RESUMO
Mary Osborn was a native Californian. She was an undergraduate at the University of California, Berkeley, where she worked in the laboratory of I.L. Chaikoff. She received her PhD at the University of Washington, where her work on the role of folic acid coenzymes in one-carbon metabolism revealed the mechanism of action of methotrexate. After postdoctoral training with Bernard Horecker in the Department of Microbiology at New York University (NYU), she embarked on her research career as a faculty member in the NYU Department of Microbiology and in the Department of Molecular Biology at Albert Einstein College of Medicine. In 1968 she moved as one of the founding faculty of the new medical school of the University of Connecticut, where she remained until her retirement in 2014. Her research was focused on the biosynthesis of the endotoxin lipopolysaccharide (LPS) of gram-negative bacteria and on the assembly of the bacterial cell envelope. She made seminal contributions in these areas. She was the recipient of numerous honors and served as president of several important scientific organizations. Later in her career she served as chair of the National Research Council Committee on Space Biology and Medicine, advisory to the National Aeronautics and Space Administration (NASA), which produced an influential report that plotted the path for NASA's space biology research program in the first decade of the twenty-first century. Dr. Osborn died on Jan. 17, 2019.
Assuntos
Bacteriologia/história , Bactérias Gram-Negativas/metabolismo , Lipopolissacarídeos/biossíntese , Bacteriologia/tendências , Bactérias Gram-Negativas/genética , História do Século XX , História do Século XXI , Estados UnidosRESUMO
BACKGROUND: Mortuaries are predominantly staffed by anatomical pathology technologists (APTs) and pathologists, and the work they undertake carries implicit health risk due to its nature. Until now there has not been a nationwide assessment of the occupational health of these essential workers in the UK. AIMS: To assess the current occupational health status and needs of the mortuary workforce in the UK. METHODS: We created a bespoke, brief online survey which was approved by the professional bodies representing APTs and pathologists in the UK. The survey was disseminated electronically using these organizations' targeted mailing lists. RESULTS: Two hundred and thirty participants completed the survey, comprising 108 (47%) APTs and 122 (53%) pathologists. Most (89%) respondents reported that they have suffered from occupational health issues, the largest subcategory being musculoskeletal problems (77%). Almost half (48%) of APTs and around one-quarter (26%) of pathologists who responded have taken time off work in the last year because of occupational health problems, with almost one-fifth (19%) of the APTs having taken at least 4 weeks off. CONCLUSIONS: A significant number of workhours are lost per year to sick leave resulting from occupational health problems. Respondents' comments highlight issues in workspaces, rest facilities and staffing, and variability in working conditions across the country. We suggest that future workforce planning should prioritize good occupational health, with nationwide improvements in mortuary design.
Assuntos
Doenças Profissionais , Saúde Ocupacional , Humanos , Inquéritos e Questionários , Inquéritos Epidemiológicos , Recursos Humanos , Doenças Profissionais/epidemiologia , Reino UnidoRESUMO
There is increasing evidence that patients with Coronavirus disease 19 (COVID-19) present with neurological and psychiatric symptoms. Anosmia, hypogeusia, headache, nausea and altered consciousness are commonly described, although there are emerging clinical reports of more serious and specific conditions such as acute cerebrovascular accident, encephalitis and demyelinating disease. Whether these presentations are directly due to viral invasion of the central nervous system (CNS) or caused by indirect mechanisms has yet to be established. Neuropathological examination of brain tissue at autopsy will be essential to establish the neuro-invasive potential of the SARS-CoV-2 virus but, to date, there have been few detailed studies. The pathological changes in the brain probably represent a combination of direct cytopathic effects mediated by SARS-CoV-2 replication or indirect effects due to respiratory failure, injurious cytokine reaction, reduced immune response and cerebrovascular accidents induced by viral infection. Further large-scale molecular and cellular investigations are warranted to clarify the neuropathological correlates of the neurological and psychiatric features seen clinically in COVID-19. In this review, we summarize the current reports of neuropathological examination in COVID-19 patients, in addition to our own experience, and discuss their contribution to the understanding of CNS involvement in this disease.
