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2.
Mar Pollut Bull ; 178: 113500, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35427814

RESUMO

Marine debris, directly and indirectly, threatens marine habitat and biota. Fishing activity is generally recognised as a contributor to marine debris, but the relative input from recreational fishing remains unassessed. Here we provide the first comprehensive literature review of recreational fishing marine debris (RFMD) on a global scale. A systematic literature review identified 70 studies related to RFMD, and plastic and metal respectively were the dominant debris materials found. Nearshore coastal areas and reefs, acted as both sources and sinks of RFMD and a diverse suite of potential impacts such as ghost fishing and entanglement were identified at local scales. Overall, research of RFMD is lacking globally, however, its role in marine debris input is likely underestimated. We recommend more research on the volumes and risks, using a standardised classification approach. Where intervention is required, we suggest cooperative approaches between the sector and authorities.


Assuntos
Caça , Resíduos , Ecossistema , Monitoramento Ambiental , Plásticos , Resíduos/análise
3.
Clin Anat ; 24(6): 741-7, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21400610

RESUMO

The human costotransverse joint (CTJ) is the articulation between the posterior tubercle of the ribs with the first through tenth costal facet of the thoracic transverse processes. While the CTJ is well defined anatomically and considered a synovial joint, the human CTJ as a pain generating structure is controversial and not supported from a histological perspective. The objective of the present study was to investigate the histological pain producing properties of CTJ capsule tissue. Ten micron cross-sections at each level (1-10) were stained with H & E or immunostained with antisera against Substance P (SP), calcitonin-gene-related peptide (CGRP), and neuropeptide Y (NPY). Immunoreactivity was confirmed for SP, CGRP, and NPY within the CTJ tissue samples of two unembalmed male cadavers. The presence of previously mentioned neuropeptides suggests that human CTJ is capable of producing pain through somatic and autonomic nervous systems. Therefore, clinicians should consider the CTJ as a differential diagnostic possibility when examining and treating painful thoracic conditions.


Assuntos
Cápsula Articular/anatomia & histologia , Costelas/anatomia & histologia , Vértebras Torácicas/anatomia & histologia , Humanos , Imuno-Histoquímica , Masculino
4.
Acta Paediatr ; 99(7): 1104-7, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20151952

RESUMO

UNLABELLED: A case of extensive deep venous thrombosis in a four a day old infant was presented. Unusually this patient was shown to be heterozygous for three thrombophilia genes; Factor V Leiden, prothrombin and antithrombin gene mutations, the latter being novel. CONCLUSION: There are no randomized controlled trials to guide management in deep venous thrombosis in the newborn but knowledge of the prothrombotic risk factors may help direct treatment.


Assuntos
Protrombina/genética , Veias Renais/diagnóstico por imagem , Trombose Venosa/diagnóstico , Humanos , Recém-Nascido , Mutação , Fatores de Risco , Ultrassonografia , Trombose Venosa/genética
5.
Science ; 256(5061): 1308-11, 1992 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-1598573

RESUMO

From histological investigations into the microstructure of conodont elements, a number of tissue types characteristic of the phosphatic skeleton of vertebrates have been identified. These include cellular bone, two forms of hypermineralized enamel homologs, and globular calcified cartilage. The presence of cellular bone in conodont elements provides unequivocal evidence for their vertebrate affinities. Furthermore, the identification of vertebrate hard tissues in the oral elements of conodonts extends the earliest occurrence of vertebrate hard tissues back by around 40 million years, from the Middle Ordovician (475 million years ago) to the Late Cambrian (515 million years ago).


Assuntos
Osso e Ossos/ultraestrutura , Cartilagem/ultraestrutura , Fósseis , Vertebrados/anatomia & histologia , Animais , Microscopia Eletrônica de Varredura , Paleontologia , Filogenia
6.
Br J Pharmacol ; 154(4): 882-9, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18414379

