RESUMO
Fibrin sealants have long been used as carrier for osteogenic cells in bone regeneration. However, it has not been demonstrated whether fibrin's role is limited to delivering cells to the bone defect or whether fibrin enhances osteogenesis. This study investigated fibrin's influence on the behaviour of human periosteum-derived cells (hPDCs) when cultured in vitro under osteogenic conditions in two-dimensional (fibrin substrate) and three-dimensional (fibrin carrier) environments. Tranexamic acid (TEA) was used to reduce fibrin degradation after investigating its effect on hPDCs in monolayer culture on plastic.TEA did not affect proliferation nor calcium deposition of hPDCs under these conditions. Expression profiles of specific osteogenic markers were also maintained within the presence of TEA, apart from reduced alkaline phosphatase (ALP) expression (day 14). Compared to plastic, proliferation was upregulated on 2D fibrin substrates with a 220% higher DNA content by day 21. Gene expression was also altered, with significantly (p<0.05) decreased Runx2 (day 7) and ALP (day 14) expression and increased collagen I expression (day 14 and 21). In contrast to plastic, mineralisation was absent on fibrin substrates. Inside fibrin carriers, hPDCs were uniformly distributed. Moderate cell growth and reduced osteogenic marker expression was observed inside fibrin carriers. After 2 weeks, increased cell death was present in the carrier's centre. In conclusion, fibrin negatively influences osteogenic differentiation, compared to culture plastic, but enhanced proliferation (at least in 2D cultures) for hPDCs cultured in osteogenic conditions. TEA maintained the integrity of fibrin-based constructs, with minor effects on the osteogenic differentiation of hPDCs.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Fibrina/farmacologia , Osteogênese/efeitos dos fármacos , Periósteo/citologia , Ácido Tranexâmico/farmacologia , Adulto , Antraquinonas/metabolismo , Biomarcadores/metabolismo , Cálcio/metabolismo , Contagem de Células , Diferenciação Celular/genética , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Criança , Pré-Escolar , DNA/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Osteogênese/genética , Estabilidade Proteica/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Coloração e Rotulagem , Adulto JovemRESUMO
In this study, we investigated a clinically relevant model of in vivo ectopic bone formation utilizing human periosteum derived cells (HPDCs) seeded in a Collagraft carrier and explored the mechanisms by which this process is driven. Bone formation occurred after eight weeks when a minimum of one million HPDCs was loaded on Collagraft carriers and implanted subcutaneously in NMRI nu/nu mice. De novo bone matrix, mainly secreted by the HPDCs, was found juxta-proximal of the calcium phosphate (CaP) granules suggesting that CaP may have triggered the 'osteoinductive program'. Indeed, removal of the CaP granules by ethylenediaminetetraacetic acid decalcification prior to cell seeding and implantation resulted in loss of bone formation. In addition, inhibition of endogenous bone morphogenetic protein and Wnt signalling by overexpression of the secreted antagonists Noggin and Frzb, respectively, also abrogated osteoinduction. Proliferation of the engrafted HPDCs was strongly reduced in the decalcified scaffolds or when seeded with adenovirus-Noggin/Frzb transduced HPDCs indicating that cell division of the engrafted HPDCs is required for the direct bone formation cascade. These data suggest that this model of bone formation is similar to that observed during physiological intramembranous bone development and may be of importance when investigating tissue engineering strategies.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Fosfatos de Cálcio/farmacologia , Modelos Biológicos , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/metabolismo , Adolescente , Adulto , Animais , Proteínas de Transporte/metabolismo , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Coristoma/patologia , Colágeno/farmacologia , Regulação para Baixo/efeitos dos fármacos , Durapatita/farmacologia , Feminino , Glicoproteínas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Periósteo/citologia , Adulto JovemRESUMO
Delineation of apical and basolateral membrane domains is a critical step in the epithelialization of the outer layer of cells in the embryo. We have examined the initiation of polarized membrane traffic in Xenopus and show that membrane traffic is not polarized in oocytes but polarized membrane domains appear at first cleavage. The following proteins encoded by injected RNA transcripts were used as markers to monitor membrane traffic: (a) VSV G, a transmembrane glycoprotein preferentially inserted into the basolateral surface of polarized epithelial cells; (b) GThy-1, a fusion protein of VSV G and Thy-1 that is localized to the apical domains of polarized epithelial cells; and (c) prolactin, a peptide hormone that is not polarly secreted. In immature oocytes, there is no polarity in the expression of VSV G or GThy-1, as shown by the constitutive expression of both proteins at the surface in the animal and vegetal hemispheres. At meiotic maturation, membrane traffic to the surface is blocked; the plasma membrane no longer accepts the vesicles synthesized by the oocyte (Leaf, D. L., S. J. Roberts, J. C. Gerhart, and H.-P. Moore. 1990. Dev. Biol. 141:1-12). When RNA transcripts are injected after fertilization, VSV G is expressed only in the internal cleavage membranes (basolateral orientation) and is excluded from the outer surface (apical orientation, original oocyte membrane). In contrast, GThy-1 and prolactin, when expressed in embryos, are inserted or released at both the outer membrane derived from the oocyte and the inner cleavage membranes. Furthermore, not all of the cleavage membrane comes from an embryonic pool of vesicles--some of the cleavage membrane comes from vesicles synthesized during oogenesis. Using prolactin as a marker, we found that a subset of vesicles synthesized during oogenesis was only released after fertilization. However, while embryonic prolactin was secreted from both apical and basolateral surfaces, the secretion of oogenic prolactin was polarized. Oogenic prolactin was secreted only into the blastocoel (from the cleavage membrane), none could be detected in the external medium (from the original oocyte membrane). These results provide the first direct evidence that the oocyte synthesizes a cache of vesicles for specific recruitment to the embryonic cleavage membranes which are polarized beginning with the first cleavage division.