Assuntos
COVID-19/complicações , COVID-19/patologia , Doenças do Sistema Nervoso/patologia , Doenças do Sistema Nervoso/virologia , Feminino , Humanos , Masculino , SARS-CoV-2RESUMO
The need to evacuate an ICU or operating theatre complex during a fire or other emergency is a rare event but one potentially fraught with difficulty: Not only is there a risk that patients may come to harm but also that staff may be injured and unable to work. Designing newly-built or refurbished ICUs and operating theatre suites is an opportunity to incorporate mandatory fire safety features and improve the management and outcomes of such emergencies: These include well-marked manual fire call points and oxygen shut off valves (area valve service units); the ability to isolate individual zones; multiple clear exit routes; small bays or side rooms; preference for ground floor ICU location and interconnecting routes with operating theatres; separate clinical and non-clinical areas. ICUs and operating theatre suites should have a bespoke emergency evacuation plan and route map that is readily available. Staff should receive practical fire and evacuation training in their clinical area of work on induction and annually as part of mandatory training, including 'walk-through practice' or simulation training and location of manual fire call points and fire extinguishers, evacuation routes and location and operation of area valve service units. The staff member in charge of each shift should be able to select and operate fire extinguishers and lead an evacuation. Following an emergency evacuation, a network-wide response should be activated, including retrieval and transport of patients to other ICUs if needed. A full investigation should take place and ongoing support and follow-up of staff provided.
Assuntos
Desastres , Incêndios , Unidades de Terapia Intensiva , Salas Cirúrgicas , Gestão da Segurança/métodos , Emergências , Inundações , HumanosRESUMO
BACKGROUND: While several studies have examined the treatment of adolescents and young adults (AYAs) with acute lymphoblastic leukemia (ALL), studies of acute myeloid leukemia (AML) are rare. Using national data for Australia, we describe (i) the number and type of treatment centers caring for AYAs, (ii) induction/first-line treatments, and (iii) survival outcomes. PROCEDURE: National population-based study assessing treatment of 15- to 24-year-olds diagnosed with ALL or AML between 2007 and 2012. Treatment details were abstracted from hospital medical records. Treatment centers were classified as pediatric or adult (adult AYA-focused or other adult; and by AYA volume [high/low]). Cox proportional hazard regression analyses examined associations between treatment and overall, event-free, and relapse-free survival outcomes. RESULTS: Forty-seven hospitals delivered induction therapy to 351 patients (181 ALL and 170 AML), with 74 (21%) treated at pediatric centers; 70% of hospitals treated less than two AYA leukemia patients per year. Regardless of treatment center, 82% of ALL patients were on pediatric protocols. For AML, pediatric protocols were not used in adult centers, with adult centers using a non-COG 7+3-type induction protocol (51%, where COG is Cooperative Oncology Group) or an ICE-type protocol (39%, where ICE is idarubicin, cytarabine, etoposide). Exploratory analyses suggested that for both ALL and AML, AYAs selected for adult protocols have worse overall, event-free, and relapse-free survival outcomes. CONCLUSIONS: Pediatric protocols were commonly used for ALL patients regardless of where they are treated, indicating rapid assimilation of recent evidence by Australian hematologists. For AML, pediatric protocols were only used at pediatric centers. Further investigation is warranted to determine the optimal treatment approach for AYA AML patients.
Assuntos
Quimioterapia de Indução/métodos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Adolescente , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Austrália , Feminino , Humanos , Masculino , Oncologia/métodos , Pediatria/métodos , Modelos de Riscos Proporcionais , Resultado do Tratamento , Adulto JovemRESUMO
Cronkhite-Canada syndrome is an acquired inflammatory polyposis syndrome in which alopecia, onychomadesis and hyperpigmentation occur concurrently with gastrointestinal symptoms. The pathophysiology of alopecia in Cronkhite-Canada syndrome has not been definitively elucidated. We present evidence for alopecia areata incognita as a possible mechanism of hair loss.