RESUMO

BACKGROUND AND PURPOSE: Emerging evidence suggests that activation of G-protein-coupled receptors (GPCRs) can be directly regulated by membrane voltage. However, the physiological and pharmacological relevance of this effect remains unclear. We have further examined this phenomenon for P2Y1 receptors in the non-excitable megakaryocyte using a range of agonists and antagonists. EXPERIMENTAL APPROACH: Simultaneous whole-cell patch clamp and fura-2 fluorescence recordings of rat megakaryocytes, which lack voltage-gated Ca2+ influx, were used to examine the voltage-dependence of P2Y1 receptor-evoked IP3-dependent Ca2+ mobilization. RESULTS: Depolarization transiently and repeatedly enhanced P2Y1 receptor-evoked Ca2+ mobilization across a wide concentration range of both weak, partial and full, potent agonists. Moreover, the amplitude of the depolarization-evoked [Ca2+]i increase displayed an inverse relationship with agonist concentration, such that the greatest potentiating effect of voltage was observed at near-threshold levels of agonist. Unexpectedly, depolarization also stimulated an [Ca2+]i increase in the absence of agonist during exposure to the competitive antagonists A3P5PS and MRS2179, or the allosteric enhancer 2,2'-pyridylisatogen tosylate. A further effect of some antagonists, particularly suramin, was to enhance the depolarization-evoked Ca2+ responses during co-application of an agonist. Of several P2Y1 receptor inhibitors, only SCH202676, which has a proposed allosteric mechanism of action, could block ADP-induced voltage-dependent Ca2+ release. CONCLUSIONS AND IMPLICATIONS: The ability of depolarization to potentiate GPCRs at near-threshold agonist concentrations represents a novel mechanism for coincidence detection. Furthermore, the induction and enhancement of voltage-dependent GPCR responses by antagonists has implications for the design of therapeutic compounds.


Assuntos
Canais de Cálcio/metabolismo , Megacariócitos/metabolismo , Receptores Purinérgicos P2/metabolismo , Difosfato de Adenosina/metabolismo , Regulação Alostérica , Animais , Canais de Cálcio/efeitos dos fármacos , Relação Dose-Resposta a Droga , Corantes Fluorescentes , Fura-2 , Masculino , Técnicas de Patch-Clamp , Agonistas do Receptor Purinérgico P2 , Antagonistas do Receptor Purinérgico P2 , Ratos , Ratos Wistar , Receptores Purinérgicos P2Y1 , Transdução de Sinais
7.
Herit Sci ; 6(1): 58, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-31258911

RESUMO

This paper demonstrates the combined use of X-ray computed tomography (XCT), energy dispersive X-ray spectroscopy (EDX) and X-ray fluorescence (XRF) to evaluate the conservational history of the dentary (lower jaw) of Megalosaurus bucklandii Mantell, 1827, the first scientifically described dinosaur. Previous analysis using XCT revealed that the specimen had undergone at least two phases of repair using two different kinds of plaster, although their composition remained undetermined. Additional chemical analysis using EDX and XRF has allowed the determination of the composition of these unidentified plasters, revealing that they are of similar composition, composed dominantly of 'plaster of Paris' mixed with quartz sand and calcite, potentially from the matrix material of the Stonesfield Slate, with the trace presence of chlorine. One of the plasters unusually contains the pigment minium (naturally occurring lead tetroxide; Pb2 2+Pb4+O4) whilst the other seems to have an additional coating of barium hydroxide (Ba(OH)2), indicating that these likely represent two separate stages of repair. The potential of this combined approach for evaluating problematic museum objects for conservation is further discussed as is its usage in cultural heritage today.

8.
J Thromb Haemost ; 5(5): 910-7, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17362227

RESUMO

In the platelet, it is well established that many G-protein- and tyrosine kinase-coupled receptors stimulate phospholipase-C-dependent Ca(2+) mobilization; however, the extent to which secondary activation of adenosine 5'-triphosphate (ATP)-gated P2X(1) receptors contributes to intracellular Ca(2+) responses remains unclear. We now show that selective inhibition of P2X(1) receptors substantially reduces the [Ca(2+)](i) increase evoked by several important agonists in human platelets; for collagen, thromboxane A(2), thrombin, and adenosine 5'-diphoshate (ADP) the maximal effect was a reduction to 18%, 34%, 52%, and 69% of control, respectively. The direct contribution of P2X(1) to the secondary Ca(2+) response was far greater than that of either P2Y receptors activated by co-released ADP, or via synergistic P2X(1):P2Y interactions. The relative contribution of P2X(1) to the peak Ca(2+) increase varied with the strength of the initial stimulus, being greater at low compared to high levels of stimulation for both glycoprotein VI and PAR-1, whereas P2X(1) contributed equally at both low and high levels of stimulation of thromboxane A(2) receptors. In contrast, only strong stimulation of P2Y receptors resulted in significant P2X(1) receptor activation. ATP release was detected by soluble luciferin:luciferase in response to all agonists that stimulated secondary P2X(1) receptor activation. However, P2X(1) receptors were stimulated earlier and to a greater extent than predicted from the average ATP release, which can be accounted for by a predominantly autocrine mechanism of activation. Given the central role of [Ca(2+)](i) increases in platelet activation, these studies indicate that ATP should be considered alongside ADP and thromboxane A(2) as a significant secondary platelet agonist.