Assuntos
Membrana Celular/metabolismo , Polaridade Celular/fisiologia , Embrião não Mamífero/metabolismo , Proteínas de Membrana/metabolismo , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Transporte Biológico Ativo , Embrião não Mamífero/citologia , Desenvolvimento Embrionário , Imunofluorescência , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Plasmídeos/genética , Prolactina/genética , Prolactina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Antígenos Thy-1 , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Xenopus laevisRESUMO
Osteosarcoma management continues to lack the appropriate prognostic tools to assign personalised treatment. This leaves non-responders to standard care vulnerable to recurring disease and pulmonary metastases. Developing 3D in vitro disease models to serve as a test bed for personalised treatment is a promising approach to address this issue. This study describes the generation of 3D osteosarcoma models termed "tumouroids", which are geometrically compartmentalised to reproduce the bone cancer mass and its surrounding. Although the tumour microenvironment impacts osteosarcoma in many ways, this model focussed on interrogating the influence of a biomimetic matrix on tumour cell behaviour. The 3D matrix was supplemented with the bone-marrow proteins laminin, fibronectin and NuOss® bone granules. This led to increased invasion of osteosarcoma cell aggregates from within the bone-like matrix into the surrounding acellular bone marrow-like ECM. The presence of bone granules also yielded an atypical molecular profile of osteosarcoma cells, suggesting malignant metabolic reprogramming. Changes include decreased MMP-9 (pâ¯<â¯0.05) and increased PTEN (pâ¯<â¯0.05), MCP-1 (pâ¯<â¯0.01) and MCT-4 (pâ¯<â¯0.05) gene expression. This complex 3D biomimetic composition also changed cellular responses to doxorubicin, a common chemotherapeutic agent used to treat osteosarcoma, and reproduced key issues of in vivo treatment like drug penetrance and doxorubicin-induced bone toxicity. This work highlights the importance of a biomimetic matrix in 3D osteosarcoma models for both basic and translational research. STATEMENT OF SIGNIFICANCE: This study describes the generation of 3D osteosarcoma models termed "tumouroids", which are geometrically compartmentalised to reproduce the bone cancer mass and its environment. Utilising this novel model, specific parameters of osteosarcoma growth and invasion were investigated. Osteosarcoma cell lines proliferate at a slower rate, exhibit malignant metabolic reprogramming, and respond to drug intervention at lower concentrations of doxorubicin hydrochloride in matrix-complex compared to basic tumouroids. As such, this study provides evidence that the tumour microenvironment impacts osteosarcoma in many ways. The osteosarcoma tumouroid described herein may form the basis of a personalised-medicine strategy, which will allow the testing of drug effectiveness similar to that used for antibiotic selection for pathogenic bacteria.
Assuntos
Materiais Biomiméticos/química , Matriz Óssea/química , Neoplasias Ósseas , Matriz Extracelular/química , Modelos Biológicos , Osteossarcoma , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Humanos , Invasividade Neoplásica , Osteossarcoma/metabolismo , Osteossarcoma/patologiaRESUMO
The Antarctic Peninsula Ice Sheet is currently experiencing sustained and accelerating loss of ice. Determining when these changes were initiated and identifying the main drivers is hampered by the short instrumental record (1992 to present). Here we present a 6,250 year record of glacial discharge based on the oxygen isotope composition of diatoms (δ18Odiatom) from a marine core located at the north-eastern tip of the Antarctic Peninsula. We find that glacial discharge - sourced primarily from ice shelf and iceberg melting along the eastern Antarctic Peninsula - remained largely stable between ~6,250 to 1,620 cal. yr BP, with a slight increase in variability until ~720 cal. yr. BP. An increasing trend in glacial discharge occurs after 550 cal. yr BP (A.D. 1400), reaching levels unprecedented during the past 6,250 years after 244 cal. yr BP (A.D. 1706). A marked acceleration in the rate of glacial discharge is also observed in the early part of twentieth century (after A.D. 1912). Enhanced glacial discharge, particularly after the 1700s is linked to a positive Southern Annular Mode (SAM). We argue that a positive SAM drove stronger westerly winds, atmospheric warming and surface ablation on the eastern Antarctic Peninsula whilst simultaneously entraining more warm water into the Weddell Gyre, potentially increasing melting on the undersides of ice shelves. A possible implication of our data is that ice shelves in this region have been thinning for at least ~300 years, potentially predisposing them to collapse under intensified anthropogenic warming.