Assuntos
Alopecia em Áreas/complicações , Polipose Intestinal/complicações , Transtornos da Pigmentação/complicações , Anti-Inflamatórios não Esteroides/administração & dosagem , Feminino , Glucocorticoides/administração & dosagem , Humanos , Mesalamina/administração & dosagem , Pessoa de Meia-Idade , Prednisolona/administração & dosagem , Síndrome , Vitaminas/administração & dosagemRESUMO
OBJECTIVES: The objective of this study was to evaluate possible nonlinear lamotrigine (LTG) pharmacokinetics at elevated concentration. LTG is reported to have linear kinetics, so that elimination rate is linearly proportional to blood concentration and a change in dose is accompanied by a proportionate change in serum concentration. We encountered patients in whom LTG serum concentration increased dramatically in response to minor or no change in LTG dose. We studied this phenomenon in patients with LTG toxicity in one clinic. MATERIALS AND METHODS: Using electronic medical records from 1997 to 2014, we identified patients who developed clinical LTG toxicity with LTG serum concentrations >20 mg/l, after tolerating lamotrigine at lower serum concentrations. We reviewed LTG dose change and other changes that preceded the episode of toxicity. RESULTS: Twenty-two patients had at least one episode of LTG toxicity with levels higher than 20 mg/l (of 922 patients with available levels). The peak serum concentration varied from 21.1 to 40.3 mg/l (mean 28.7). The increase in level was explained in three patients (post-delivery in one, addition of valproate in two). In the 18 others, the increase was not explained or it was disproportionate to an increase in LTG dose. CONCLUSIONS: Spikes in LTG levels and associated clinical toxicity may occur unexpectedly, suggesting that elimination kinetics may be nonlinear in some individuals at serum concentrations in the upper range. Measurement and close monitoring of LTG levels is warranted for new symptoms that could be consistent with lamotrigine toxicity, particularly when the baseline serum concentration has been >10 mg/l.
Assuntos
Anticonvulsivantes/efeitos adversos , Anticonvulsivantes/sangue , Epilepsia/sangue , Epilepsia/tratamento farmacológico , Triazinas/efeitos adversos , Triazinas/sangue , Adulto , Idoso , Anticonvulsivantes/uso terapêutico , Ataxia/sangue , Ataxia/induzido quimicamente , Tontura/sangue , Tontura/induzido quimicamente , Relação Dose-Resposta a Droga , Interações Medicamentosas/fisiologia , Registros Eletrônicos de Saúde , Feminino , Humanos , Lamotrigina , Masculino , Pessoa de Meia-Idade , Triazinas/uso terapêuticoRESUMO
BACKGROUND/AIM: The aim of this study was to describe the time and documentation needed to gain ethics and governance approvals in Australian states with and without a centralised ethical review system. METHODS: This is a prospective descriptive study undertaken between February 2012 and March 2015. Paediatric and adult hospitals (n = 67) in Australian states were approached to allow the review of their medical records. Participants included 15- to 24-year-olds diagnosed with cancer between 2008 and 2012. The main outcomes measures were time (weeks) to approval for ethics and governance and the number and type of documents submitted. RESULTS: Centralised ethics approval processes were used in five states, with approval taking between 2 and 18 weeks. One state did not use a centralised process, with ethics approval taking a median of 4.5 weeks (range: 0-15) per site. In four states using a centralised ethics process, 33 governance applications were submitted, with 20 requiring a site clinician listed as an investigator. Governance applications required the submission of 11 documents on average, including a Site-Specific Assessment form. Thirty-two governance applications required original signatures from a median of 3.5 (range: 1-10) non-research persons, which took a median of 5 weeks (range: 0-15) to obtain. Governance approval took a median of 6 weeks (range: 1-45). Twelve research study agreements were needed, each taking a median of 7.5 weeks (range: 1-20) to finalise. CONCLUSION: The benefits of centralised ethics review systems have not been realised due to duplicative, inflexible governance processes. A system that allowed the recognition of prior ethical approval and low-risk applications was more efficient than a central ethics and site-specific governance process.