Assuntos
Plaquetas/efeitos dos fármacos , Cálcio/metabolismo , Agonistas do Receptor Purinérgico P2 , Difosfato de Adenosina/farmacologia , Benzenossulfonatos/farmacologia , Plaquetas/citologia , Plaquetas/metabolismo , Humanos , Luminescência , Receptores Purinérgicos P2X , Espectrometria de Fluorescência , Tromboxano A2/farmacologia
9.
Neuroscience ; 144(3): 1057-66, 2007 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-17110046

RESUMO

Although the etiology of Parkinson's disease (PD) is unknown, a common element of most theories is the involvement of oxidative stress, either as a cause or effect of the disease. There have been relatively few studies that have characterized oxidative stress in animal models of PD. In the present study a 6-hydroxydopamine (6-OHDA) rodent model of PD was used to investigate the in vivo production of oxidative stress after administration of the neurotoxin. 6-OHDA was injected into the striatum of young adult rats and the production of protein carbonyls and 4-hydroxynonenal (HNE) was measured at 1, 3, 7, and 14 days after administration. A significant increase in both markers was found in the striatum 1 day after neurotoxin administration, and this increase declined to basal levels by day 7. There was no significant increase found in the substantia nigra at any of the time points investigated. This same lesion paradigm produced dopamine depletions of 90-95% in the striatum and 63-80% in the substantia nigra by 14-28 days post-6-OHDA. Protein carbonyl and HNE levels were also measured in middle-aged and aged animals 1 day after striatal 6-OHDA. Both protein carbonyl and HNE levels were increased in the striatum of middle-aged and aged animals treated with 6-OHDA, but the increases were not as great as those observed in the young adult animals. Similar to the young animals, there were no increases in either marker in the substantia nigra of the middle-aged and aged animals. There was a trend for an age-dependent increase in basal amounts of oxidative stress markers when comparing the non-lesioned side of the brains of the three age groups. These results support that an early event in the course of dopamine depletion following intrastriatal 6-OHDA administration is the generation of oxidative stress.


Assuntos
Corpo Estriado/metabolismo , Dopamina/deficiência , Estresse Oxidativo/fisiologia , Transtornos Parkinsonianos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores Etários , Envelhecimento/fisiologia , Aldeídos/metabolismo , Animais , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/fisiopatologia , Masculino , Oxidopamina , Transtornos Parkinsonianos/fisiopatologia , Ratos , Ratos Endogâmicos F344 , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismo , Substância Negra/fisiopatologia
10.
Mol Endocrinol ; 20(2): 379-88, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16179383

RESUMO

Oxytocin (OT) has long been used as an uterotonic during labor management in women, and yet responses to OT infusion remain variable and unpredictable among patients. The investigation of oxytocin receptor (OTR) regulation will benefit labor management, because the clinical practice of continuous iv infusion of OT is not optimal. As with other G protein-coupled receptors, it is likely that the OTR internalizes and/or desensitizes upon continuous agonist exposure. The mechanisms by which this might occur, however, are unclear. Here we explore OTR internalization and desensitization in human embryonic kidney cells by utilizing inhibitors of heterologous second messenger systems and recently available mutant cDNA constructs. We report rapid and extensive internalization and desensitization of the OTR upon agonist exposure. Internalization was unaffected by inhibitors of protein kinase C or Ca(2+) calmodulin-dependant kinase II but was significantly reduced after transfection with dominant-negative mutant cDNAs of G protein-coupled receptor kinase 2, beta-Arrestin2, Dynamin, and Eps15 (a component of clathrin-coated pits). Moreover, desensitization of the OTR, measured by a calcium mobilization assay, was also inhibited by the aforementioned cDNA constructs. Thus, our data demonstrate, for the first time, the importance of the classical clathrin-mediated pathway during agonist-induced OTR internalization and desensitization.