RESUMO
BACKGROUND: Chondrogenic mesenchymal stem cells (MSCs) have not yet been used to address the clinical demands of large osteochondral joint surface defects. In this study, self-assembling tissue intermediates (TIs) derived from human periosteum-derived stem/progenitor cells (hPDCs) were generated and validated for stable cartilage formation in vivo using two different animal models. METHODS: hPDCs were aggregated and cultured in the presence of a novel growth factor (GF) cocktail comprising of transforming growth factor (TGF)-ß1, bone morphogenetic protein (BMP)2, growth differentiation factor (GDF)5, BMP6, and fibroblast growth factor (FGF)2. Quantitative polymerase chain reaction (PCR) and immunohistochemistry were used to study in vitro differentiation. Aggregates were then implanted ectopically in nude mice and orthotopically in critical-size osteochondral defects in nude rats and evaluated by microcomputed tomography (µCT) and immunohistochemistry. RESULTS: Gene expression analysis after 28 days of in vitro culture revealed the expression of early and late chondrogenic markers and a significant upregulation of NOGGIN as compared to human articular chondrocytes (hACs). Histological examination revealed a bilayered structure comprising of chondrocytes at different stages of maturity. Ectopically, TIs generated both bone and mineralized cartilage at 8 weeks after implantation. Osteochondral defects treated with TIs displayed glycosaminoglycan (GAG) production, type-II collagen, and lubricin expression. Immunostaining for human nuclei protein suggested that hPDCs contributed to both subchondral bone and articular cartilage repair. CONCLUSION: Our data indicate that in vitro derived osteochondral-like tissues can be generated from hPDCs, which are capable of producing bone and cartilage ectopically and behave orthotopically as osteochondral units.
Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Proteína Morfogenética Óssea 6/farmacologia , Cartilagem/metabolismo , Diferenciação Celular/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Periósteo/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/metabolismo , Engenharia Tecidual , Fator de Crescimento Transformador beta1/farmacologia , Animais , Antígenos de Diferenciação/biossíntese , Cartilagem/química , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Periósteo/citologia , Transplante de Células-Tronco , Células-Tronco/citologiaRESUMO
Footpad dermatitis and hockburn are serious welfare and economic issues for the production of broiler (meat) chickens. The authors here describe the use of an inexpensive camera system that monitors the movements of broiler flocks throughout their lives and suggest that it is possible to predict, even in young birds, the cross-sectional prevalence at slaughter of footpad dermatitis and hockburn before external signs are visible. The skew and kurtosis calculated from the authors' camera-based optical flow system had considerably more power to predict these outcomes in the 50 flocks reported here than water consumption, bodyweight or mortality and therefore have the potential to inform improved flock management through giving farmers early warning of welfare issues. Further trials are underway to establish the generality of the results.
Assuntos
Dermatite/veterinária , Doenças do Pé/veterinária , Doenças das Aves Domésticas/diagnóstico , Tarso Animal/patologia , Bem-Estar do Animal , Animais , Peso Corporal , Galinhas , Estudos Transversais , Dermatite/diagnóstico , Ingestão de Líquidos , Doenças do Pé/diagnóstico , Fenômenos Ópticos , Valor Preditivo dos TestesRESUMO
ABSTRACT Twenty-five Xanthomonas isolates, including some isolates received as either X. campestris pv. armoraciae or pv. raphani, caused discrete leaf spot symptoms when spray-inoculated onto at least one Brassica oleracea cultivar. Twelve of these isolates and four other Xanthomonas isolates were spray- and pin-inoculated onto 21 different plant species/cultivars including horseradish (Armoracia rusticana), radish (Raphanus sativus), and tomato (Lycopersicon esculentum). The remaining 13 leaf spot isolates were spray-inoculated onto a subset of 10 plant species/cultivars. The leaf spot isolates were very aggressive on several Brassica spp., radish, and tomato causing leaf spots and dark sunken lesions on the middle vein, petiole, and stem. Based on the differential reactions of several Brassica spp. and radish cultivars, the leaf spot isolates were divided into three races, with races 1 and 3 predominating. A differential series was established to determine the race-type of isolates and a gene-for-gene model based on the interaction of two avirulence genes in the pathogen races and two matching resistance genes in the differential hosts is proposed. Repetitive-DNA polymerase chain reaction-based fingerprinting was used to assess the genetic diversity of the leaf spot isolates and isolates of closely related Xanthomonas pathovars. Although there was variability within each race, the leaf spot isolates were clustered separately from the X. campestris pv. campestris isolates. We propose that X. campestris isolates that cause a nonvascular leaf spot disease on Brassica spp. should be identified as pv. raphani and not pv. armoraciae. Race-type strains and a neopathotype strain for X. campestris pv. raphani are proposed.