Assuntos
Pesquisa Biomédica/ética , Revisão Ética/normas , Comitês de Ética em Pesquisa/organização & administração , Hospitais/ética , Adolescente , Austrália , Comportamento Cooperativo , Humanos , Estudos Prospectivos , Fatores de Tempo , Adulto JovemRESUMO
Suppression of hepatitis B virus (HBV)-DNA to undetectable levels is an important goal for HIV/HBV-co-infected patients receiving anti-HBV-active antiretroviral therapy (ART), and current guidelines recommend that this outcome should be reached by 1 year of treatment. However, the proportion of patients that fail to achieve an undetectable HBV DNA at this time point and its determinants remain unknown in clinical practice. The objective of this study was to determine the incidence and risk factors for incomplete HBV suppression following 1 year of tenofovir-based ART. We performed a cohort study among tenofovir-treated HIV/HBV-co-infected patients. Patients had HBV viraemia, initiated tenofovir-based ART and had HBV DNA measured at 1 year of therapy. The primary outcome was incomplete HBV suppression (HBV DNA ≥2.6 log IU/mL) at 1 year. Logistic regression determined odds ratio (ORs) of incomplete HBV suppression for risk factors of interest. Among 133 patients, 54% (95% CI, 46-63%) had incomplete HBV suppression at 1 year. Incomplete suppression was associated with higher baseline HBV DNA (OR, 1.46 per log IU/mL increase; 95% CI, 1.1-1.94) and detectable HIV viraemia at 1 year (OR, 2.52; 95% CI, 1.19-5.32). Among 66 patients with suppressed HIV RNA at 1 year, 28 (42%) failed to achieve an undetectable HBV DNA. Failure to suppress HBV DNA by 1 year occurred in a sizeable proportion of tenofovir-treated HIV/HBV-co-infected patients. Higher HBV DNA and detectable HIV viraemia were risk factors for incomplete HBV suppression.
Assuntos
Fármacos Anti-HIV/uso terapêutico , DNA Viral/sangue , Infecções por HIV/tratamento farmacológico , Vírus da Hepatite B/fisiologia , Hepatite B/tratamento farmacológico , Adenina/análogos & derivados , Adenina/uso terapêutico , Adulto , Terapia Antirretroviral de Alta Atividade , Estudos de Coortes , Coinfecção , Farmacorresistência Viral , Feminino , Infecções por HIV/complicações , Infecções por HIV/virologia , Hepatite B/complicações , Hepatite B/virologia , Vírus da Hepatite B/genética , Humanos , Incidência , Lamivudina/uso terapêutico , Masculino , Pessoa de Meia-Idade , Organofosfonatos/uso terapêutico , Estudos Retrospectivos , Fatores de Risco , Tenofovir , Carga Viral , ViremiaRESUMO
Mechanical circulatory support emerged for the pediatric population in the late 1980s as a bridge to cardiac transplantation. The Total Artificial Heart (TAH-t) (SynCardia Systems Inc., Tuscon, AZ) has been approved for compassionate use by the Food and Drug Administration for patients with end-stage biventricular heart failure as a bridge to heart transplantation since 1985 and has had FDA approval since 2004. However, of the 1,061 patients placed on the TAH-t, only 21 (2%) were under the age 18. SynCardia Systems, Inc. recommends a minimum patient body surface area (BSA) of 1.7 m(2), thus, limiting pediatric application of this device. This unique case report shares this pediatric institution's first experience with the TAH-t. A 14-year-old male was admitted with dilated cardiomyopathy and severe biventricular heart failure. The patient rapidly decompensated, requiring extracorporeal life support. An echocardiogram revealed severe biventricular dysfunction and diffuse clot formation in the left ventricle and outflow tract. The decision was made to transition to biventricular assist device. The biventricular failure and clot formation helped guide the team to the TAH-t, in spite of a BSA (1.5 m(2)) below the recommendation of 1.7 m(2). A computed tomography (CT) scan of the thorax, in conjunction with a novel three-dimensional (3D) modeling system and team, assisted in determining appropriate fit. Chest CT and 3D modeling following implantation were utilized to determine all major vascular structures were unobstructed and the bronchi were open. The virtual 3D model confirmed appropriate device fit with no evidence of compression to the left pulmonary veins. The postoperative course was complicated by a left lung opacification. The left lung anomalies proved to be atelectasis and improved with aggressive recruitment maneuvers. The patient was supported for 11 days prior to transplantation. Chest CT and 3D modeling were crucial in assessing whether the device would fit, as well as postoperative complications in this smaller pediatric patient.