Assuntos
Clatrina/metabolismo , Dinaminas/metabolismo , Endocitose , Receptores de Ocitocina/agonistas , Receptores de Ocitocina/metabolismo , Arrestina/genética , Cálcio/metabolismo , Células Cultivadas , Clatrina/genética , Citoplasma/química , Dinaminas/genética , Quinase 2 de Receptor Acoplado a Proteína G , Hemaglutininas/análise , Humanos , Mutação , Ocitocina/farmacologia , Proteína Quinase C/antagonistas & inibidores , Receptores de Ocitocina/análise , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Transfecção , Quinases de Receptores Adrenérgicos beta/genética
11.
J Thromb Haemost ; 4(3): 587-90, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16460441

RESUMO

BACKGROUND: Hemophilia A is a severe bleeding disorder caused by almost 1000 different known mutations in the F8C gene. Direct mutation analysis is sometimes difficult for this disorder. When a mutation cannot be found, linkage analysis can be used for prenatal and carrier diagnosis. AIM: To develop a rapid and effective system for carrier detection and prenatal diagnosis of hemophilia A based on a single-multiplexed polymerase chain reaction (PCR) reaction utilizing five microsatellite markers. PATIENTS AND METHODS: Two intronic microsatellites and three other markers flanking the factor VIII gene were ascertained, and primers were designed for multiplex PCR amplification. A kindred with Hemophilia A was tested for linkage using the panel of primers, and informativity in the general population was ascertained by testing 50 unrelated females. RESULTS: Co-amplification of all microsatellites was optimized using DNA extracted by standard methods. Rapid detection and sizing of products were carried out using an automated DNA sequencer. The combined microsatellite panel was informative in each of the kindreds tested, and in 100% of the 50 unrelated females (95% CI 94.2-100%). CONCLUSIONS: This method enables the indirect detection of hemophilia A for patients in whom mutations cannot be found, facilitating carrier testing and prenatal analysis. It is rapid and straightforward compared with many other published protocols, and offers a high degree of informativity.


Assuntos
Testes Genéticos/métodos , Hemofilia A/diagnóstico , Repetições de Microssatélites/genética , Sequência de Bases , Fator VIII/genética , Feminino , Triagem de Portadores Genéticos/métodos , Ligação Genética , Marcadores Genéticos , Hemofilia A/genética , Humanos , Masculino , Dados de Sequência Molecular , Mutação , Linhagem , Reação em Cadeia da Polimerase , Gravidez , Diagnóstico Pré-Natal/métodos , Reprodutibilidade dos Testes
12.
Biochim Biophys Acta ; 1278(1): 131-6, 1996 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-8611601

RESUMO

Human platelets were studied by patch clamp recordings from inside-out membranes; there were formed by briefly dipping the platelet, in cell-attached mode, into silicone grease. At 20 degrees C in symmetrical 150 mM NaCl, spontaneous channel openings were rarely observed at negative potentials, whereas depolarised potentials (+ 60 to + 100 mV) elicited sustained channel activity in 38% of patches. The single channel conductance was 53 +A- 1 pS at + 80 mV (outward current), decreasing to 20 +/- 2 pS at -80 mV (inward current). Ion substitution experiments indicated that this channel conducts Cl- and not Na+. Furthermore, 5-nitro-2-(3-phenylpropylamino)benzoate (100 microM), a recognized inhibitor of anion channels, induced a reversible 'flickery' channel block. We estimate that each platelet possesses < or = 30 such channels. Kinetic analysis suggested at least two open channel states (tau = 0.8 +/- 0.2 ms, tau = 22 +/- 14 ms, n = 4) and two closed states (tau = 0.8 +/- 0.2 ms, tau = 12 +/- 0.6 ms, n = 4). Increasing [Ca2+]i to 10 microM, following channel activation by depolarisation, had no significant effect on channel kinetics or open probability, however, elevated [Ca2+]i (300 nM-10 microM) increased the number of anion channels activated by subsequent depolarisation. This study represents the first recordings of ionic currents in excised, inside-out membrane patches from human platelets, and provides further evidence for the existence of chloride channels in these cells.