RESUMO
A retrospective analysis was performed on 93 patients who developed recurrent endometrial carcinoma in the pelvis, vaginal vault, and lower 1/3 vagina. There were 12 lower 1/3 vaginal recurrences, 24 vault recurrences and 57 pelvic recurrences from the 1005 patients treated between 1960 and 1976. Median time to recurrence was 30 months. Twenty-six patients had distant metastases also present at the time of recurrence in the sites mentioned above. Thirty-three percent of lower 1/3 vaginal recurrences, 12.5% of vault recurrences, and 5.3% of pelvic recurrences were salvaged with further treatment. The 10-year actuarial survival rates of isolated lower 1/3 vaginal, vaginal vault, and pelvic recurrences were 50%, 45%, and 24% respectively.
Assuntos
Neoplasias Pélvicas/secundário , Neoplasias Uterinas/cirurgia , Neoplasias Vaginais/secundário , Adenocarcinoma/radioterapia , Adenocarcinoma/secundário , Carcinoma de Células Escamosas/radioterapia , Carcinoma de Células Escamosas/secundário , Terapia Combinada , Feminino , Humanos , Neoplasias Pélvicas/radioterapia , Prognóstico , Neoplasias Uterinas/radioterapia , Neoplasias Vaginais/radioterapiaRESUMO
Data were obtained retrospectively on 1005 patients with histologically proven endometrial carcinoma from January 1960 to December 1976 inclusive. The 5- and 10-year actuarial survivals for all stages were 83 and 80% respectively. Recurrent disease developed in 14% of patients. The site of first recurrence was vaginal vault in 2.5%, lower vagina in 1.1%, pelvis in 5.7%, and lungs in 1.9%. Thirty-eight patients (27%) had multiple sites of metastatic disease at the time of first relapse. The only significant independent prognostic variables for clinical Stage I adenocarcinoma treated with surgery and radiation were no myometrial penetration and poorly differentiated tumors.
Assuntos
Adenocarcinoma/terapia , Neoplasias Uterinas/terapia , Adenocarcinoma/patologia , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Diferenciação Celular , Colo do Útero/patologia , Terapia Combinada , Feminino , Humanos , Histerectomia , Pessoa de Meia-Idade , Miométrio/patologia , Metástase Neoplásica , Recidiva Local de Neoplasia , Paridade , Prognóstico , Estudos RetrospectivosRESUMO
In a retrospective analysis of 1,390 consecutive patients with carcinoma of the cervix treated by high dose radiation therapy alone at the Queensland Radium Institute, we report a quantitative relationship between the early and late gastrointestinal complications arising from such treatment. Of these 1,390 patients, 157 (11.3%) experienced early, serious complications. For geographic reasons, it was only possible to evaluate 784 patients for late post-irradiation complications. Twenty-eight (3.6%) developed one or more late bowel complications, which included adhesions, fistulae, strictures, perforation, colitis and vascular occlusion. Factors affecting the relative risk of developing either an early or late complication were analyzed and are discussed. There was an 8.2% incidence of late complications developing in those patients who had experienced early complications, compared with a 3.0% incidence of late complications developing in patients without early complications. Thus, the risk of developing a late complication was greater by a factor of 2.7 in those patients developing an early one (p less than 0.05). However, of the 28 patients developing late complications, 21 (75%) did not experience a severe acute one.