Assuntos
Cardiomiopatia Dilatada/cirurgia , Insuficiência Cardíaca/cirurgia , Transplante de Coração/métodos , Coração Artificial , Adolescente , Cardiomiopatia Dilatada/terapia , Insuficiência Cardíaca/terapia , Humanos , MasculinoRESUMO
BACKGROUND: Painful neuropathy is a pathological condition caused by numerous factors including diabetes, chemotherapy or cancer. ART26.12 is a novel fatty acid-binding protein 5 inhibitor, which our group showed could prevent and treat persistent pain in a preclinical model of oxaliplatin-induced peripheral neuropathy. METHODS: In the current study, the efficacy of orally dosed ART26.12 was tested in multiple neuropathy models of different aetiology. Paw withdrawal threshold to von Frey monofilaments and latency to escape a cold plate were used as measurements of mechanical and cold sensitivity. RESULTS: ART26.12 (25 and 50 mg/kg BID), dosed prior to the induction of paclitaxel-induced peripheral neuropathy (PIPN), reversed mechanical allodynia induced by paclitaxel in both male and female rats, and ART26.12 (50 mg/kg BID) prevented the induction of PIPN in female rats. ART26.12 (50 mg/kg BID) also had a protective effect on body weight in the PIPN model. ART26.12 (25 and 100 mg/kg BID) reversed mechanical allodynia when treating established streptozotocin-induced diabetic neuropathy in male rats. In a model of breast cancer-induced bone pain in female rats, ART26.12 (100 mg/kg BID) reversed mechanical allodynia within 1 h of dosing. In the same model, ART26.12 (25 mg/kg BID) reversed mechanical allodynia from day 4 of treatment. CONCLUSION: Overall, these preclinical data suggest that ART26.12 is a safe and efficacious therapeutic drug for continued development towards the prevention and treatment of peripheral neuropathy. SIGNIFICANCE STATEMENT: This work now shows that ART26.12, a novel and selective inhibitor of FABP5, can prevent and treat multiple preclinical models of peripheral neuropathy. Given its excellent safety profile, further work is warranted to develop ART26.12 as a potential therapeutic tool for pain management.
RESUMO
BACKGROUND: The human organic cation transporter-1 (OCT-1) is the primary active protein for imatinib uptake into target BCR-ABL-positive cells. Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used by chronic myeloid leukaemia (CML) patients on imatinib to manage musculoskeletal complaints. METHODS: Here we investigated the impact of NSAIDs on functional activity of the OCT-1 (OCT-1 activity; OA) in CML cells. RESULTS: Although ten of twelve NSAIDs tested had no significant impact on OA (P>0.05), we observed increased OA (27% increase in K562; 22% increase in KU812 cells, P<0.05) and reduced IC50(imatinib) when treated with diclofenac. Co-incubation with imatinib and diclofenac resulted in a significantly lower viable cell number compared with imatinib alone. In contrast, ibuprofen led to a significant decrease in OA, an increase in IC50(imatinib) and thus reduced the cytotoxicity of imatinib. In primary CML samples, diclofenac significantly increased OA, particularly in patients with low OA (<4 ng per 200 000 cells), and significantly decreased IC50(imatinib). Ibuprofen induced significant decreases in OA in CML samples and healthy donors. CONCLUSION: On the basis of the expected impact of these two drugs on OA, ibuprofen should be avoided in combination with imatinib. Further studies are warranted regarding the potential benefit of diclofenac to improve OA in a clinical setting.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/metabolismo , Diclofenaco/farmacologia , Ibuprofeno/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Transportador 1 de Cátions Orgânicos/efeitos dos fármacos , Piperazinas/metabolismo , Pirimidinas/metabolismo , Benzamidas , Linhagem Celular Tumoral , Interações Medicamentosas/fisiologia , Humanos , Mesilato de Imatinib , Concentração Inibidora 50RESUMO