Assuntos
Plaquetas/metabolismo , Cálcio/fisiologia , Canais de Cloreto/sangue , Cálcio/farmacologia , Membrana Celular/metabolismo , Canais de Cloreto/efeitos dos fármacos , Cloretos/sangue , Eletrofisiologia , Gluconatos/farmacologia , Humanos , Líquido Intracelular/metabolismo , Nitrobenzenos/farmacologia , Técnicas de Patch-Clamp , Sódio/sangue
13.
Curr Med Res Opin ; 31(11): 1985-91, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26296234

RESUMO

BACKGROUND: Health and life expectancy for people with hemophilia have improved significantly in recent years, but we face new challenges, especially in the context of resource-constrained health services. AIM: This paper aims to highlight such challenges and propose practical solutions. METHODS: Nine hemophilia specialists from Australia and New Zealand reached consensus on areas of greatest need for improvement in hemophilia care in these countries, based on clinical experience and published data, and agreed on how to address these. RESULTS: Demography, optimizing treatment and assessing treatment success were identified as broad areas of challenge which included: comorbidities in ageing patients; transitioning from pediatric to adult care; equity of care for remote populations; weight-based dosing in obese patients; tailoring prophylaxis; accurate diagnosis of acute joint pain; managing chronic arthropathy; providing psychosocial support; consistency in definitions and assessment; and quantifiable outcome measures. Practice points included increased cross-specialty coordination and including psychologists and rheumatologists as part of comprehensive care teams; close collaboration between pediatric and adult centers to facilitate transition of care; systems such as telehealth that ensure continuity of care for remote populations; using pharmacokinetic data to tailor therapy; rapid and accurate diagnosis of acute joint pain; using data from bleeding registries to assess treatment effects and help with service planning; and ensuring consistency through benchmarking and standardization of HTCs. SUMMARY: Achieving treatment equity, optimal outcomes and cost savings may be possible through investing in national governance structures, expanding the comprehensive model of care and implementing innovative solutions tailored to local needs.


Assuntos
Hemofilia A/terapia , Transição para Assistência do Adulto , Adulto , Austrália , Criança , Consenso , Humanos , Nova Zelândia , Pediatria
14.
AJNR Am J Neuroradiol ; 36(1): 14-23, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25300984

RESUMO

BACKGROUND AND PURPOSE: Gadobutrol (Gadavist) and gadoteridol (ProHance) have similar macrocyclic molecular structures, but gadobutrol is formulated at a 2-fold higher (1 mol/L versus 0.5 mol/L) concentration. We sought to determine whether this difference impacts morphologic contrast-enhanced MR imaging. MATERIALS AND METHODS: Two hundred twenty-nine adult patients with suspected or known brain tumors underwent two 1.5T MR imaging examinations with gadoteridol or gadobutrol administered in randomized order at a dose of 0.1 mmol/kg of body weight. Imaging sequences and T1 postinjection timing were identical for both examinations. Three blinded readers evaluated images qualitatively and quantitatively for lesion detection and for accuracy in characterization of histologically confirmed brain tumors. Data were analyzed by using the Wilcoxon signed rank test, the McNemar test, and a mixed model. RESULTS: Two hundred nine patients successfully completed both examinations. No reader noted a significant qualitative or quantitative difference in lesion enhancement, extent, delineation, or internal morphology (P values = .69-1.00). One hundred thirty-nine patients had at least 1 histologically confirmed brain lesion. Two readers found no difference in the detection of patients with lesions (133/139 versus 135/139, P = .317; 137/139 versus 136/139, P = .564), while 1 reader found minimal differences in favor of gadoteridol (136/139 versus 132/139, P = .046). Similar findings were noted for the number of lesions detected and characterization of tumors (malignant/benign). Three-reader agreement for characterization was similar for gadobutrol (66.4% [κ = 0.43]) versus gadoteridol (70.3% [κ = 0.45]). There were no significant differences in the incidence of adverse events (P = .199). CONCLUSIONS: Gadoteridol and gadobutrol at 0.1 mmol/kg of body weight provide similar information for visualization and diagnosis of brain lesions. The 2-fold higher gadolinium concentration of gadobutrol provides no benefit for routine morphologic imaging.