Assuntos
Carcinoma/radioterapia , Gastroenteropatias/etiologia , Lesões por Radiação/etiologia , Neoplasias do Colo do Útero/radioterapia , Adulto , Idoso , Austrália , Braquiterapia/efeitos adversos , Carcinoma/complicações , Carcinoma/mortalidade , Feminino , Gastroenteropatias/mortalidade , Humanos , Pessoa de Meia-Idade , Lesões por Radiação/mortalidade , Radioterapia de Alta Energia/efeitos adversos , Estudos Retrospectivos , Fatores de Tempo , Neoplasias do Colo do Útero/complicações , Neoplasias do Colo do Útero/mortalidadeRESUMO
1. The characteristics of a propranolol-resistant (-)-[125I]-cyanopindolol (CYP) binding site in rat soleus muscle were determined. 2. Saturation studies performed on homogenates of rat soleus muscle showed two phases of (-)-[125I]-CYP binding, a high affinity site (KD1 30.5 +/- 16.3 pM, Bmax 9.4 +/- 1.38 fmol mg-1 protein) and a lower affinity site (KD2 522.5 +/- 29.1 pM, Bmax 62.19 +/- 11.76 fmol mg-1 protein, n = 4). 3. In rat soleus muscle homogenates labelled with (-)-[125I]-CYP (500 pM), (-)-propranolol competition curves were biphasic with pKD values of 8.30 +/- 0.19, and 5.33 +/- 0.08, n = 7. 4. Competition between (-)-[125I]-CYP (500 pM) and (+/-)-tertatolol, (+/-)-nadolol, (+/-)-alprenolol, (+/-)-CYP, and (-) and (+)-pindolol showed that these compounds competed for binding at the propranolol-resistant site with affinities lower than those displayed at typical beta-adrenoceptors. The atypical beta-adrenoceptor agonists BRL 37344, SR58611A and ICI D7114 and the partial agonist (+/-)-CGP 12177 also competed for (-)-[125I]-CYP binding. 5. Stereoselectivity was demonstrated for the stereoisomers of alprenolol and tertalolol. The (-)-isomers of alprenolol and tertalolol had higher affinity than their corresponding (+)-isomers (3.1 and 2.6 fold respectively). These low stereoselectivity values are a characteristic of atypical beta-adrenoceptors. 6. The beta-adrenoceptor agonists, (-)-adrenaline, (-)-isoprenaline and (-)-noradrenaline, all showed lower affinity than the atypical beta-adrenoceptor agonists and competition curves appeared biphasic in nature. 7. These results confirm the presence of a propranolol-resistant (- )-[125I]-CYP binding site in rat soleus muscle. The affinities of the tested compounds at the propranolol-resistant (- )-[125I]-CYP binding site show similarities to their affinities at 'atypical' beta-adrenoceptors in adipocytes and gastrointestinal tissues and at the cloned beta 3-adrenoceptor.
Assuntos
Pindolol/análogos & derivados , Propranolol/farmacologia , Receptores Adrenérgicos beta/metabolismo , Agonistas Adrenérgicos beta/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/metabolismo , Antagonistas Adrenérgicos beta/farmacologia , Animais , Ligação Competitiva/efeitos dos fármacos , Feminino , Técnicas In Vitro , Radioisótopos do Iodo , Iodocianopindolol , Masculino , Pindolol/metabolismo , Propranolol/farmacocinética , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos beta/efeitos dos fármacosRESUMO
1. Relaxation of carbachol pre-contracted human colonic muscle to (-)-isoprenaline was examined in circular, longitudinal and taenia coli preparations to determine the beta-adrenoceptor subtypes involved. beta 1-, beta 2- and beta 3-Adrenoceptor mRNAs were also measured in colonic muscle and mucosa. 2. (-)-isoprenaline caused relaxation of longitudinal smooth muscle preparations with pEC50-7.39 +/- 0.12, and this response was inhibited by both propranolol (0.1 microM, pKB 8.55 +/- 0.12) and the selective beta 1-antagonist, CGP 20712A (0.1 microM, pKB 8.80 +/- 0.20), while the selective beta 2-antagonist, ICI 118551 (0.1 microM) failed to inhibit isoprenaline relaxation consistently. 3. (-)-Isoprenaline caused relaxation of taenia coli with a pEC50 of 6.70 +/- 0.17. Propranolol (0.1 microM). CGP 20712A (0.1 microM) and ICI 118551 (0.1 microM) inhibited the isoprenaline response with similar low affinities (pKB values 7.93, 7.71 and 7.54, respectively). Carbachol pre-contracted circular smooth muscle preparations failed to relax consistently to isoprenaline and these responses were not characterized. 4. beta 1- and beta 2-Adrenoceptor mRNAs were present in circular longitudinal muscle samples and taenia coli samples, and lower levels were detected in mucosa. beta 3-mRNA was also present in both muscle preparations but was not detected in human colonic mucosa. 5. In summary, beta 1-adrenoceptors are the predominant subtype mediating isoprenaline-induced relaxation of the thin longitudinal smooth muscle of human colon, while beta 1-receptors do not appear to be involved in these responses. However, beta 3-adrenoceptors may play a role in relaxation of the taenia coli as conventional antagonist affinities are low. beta 3-Adrenoceptor mRNA was present in taenia coli and circular/longitudinal smooth muscle but absent from human colonic mucosa.
