/farmacologia , Receptores ErbB , Radioisótopos do Iodo , Fígado/ultraestrutura , Lisossomos/metabolismo , Microscopia Eletrônica , Ratos , Receptores de Superfície Celular/efeitos dos fármacos , Frações Subcelulares/metabolismoRESUMO
Morphometric analysis demonstrated a twofold increase in the surface area of the hepatic endoplasmic reticulum in Fischer 344 rats between 1 and 20 months of age, followed by a significant decrease in this parameter between 20 and 30 months. These changes are attributed to the smooth-surfaced endoplasmic reticulum, since neither the rough-surfaced variety nor the Golgi membranes underwent any significant change in surface area as a function of the age of the animal.
Assuntos
Envelhecimento , Retículo Endoplasmático/ultraestrutura , Fígado/ultraestrutura , Animais , Complexo de Golgi/ultraestrutura , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
This new enzymatic method disperses rabbit lung into morphologically intact, viable individual cells. The scattered cells constitute more than 50 percent of the original tissue. At least 90 percent of the cells exclude trypan blue from the nucleus. The dispersed lung cells consumed 6.2 microliters of oxygen per hour per milligram, dry weight. They incorporated [1-(14)C]palmitate into lecithin.
Assuntos
Pulmão/metabolismo , Animais , Isótopos de Carbono , Catalase/metabolismo , Células Cultivadas , Desoxirribonucleases/metabolismo , Pulmão/citologia , Métodos , Colagenase Microbiana/metabolismo , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Consumo de Oxigênio , Ácidos Palmíticos/metabolismo , Elastase Pancreática/metabolismo , Papaína/metabolismo , Fosfatidilcolinas/biossíntese , Pronase/metabolismo , Coelhos , Coloração e Rotulagem , Azul TripanoRESUMO
Quantitative electron microscopic autoradiography and diaminobenzidine cytochemistry provide evidence for an uptake and vesicular transport mechanism for iodine-125-labeled immunoglobulin A from plasma to bile by hepatocytes in vivo. The data confirm the existence of a hepatobiliary pathway for secretion of immunoglobulin A into the intestine and are consistent with a vesicular transport mechanism for biliary proteins within liver parenchymal cells.