BACKGROUND: Paediatric oncology patients with febrile neutropenia are usually hospitalised and treated with empirical broad-spectrum antibiotic therapy to counter the risk of infection. However, there is currently no method available to rapidly identify bacteremia in these patients. T-helper-type-1 (Th1) cytokines are required for effective immune response to many pathogenic organisms and T regulatory cells are known suppressors of Th1 cells. We hypothesized that characterization of reduced intracellular Th1 cytokines and increased T regulatory cells (Tregs) may prove useful in identifying infection in childhood oncology patients with febrile neutropenia. METHODS: Intracellular Th 1 cytokines and Tregs were enumerated in peripheral blood from a group of childhood oncology patients with febrile neutropenia using multiparameter flow cytometry. RESULTS: There was a significant increase in the percentage of CD25(+) CD127(-) CD8(-) CD3(+) Tregs and a significant decrease in Th1 intracellular cytokines IFNγ, IL-2 and TNFα in the blood of culture positive patients compared with culture negative patients. CONCLUSIONS: Enumeration of Tregs and intracellular Th1 cytokines may provide a sensitive, specific test for determining infection in childhood oncology patients before blood culture results become available.
Assuntos
Citocinas/sangue , Neoplasias/sangue , Neutropenia/sangue , Linfócitos T Reguladores/metabolismo , Bacteriemia/sangue , Bacteriemia/etiologia , Complexo CD3/metabolismo , Antígenos CD8/metabolismo , Células Cultivadas , Criança , Febre/sangue , Febre/etiologia , Citometria de Fluxo , Humanos , Mediadores da Inflamação/sangue , Interferon gama/sangue , Interleucina-2/sangue , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Contagem de Linfócitos , Neoplasias/complicações , Neutropenia/etiologia , Células Th1/metabolismo , Fator de Necrose Tumoral alfa/sangueRESUMO
BACKGROUND: recent decades have seen combination chemoradiotherapy become the standard treatment for anal squamous cell carcinoma (SCC). However, the burden of this disease continues to rise, with only 10% of patients with metastatic disease surviving >2 years. Further insight into tumour characteristics and molecular biology may identify novel therapeutic targets. This systematic review examines current prognostic markers in SCC of the anus. METHODS: an extensive literature search was performed to identify studies reporting on biomarkers in anal cancer in the context of clinical outcome following treatment primarily with chemoradiotherapy. RESULTS: in all, 21 studies were included. A total of 29 biomarkers were studied belonging to 9 different functional classes. Of these biomarkers, 13 were found to have an association with outcome in at least one study. The tumour-suppressor genes p53 and p21 were the only markers shown to be of prognostic value in more than one study. CONCLUSIONS: an array of biomarkers have been identified that correlate with survival following chemoradiotherapy in anal cancer. However, investigators are yet to identify a biomarker that has the ability to consistently predict outcome in this disease. Further studies are needed to elucidate whether these candidate biomarkers demonstrate their optimum value when they serve as targets for new therapeutic strategies.
Assuntos
Neoplasias do Ânus/mortalidade , Biomarcadores Tumorais , Carcinoma de Células Escamosas/mortalidade , Neoplasias do Ânus/genética , Neoplasias do Ânus/patologia , Apoptose , Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Inibidor de Quinase Dependente de Ciclina p21/análise , Inibidor de Quinase Dependente de Ciclina p27 , Genes Supressores de Tumor , Genes p53 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , PrognósticoRESUMO
Eggs of the sea urchin Strongylocentrotus purpuratus were examined by indirect immunofluorescence microscopy for tubulin-containing structures at intervals from fertilization through first cleavage. The staining revealed that the monaster is made up not only of the sperm aster but also of tubulin-staining fibers originating elsewhere in the egg. The monaster does not divide directly but is broken down first before the amphiaster or interphase asters begin to form. The interphase asters reach a peak of development at the streak stage and are in turn broken down before the formation of the mitotic apparatus. The breakdown of the monaster, interphase asters, as well as the asters of the mitotic apparatus proceeds from the cell center or aster centers to the periphery of the cell and is followed by growth of new asters, also proceeding outward from the aster centers. The pattern suggests a transient wavelike movement of some condition, or factor, which favors microtubule depolymerization.