Assuntos
Colo/fisiologia , Receptores Adrenérgicos beta/fisiologia , Agonistas Adrenérgicos beta/farmacologia , Carbacol/farmacologia , Colo/efeitos dos fármacos , Humanos , Técnicas In Vitro , Isoproterenol/farmacologia , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , RNA Mensageiro/genética , Receptores Adrenérgicos beta/classificação , Receptores Adrenérgicos beta/genéticaRESUMO
1. Homogenate binding studies and receptor autoradiography have been used to examine the binding characteristics and localization of propranolol-resistant (-)-[125I]-cyanopindolol (CYP) binding sites in rat ileum. 2. Saturation studies with (-)-[125I]-CYP and homogenates of rat ileum identified a site with pKD 8.89 +/- 0.08 and Bmax = 50.3 +/- 4.1 fmol mg-1 protein (n = 6). Both beta 1- and beta 2-adrenoceptors (AR) were not detected in these preparations. 3. (-)-Isoprenaline infusion (400 micrograms kg-1 h-1) for 14 days caused no significant change in the density of (-)-[125I]-CYP binding which was 48.9 +/- 12.8 and 40.6 +/- 12.3 fmol mg-1 protein in control and isoprenaline-treated animals respectively (n = 6) (P = 0.97). 4. Competition for (-)-[125I]-CYP binding in the presence of 0.1 microM (-)-propranolol gave affinity values for CYP, tertatolol, alprenolol, ICI 118551 and CGP 20712A that correspond to known affinities at atypical beta-ARs. Stereoselectivity ratios for tertatolol and alprenolol were low. 5. Autoradiographic localization of propranolol resistant (-)-[125I]-CYP binding showed sites associated with the mucosa and to a lesser extent to the muscularis. A small population of beta 2-ARs were detected located predominantly in the longitudinal and circular smooth muscle layers. 6. This study identifies an (-)-[125I]-CYP binding site in rat ileum that is resistant to blockade by propranolol (0.1 microM), is located predominantly in the mucosa, shows resistance to downregulation by isoprenaline and has binding characteristics of the atypical beta-AR.
Assuntos
Íleo/ultraestrutura , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Agonistas Adrenérgicos beta/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/metabolismo , Antagonistas Adrenérgicos beta/farmacologia , Animais , Autorradiografia , Sítios de Ligação , Ligação Competitiva , Feminino , Íleo/metabolismo , Radioisótopos do Iodo , Isoproterenol/metabolismo , Cinética , Masculino , Pindolol/análogos & derivados , Pindolol/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
1. Functional and molecular approaches were used to characterize the beta-AR subtypes mediating relaxation of rat ileal smooth muscle. 2. In functional studies, (-)-isoprenaline relaxation was unchanged by CGP20712A (beta1-AR antagonist) or ICI118551 (beta2-AR antagonist) but shifted by propranolol (pKB=6.69). (+/-)-Cyanopindolol, CGP12177 and ICID7114 did not cause relaxation but antagonized (-)-isoprenaline relaxation. 3. BRL37344 (beta3-AR agonist) caused biphasic relaxation. The high affinity component was shifted with low affinity by propranolol, (+/-)-cyanopindolol, tertatolol and alprenolol. CL316243 (beta3-AR agonist) relaxation was unaffected by CGP20712A or ICI118551 but blocked by SR58894A (beta3-AR antagonist; pA2 = 7.80). Enhanced relaxation after exposure to forskolin and pertussis toxin showed that beta3-AR relaxation can be altered by manipulation of components of the adenylate cyclase signalling pathway. 4. The beta-AR agonist RO363 relaxed the ileum (pEC50=6.18) and was blocked by CGP20712A. Relaxation by the beta2-AR agonist zinterol (pEC50=5.71) was blocked by SR58894A but not by ICI118551. 5. In rat ileum, beta1-, beta2- and beta3-AR mRNA was detected. Comparison of tissues showed that beta3-AR mRNA expression was greatest in WAT>colon=ileum >cerebral cortex>soleus; beta1-AR mRNA was most abundant in cerebral cortex > WAT > ileum = colon > soleus; beta2-AR mRNA was expressed in soleus > WAT > ileum = colon > cerebral cortex. 6. These results show that beta3-ARs are the predominant beta-AR subtype mediating rat ileal relaxation while beta1-ARs may produce a small relaxation. The beta2-AR agonist zinterol produces relaxation through beta3-ARs and there was no evidence for the involvement of beta2-ARs in relaxation despite the detection of beta2-AR mRNA.