Assuntos
Neoplasias Encefálicas/diagnóstico , Meios de Contraste/administração & dosagem , Compostos Heterocíclicos/administração & dosagem , Imageamento por Ressonância Magnética/métodos , Compostos Organometálicos/administração & dosagem , Adulto , Idoso , Estudos Cross-Over , Feminino , Gadolínio/administração & dosagem , Humanos , Masculino , Pessoa de Meia-Idade , Neuroimagem/métodos
15.
AJNR Am J Neuroradiol ; 36(9): 1589-98, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26185325

RESUMO

BACKGROUND AND PURPOSE: Gadobenate dimeglumine (MultiHance) has higher r1 relaxivity than gadoterate meglumine (Dotarem) which may permit the use of lower doses for MR imaging applications. Our aim was to compare 0.1- and 0.05-mmol/kg body weight gadobenate with 0.1-mmol/kg body weight gadoterate for MR imaging assessment of brain tumors. MATERIALS AND METHODS: We performed crossover, intraindividual comparison of 0.1-mmol/kg gadobenate with 0.1-mmol/kg gadoterate (Arm 1) and 0.05-mmol/kg gadobenate with 0.1-mmol/kg gadoterate (Arm 2). Adult patients with suspected or known brain tumors were randomized to Arm 1 (70 patients) or Arm 2 (107 patients) and underwent 2 identical examinations at 1.5 T. The agents were injected in randomized-sequence order, and the 2 examinations were separated by 2-14 days. MR imaging scanners, imaging sequences (T1-weighted spin-echo and T1-weighted high-resolution gradient-echo), and acquisition timing were identical for the 2 examinations. Three blinded readers evaluated images for diagnostic information (degree of definition of lesion extent, lesion border delineation, visualization of lesion internal morphology, contrast enhancement) and quantitatively for percentage lesion enhancement and lesion-to-background ratio. Safety assessments were performed. RESULTS: In Arm 1, a highly significant superiority (P < .002) of 0.1-mmol/kg gadobenate was demonstrated by all readers for all end points. In Arm 2, no significant differences (P > .1) were observed for any reader and any end point, with the exception of percentage enhancement for reader 2 (P < .05) in favor of 0.05-mmol/kg gadobenate. Study agent-related adverse events were reported by 2/169 (1.2%) patients after gadobenate and by 5/175 (2.9%) patients after gadoterate. CONCLUSIONS: Significantly superior morphologic information and contrast enhancement are demonstrated on brain MR imaging with 0.1-mmol/kg gadobenate compared with 0.1-mmol/kg gadoterate. No meaningful differences were recorded between 0.05-mmol/kg gadobenate and 0.1-mmol/kg gadoterate.


Assuntos
Neoplasias Encefálicas/patologia , Imageamento por Ressonância Magnética/métodos , Adulto , Idoso , Meios de Contraste , Estudos Cross-Over , Feminino , Humanos , Masculino , Meglumina/análogos & derivados , Pessoa de Meia-Idade , Compostos Organometálicos
16.
J Bone Miner Res ; 9(9): 1407-17, 1994 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-7817824