Assuntos
Fertilização , Microtúbulos/ultraestrutura , Tubulina (Proteína)/análise , Zigoto/ultraestrutura , Animais , Divisão Celular , Núcleo Celular/ultraestrutura , Feminino , Imunofluorescência , Interfase , Microscopia de Fluorescência , Mitose , Organoides/ultraestrutura , Ouriços-do-MarRESUMO
To screen invertebrate tissues for the possible expression of intermediate filaments (IFs), immunofluorescence microscopy with the monoclonal antibody anti-IFA known to detect all mammalian IF proteins was used (Pruss, R. M., R. Mirsky, M. C. Raff, R. Thorpe, A. J. Dowding, and B. H. Anderton. 1981. Cell, 27:419-428). In a limited survey, the lower chordate Branchiostoma as well as the invertebrates Arenicola, Lumbricus, Ascaris, and Helix pomatia revealed a positive reaction primarily on epithelia and on nerves, whereas certain other invertebrates appeared negative. To assess the nature of the positive reaction, Helix pomatia was used since a variety of epithelia was strongly stained by anti-IFA. Fixation-extraction procedures were developed that preserve in electron micrographs of esophagus impressive arrays of IFs as tonofilament bundles. Fractionation procedures performed on single cell preparations document large meshworks of long and curvilinear IF by negative stain. These structures can be purified. One- and two-dimensional gels show three components, all of which are recognized by anti-IFA in immunoblotting: 66 kD/pl 6.35, 53 kD/pl 6.05, and 52 kD/pl 5.95. The molar ratio between the larger and more basic polypeptide and the sum of the two more acidic forms is close to 1. After solubilization in 8.5 M urea, in vitro filament reconstitution is induced when urea is removed by dialysis against 2-50 mM Tris buffer at pH 7.8. The reconstituted filaments contain all three polypeptides. The results establish firmly the existence of invertebrate IFs outside neurones and demonstrate that the esophagus of Helix pomatia displays IFs which in line with the epithelial morphology of the tissue could be related to keratin IF of vertebrates.
Assuntos
Citoesqueleto/ultraestrutura , Caracois Helix/anatomia & histologia , Aminoácidos/análise , Animais , Anticorpos Monoclonais , Citoesqueleto/análise , Epitélio/análise , Epitélio/ultraestrutura , Esôfago/análise , Esôfago/ultraestrutura , Imunofluorescência , Proteínas de Filamentos Intermediários/análise , Camundongos , Camundongos Endogâmicos C3H , Camundongos Nus , Peptídeos/análise , Frações Subcelulares/análise , Frações Subcelulares/ultraestruturaRESUMO
Current concepts of the developmentally controlled multigene family of intermediate filament (IF) proteins expect the origin of their complexity in evolutionary precursors preceding all vertebrate classes. Among invertebrates, however, firm ultrastructural as well as molecular documentation of IFs is restricted to some giant axons and to epithelia of a few molluscs and annelids. As Ascaris lumbricoides is easily dissected into clean tissues, IF expression in this large nematode was analyzed by electron microscopic and biochemical procedures and a monoclonal antibody reacting with all mammalian IF proteins. We document for the first time the presence of IFs in muscle cells of an invertebrate. They occur in three muscle types (irregular striated pharynx muscle, obliquely striated body muscle, uterus smooth muscle). IFs are also found in the epithelia studied (syncytial epidermis, intestine, ovary, testis). Immunoblots on muscles, pharynx, intestine, uterus, and epidermis identify a pair of polypeptides (with apparent molecular masses of 71 and 63 kD) as IF constituents. In vitro reconstitution of filaments was obtained with the proteins purified from body muscle. In the small nematode Caenorhabditis elegans IF proteins are so far found only in the massive desmosome-anchored tonofilament bundles which traverse a special epithelial cell type, the marginal cells of the pharynx. We speculate that IFs may occur in most but perhaps not all invertebrates and that they may not occur in all cells in large amounts. As electron micrographs of the epidermis of a planarian--a member of the Platyhelminthes--reveal IFs, the evolutionary origin of this cytoplasmic structure can be expected either among the lowest metazoa or already in some unicellular eukaryotes.