Assuntos
Íleo/fisiologia , Relaxamento Muscular/efeitos dos fármacos , RNA Mensageiro/análise , Receptores Adrenérgicos beta 1/fisiologia , Receptores Adrenérgicos beta 2/fisiologia , Receptores Adrenérgicos beta/fisiologia , Toxina Adenilato Ciclase , Animais , Colforsina/farmacologia , Dioxóis/farmacologia , Relação Dose-Resposta a Droga , Etanolaminas/farmacologia , Isoproterenol/farmacologia , Masculino , Toxina Pertussis , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 3 , Fatores de Virulência de Bordetella/farmacologiaRESUMO
BACKGROUND: This study describes the pharmaco-epidemiology of ulcer-healing therapies in primary care in the north of England. METHODS: Anonymous patient-specific prescribing data were extracted from computerized general practice records for 41 practices. Prescribing prevalences were determined according to patient age, gender and recorded prescription indication, for both antacids and H2-receptor antagonists or omeprazole. RESULTS: During the year of the study, antacids were prescribed for 3.9% of the study population, and H2-receptor antagonists or omeprazole for 3.7%. Rates increased with age, peaking at 99 (antacids) and 87 (H2-receptor antagonists) per 1000 population aged 65-84 years. Antacid prescribing rates for women were over twice those for men amongst those aged 15-34 years. For H2-receptor antagonists, rates were higher in men than women, with the excess attributable to prescribing for ulcer indications, although at all ages prescribing for oesophagitis was more prevalent among women. Of the patients prescribed H2-receptor antagonists or omeprazole, 45% (accounting for 51% of prescription items) had indications which included peptic ulcer or oesophagitis, 42% had gastritis or dyspepsia only (35% items), and in 13% no peptic indication was recorded. For each of antacids and H2-receptor antagonists, the practices had similar prescribing profiles according to patient age and gender, but their absolute levels differed up to two-fold. CONCLUSIONS: Prescribing of antacids and ulcer-healing drugs varies systematically with patient age and gender. Consequently, evaluation of crude prescribing rates, without reference to patient demography, is unreliable as a guide to levels of usage. In general practice, H2-receptor antagonists and omeprazole appear to be over-prescribed for minor indications.
Assuntos
Antiácidos/administração & dosagem , Atenção Primária à Saúde , Agonistas do Receptor de Serotonina/administração & dosagem , Úlcera Gástrica/tratamento farmacológico , Adolescente , Adulto , Distribuição por Idade , Idoso , Criança , Pré-Escolar , Prescrições de Medicamentos/estatística & dados numéricos , Uso de Medicamentos , Dispepsia/tratamento farmacológico , Inglaterra , Feminino , Gastrite/tratamento farmacológico , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Omeprazol/administração & dosagem , Receptores Histamínicos H2/efeitos dos fármacos , Fatores SexuaisRESUMO
Renal replacement therapy relies predominantly on the use of cellulose-based membranes. Such membranes have a biocompatibility profile which is inferior to membranes manufactured from synthetic polymers. Synthetically modified cellulose (SMC) is a new, low-flux haemodialysis membrane in which hydroxyl groups have been replaced with benzyl groups. The biocompatibility profile characterized by changes in white cell and platelet counts and the activation of complement components (C3a, C5a and C5b-9) have been studied in vivo and compared with those of cellulose acetate, unmodified cellulose (Cuprophan ) and low-flux polysulphone (Fresenius Polysulfone) in the same group of patients. For SMC, the white cell count at 15 min declined to 65.6% of pretreatment level, compared with 63.8% for the cellulose acetate, 79.6% for low-flux polysulphone and 28.1% for Cuprophan, thereafter returning to pretreatment levels. Both modified cellulose membranes were superior to unmodified cellulose (P = 0.001); the differences between the modified cellulose membranes were not significant statistically. The changes induced by all three cellulose-based membranes exceeded those for low-flux polysulphone (P = 0.001). Associated with the neutropenia was a reduction in platelet count, but this was independent of membrane type. The mean time-averaged concentrations of C3a(des Arg) over 150 min were 1168 ng ml(-1) (SMC), 1030 ng ml(-1) (cellulose acetate), 1297 ng ml(-1) (Cuprophan) and 790 ng ml(-1) (low-flux polysulphone). Equivalent values for C5a(des Arg) were 6.12 (SMC), 2.98 (cellulose acetate), 11.03 (Cuprophan) and 1.33 ng ml(-1) (low-flux polysulphone). C5b-9 values were 385 (SMC), 386 (cellulose acetate), 177 (Cuprophan) and 185 ng ml(-1) (low-flux polysulphone). For each of the complement components the differences between the membranes were significant [P = 0.0009 (C3a(des Arg)), P = 0.0001 (c5a(des Arg) and C5b-9)]. The levels of C5b-9 generated during dialysis also showed a significant positive correlation compared to C5a for all membranes considered as a single group (Pearson's correlation coefficient = 0.870, P = 0.0001). It is concluded that the modification of the cellobiosic unit is a promising approach to improve the biocompatibility profile of cellulose-based membranes. The two different methods of modification lead to similar improvements in biocompatibility compared with unmodified cellulose, but as yet do not match that of low-flux polysulphone.