RESUMO

We previously demonstrated that the [Ca2+]i response to PTH is heterogeneous in single UMR-106-01 osteogenic sarcoma cells. To verify whether response heterogeneity is a universal feature of PTH signal transduction, cAMP production was monitored in monolayer cultures of UMR-106-01 cells and human trabecular bone osteoblasts (HOB) using the cAMP-sensitive fluorescent indicator FlCRhR. FlCRhR was microinjected into single cells, and the 500-530/> 560 nm fluorescence ratio was monitored by confocal laserscanning video imaging as a measure of cAMP concentration ([cAMP]). Virtually all UMR-106-01 cells exposed to bovine PTH(1-34) (10(-7) M) exhibited an increase in intracellular [cAMP], with an average fluorescence ratio change of 145 +/- 17% of baseline (n = 15), corresponding to nearly maximal dissociation of protein kinase A. In the continued presence of the hormone (10(-7) M), [cAMP] remained elevated for at least 30 minutes. This effect was accompanied by a slow translocation of the fluorescein-labeled catalytic subunit of protein kinase A from the cytoplasm to the nucleus. In contrast, PTH(1-34) caused no detectable increase in [cAMP] in HOB cells, although PGE2 (3 x 10(-6) M) stimulation was able to increase the FlCRhR ratio (154 +/- 27%, n = 10). The truncated fragment PTH(2-34) was only 67% as potent at PTH(1-34), but deletion of the first two amino acids at the N terminus abolished the hormone's ability to stimulate cAMP production in UMR-106-01 cells. Brief exposure to 10(-7) M of either PTH(3-34) or PTH(7-34) did not affect the amplitude of the fluorescence ratio change induced by equimolar doses of PTH(1-34). Thus, in osteoblast-like cells stimulated with PTH, the [cAMP] response is much more homogeneous from cell to cell than the [Ca2+]i response.


Assuntos
AMP Cíclico/metabolismo , Osteoblastos/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Fragmentos de Peptídeos/farmacologia , Animais , Neoplasias Ósseas/patologia , Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Corantes Fluorescentes/administração & dosagem , Corantes Fluorescentes/química , Humanos , Processamento de Imagem Assistida por Computador , Microinjeções , Microscopia Confocal , Microscopia de Fluorescência , Osteossarcoma/patologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Teriparatida , Células Tumorais Cultivadas
17.
Cell Calcium ; 18(5): 377-89, 1995 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8581966

RESUMO

We have investigated, at the single cell level, intracellular Ca2+ ([Ca2+]i) modulations triggered by the high affinity receptor for IgG, Fc gamma RI, in the monocytic cell line, U937. Cells were co-loaded with the Ca(2+)-sensitive dyes, Fluo-3 and Fura-Red, by incubation with their acetoxymethyl (AM) esters and confocal ratio imaging was used to monitor the [Ca2+]i changes induced by antibody cross-linking of IgG-loaded Fc gamma RI. A single Ca2+ spike was observed in 81% of untreated cells whereas dibutyryl cAMP-induced differentiation into a more macrophage cell type resulted in a sub-population of cells (44%) responding to receptor cross-linking with calcium oscillations. This change in calcium signalling may explain the difference in functional responses triggered by Fc gamma RI in monocytes and macrophages. Analysis of the Fluo-3 and Fura-Red fluorescence, after AM-ester loading, showed that both dyes have similar photobleach rates and intracellular localization allowing compensation for shifts in focal plane, dye photobleaching and non-uniformity of dye loading. In addition, because the binding kinetics of both dyes are equivalent, accurate temporal information can be gained about [Ca2+] changes. There are, however, two major problems with this dual indicator technique. Firstly, loading from AM esters results in considerable variation between cells in the intracellular concentration ratio of the two dyes, making calibration difficult. Secondly, the fluorescence ratio, Fluo-3/Fura-Red, behaves non-linearly at Ca2+ concentrations less than approximately 500 nM and comparison with Fura-2-loaded single cell photometry studies suggests there is considerable amplitude distortion of the signal when the ratios are displayed on a linear scale. These problems may considerably limit the application of Fluo-3/Fura-Red ratiometric measurements.


Assuntos
Cálcio/metabolismo , Imunoglobulina G/farmacologia , Receptores Fc/metabolismo , Trifosfato de Adenosina/farmacologia , Compostos de Anilina , Benzofuranos , Diferenciação Celular , Corantes Fluorescentes , Humanos , Imidazóis , Microscopia Confocal , Células Tumorais Cultivadas , Xantenos
18.
Hum Mutat ; 19(3): 274-8, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11857744

RESUMO

Hemophilia A is a bleeding disorder caused by a quantitative or qualitative deficiency in the coagulation factor VIII. Causative mutations are heterogeneous in nature and are distributed throughout the FVIII gene. With the exception of mutations that result in prematurely truncated protein, it has proved difficult to correlate mutation type/amino acid substitution with severity of disease. We have identified 81 mutations in 96 unrelated patients, all of whom have typed negative for the common IVS-22 inversion mutation. Forty-one of these mutations are not recorded on F8C gene mutation databases. We have analyzed these 41 mutations with regard to location, whether or not each is a cross-species conserved region, and type of substitution and correlated this information with the clinical severity of the disease. Our findings support the view that the phenotypic result of a mutation in the FVIII gene correlates more with the position of the amino acid change within the 3D structure of the protein than with the actual nature of the alteration.