Assuntos
Citoesqueleto/ultraestrutura , Filamentos Intermediários/ultraestrutura , Músculos/ultraestrutura , Nematoides/ultraestrutura , Animais , Ascaris , Caenorhabditis , Epitélio/análise , Epitélio/ultraestrutura , Imunofluorescência , Proteínas de Filamentos Intermediários/análise , Filamentos Intermediários/análise , Microscopia Eletrônica , Músculos/análise , Nematoides/análise , PlanáriasRESUMO
Antibodies to muscle-specific proteins were used in immunofluorescence to monitor the development of skeletal muscle during mouse embryogenesis. At gestation day (g.d.) 9 a single layer of vimentin filament containing cells in the myotome domain of cervical somites begins to stain positively for myogenic proteins. The muscle-specific proteins are expressed in a specific order between g.d. 9 and 9.5. Desmin is detected first, then titin, then the muscle specific actin and myosin heavy chains, and finally nebulin. At g.d. 9.5 fibrous desmin structures are already present, while for the other myogenic proteins no structure can be detected. Some prefusion myoblasts display at g.d. 11 and 12 tiny and immature myofibrils. These reveal a periodic pattern of myosin, nebulin, and those titin epitopes known to occur at and close to the Z line. In contrast titin epitopes, which are present in mature myofibrils along the A band and at the A-I junction, are still randomly distributed. We propose, that the Z line connected structures and the A bands (myosin filaments) assemble independently, and that the known interaction of the I-Z-I brushes with the A bands occurs at a later developmental stage. After fusion of myoblasts to myotubes at g.d. 13 and 14 all titin epitopes show the myofibrillar banding pattern. The predominantly longitudinal orientation of desmin filaments seen in myoblasts and in early myotubes is transformed at g.d. 17 and 18 to distinct Z line connected striations. Vimentin, still present together with desmin in the myoblasts, is lost from the myotubes. Our results indicate that the putative elastic titin filaments act as integrators during skeletal muscle development. Some developmental aspects of eye and limb muscles are also described.
Assuntos
Actinas/metabolismo , Desmina/metabolismo , Proteínas Musculares/metabolismo , Músculos/metabolismo , Miosinas/metabolismo , Proteínas Quinases , Vimentina/metabolismo , Actinas/genética , Animais , Diferenciação Celular , Conectina , Desmina/genética , Epitopos/análise , Feminino , Imunofluorescência , Regulação da Expressão Gênica , Camundongos , Proteínas Musculares/análise , Proteínas Musculares/genética , Músculos/citologia , Músculos/embriologia , Miosinas/genética , Gravidez , Vimentina/genéticaRESUMO
The microtubule system of melanophores of the angelfish, Pterophyllum scalare, has been studied using antibodies prepared against purified porcine brain tubulin in indirect immunofluorescence microscopy. Melanophores were freed from the surrounding tissue components of isolated scales by mild enzymatic digestion and then allowed to settle on a glass cover slip. In both the dispersed and the aggregated states large numbers of fluorescent fibers are seen. The number and the astral arrangement of these fibers, which run from the central region to the periphery of the cell, are striking. The system of fluorescent fibers is replaced by diffuse fluorescence of moderate intensity after cold treatment, but is restored after rewarming the cells. Differences in the immunofluorescence profiles between cells with dispersed and aggregated pigment are discussed in relation to electron microscopic data available for this system.