Assuntos
Materiais Biocompatíveis/química , Celulose/química , Membranas Artificiais , Diálise Renal/instrumentação , Celulose/análogos & derivados , Ativação do Complemento , Humanos , Contagem de Leucócitos , Neutropenia/etiologia , Contagem de Plaquetas , Polímeros/química , Estudos Prospectivos , Diálise Renal/efeitos adversos , Sulfonas/químicaRESUMO
During obstructive sleep apnea, transient arousal at the resumption of breathing is coincident with a substantial rise in blood pressure. To assess the hemodynamic effect of arousal alone, 149 transient stimuli were administered to five normal subjects. Two electroencephalograms (EEG), an electrooculogram, a submental electromyogram (EMG), and beat-to-beat blood pressure (Finapres, Ohmeda) were recorded in all subjects. Stimulus length was varied to produce a range of cortical EEG arousals that were graded as follows: 0, no increase in high-frequency EEG or EMG; 1, increased high-frequency EEG and/or EMG for < 10 s; 2, increased high-frequency EEG and/or EMG for > 10 s. Overall, compared with control values, average systolic pressure rose [nonrapid-eye-movement (NREM) sleep 10.0 +/- 7.69 (SD) mmHg; rapid-eye-movement (REM) sleep 6.0 +/- 6.73 mmHg] and average diastolic pressure rose (NREM sleep 6.1 +/- 4.43 mmHg; REM sleep 3.7 +/- 3.02 mmHg) over the 10 s following the stimulus (NREM sleep, P < 0.0001; REM sleep, P < 0.002). During NREM sleep, there was a trend toward larger blood pressure rises at larger grades of arousal (systolic: r = 0.22, 95% confidence interval 0.02-0.40; diastolic: r = 0.48, 95% confidence interval 0.31-0.62). The average blood pressure rise in response to the grade 2 arousals was approximately 75% of that during obstructive sleep apnea. Arousal stimuli that did not cause EEG arousal still produced a blood pressure rise (mean systolic rise 8.6 +/- 7.0 mmHg, P < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Nível de Alerta/fisiologia , Pressão Sanguínea/fisiologia , Sono/fisiologia , Adolescente , Adulto , Eletroencefalografia , Eletroculografia , Feminino , Frequência Cardíaca/fisiologia , Humanos , Masculino , Síndromes da Apneia do Sono/fisiopatologia , Sono REM/fisiologia , Vigília/fisiologiaRESUMO
This review outlines the theory of spectral estimation techniques based on the fast Fourier transform (FFT) and autoregressive (AR) model and their application to the analysis of human tremor data. Two FFT-based spectral estimation techniques are presented, the Blackman-Tukey and periodogram methods. Factors that influence the quality of spectral estimates are discussed including the choice of windowing function. The theory of parametric modelling is introduced and AR modelling identified as the technique best suited to the analysis of tremor data. The processes of parameter estimation and model order selection are described. The theory of AR spectral estimation is outlined and differences between the AR and FFT-based spectral estimates are summarised. A brief guide to the implementation of FFT-based and AR spectral estimation techniques is given concentrating on data analysis packages that require little or no programming expertise. This review concludes that the AR modelling approach can produce tremor spectra that are superior to those from FFT-based methods for short data sequences. Although the spectral estimates are improved, the benefits of AR modelling for providing information about the physiological mechanisms of tremor generation are not yet clear.
Assuntos
Tremor/fisiopatologia , Algoritmos , Biologia Computacional/métodos , Humanos , Modelos Neurológicos , Análise de Regressão , Tremor/diagnósticoRESUMO
The beta-adrenoceptor subtypes involved in cyclic AMP accumulation in rat soleus muscle were studied using beta1- beta2- and beta3-adrenoceptor agonists and antagonists. Responses to (-)-isoprenaline were antagonised by (-)-propranolol (p KB = 8.32 at 0.1 microM) and by erythro-DL-1(7-methylindian-4-yloxy)-3-isopropylaminobuta n-2-ol (+/-)-ICI 118551) (pKB = 9.38 at 10 nM and 9.65 at 100 nM) but not by 2-hydroxy-5(2-((2-hydroxy-3-(4-((1-methyl-4-trifluoromethyl)1H-imidazole -2-yl)-phenoxy)propyl)amino)ethoxy)-benzamide monomethane sulfonate ((+/-)-CGP 20712A at 10 nM or 100 nM). The beta3-adrenoceptor agonist sodium-4-[-2[-2-hydroxy-2-(-3-chlorophenyl)ethylamino]propyl]phenoxya cetate (BRL 37344 at 10 pM or 10 microM) caused no significant change in basal cyclic AMP levels and had no effect on the level of cyclic AMP accumulation stimulated by (-)-isoprenaline, zinterol or forskolin. (-)-Isoprenaline pretreatment (400 microg kg(-1) h(-1), 14 days) abolished responses to (-)-isoprenaline (10 microM) and zinterol (1 microM) while BRL 37344 had no effect in either isoprenaline or vehicle-treated groups. These results show that beta3-adrenoceptor agonists do not stimulate cyclic AMP accumulation in rat soleus muscle and that (-)-isoprenaline induced increases in cyclic AMP levels are mediated predominantly by beta2-adrenoceptors. This suggests that the previously reported increase in glucose uptake by beta3-adrenoceptor agonists in skeletal muscle does not involve direct stimulation of adenylate cyclase.