Assuntos
Fator VIII/genética , Mutação/genética , Animais , Códon sem Sentido/genética , Análise Mutacional de DNA/métodos , Cães , Feminino , Genótipo , Hemofilia A/sangue , Hemofilia A/genética , Humanos , Camundongos , Mutagênese Insercional/genética , Mutação de Sentido Incorreto/genética , Fenótipo , Polimorfismo Conformacional de Fita Simples , Sítios de Splice de RNA/genética , Deleção de Sequência/genética
19.
Gene ; 126(1): 1-7, 1993 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-8472949

RESUMO

Phage 21 is a temperate lambdoid coliphage, and its head-encoding genes, as well as those of phage lambda, are descended from a common ancestral phage. The head protein-encoding genes of phage 21 have been sequenced, confirming earlier genetic studies indicating that the head-encoding genes of 21 and lambda are analogous in location, size, and function. The phage 21 head-encoding genes identified (and their lambda analogues) include: 3(W), 4(B), 5(C), 6(Nu3), shp (D), 7(E), and 8(FII), respectively. An open reading frame, orf1, is analogous in position and shares some sequence identity with FI, a phage lambda gene involved in DNA packaging. The phage 21 major head protein, gp7, is predicted to have strong sequence identity (65%) with the lambda major capsid protein, gpE, including amino acids known to be important for capsid form determination. The nested genes 5/6 of phage 21 and C/Nu3 of lambda differ by several rearrangements including deletions and a triplication. The possibility that lambda genes C/Nu3 evolved from ancestal nested genes containing a triplication is discussed.


Assuntos
Colífagos/genética , Genes Virais , Proteínas Virais/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Viral , Dados de Sequência Molecular , Fases de Leitura Aberta , Homologia de Sequência de Aminoácidos , Replicação Viral/genética
20.
FEBS Lett ; 414(2): 377-80, 1997 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-9315723

RESUMO

Atrial and brain natriuretic peptides (ANP and BNP, respectively) are two cardiac natriuretic peptides (NPs) found in tetrapods from amphibians to mammals, whereas ANP and ventricular NP (VNP) have been identified in eel hearts. Because VNP has also been found in the rainbow trout ventricle, we attempted to isolate NP from trout cardiac atria in order to determine whether ANP and VNP are common cardiac NPs in teleosts. In the present experiments, we isolated VNP and a novel atrial NP consisting of 29 amino acid residues from the atria. This new trout NP exhibited similar sequence identity to mammalian ANP and BNP (50-60%). Its homology to eel ANP was low (52%) compared with high homology of trout and eel VNP (78%). Based on yield, the content of this new NP in trout atria may be even smaller than that of VNP. The new trout atrial NP exhibited low relaxant activity in the chick rectum (only 1/10 of that of trout VNP), and extremely low vasorelaxant activity in the rat aortic strip (only 1/400 of that of human ANP). However, the new trout NP was equipotent with trout VNP and human ANP in relaxing trout epibranchial artery. Based on the sequence similarity with other NPs and on atrial content, the new NP isolated from trout atria cannot yet be assigned to a known member of the NP family.


Assuntos
Fator Natriurético Atrial/química , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Miocárdio/química , Vasodilatação/efeitos dos fármacos , Vasodilatadores , Sequência de Aminoácidos , Anfíbios , Animais , Artérias , Fator Natriurético Atrial/isolamento & purificação , Fator Natriurético Atrial/farmacologia , Galinhas , Enguias , Átrios do Coração , Ventrículos do Coração , Humanos , Mamíferos , Dados de Sequência Molecular , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Músculo Liso Vascular/efeitos dos fármacos , Miocárdio/metabolismo , Oncorhynchus mykiss , Rana catesbeiana , Ratos , Reto , Alinhamento de Sequência
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