RESUMO
The presence of short dental roots can present challenges to the orthodontist both in terms of identifying its aetiology and in subsequent treatment planning. Uncommon causes include hypoparathyroidism and pseudohypoparathyroidism, where short roots may be seen in combination with other oral manifestations including enamel hypoplasia secondary to low calcium levels. This case report highlights these features and the orthodontic treatment proposed.
Assuntos
Hipoplasia do Esmalte Dentário , Hipoparatireoidismo , Anormalidades Dentárias , Criança , Humanos , Ortodontistas , Planejamento de Assistência ao PacienteRESUMO
Introduction With an increasing demand to improve patient safety within the NHS, it is important to ensure that measures are undertaken to continually improve patient care. Wrong site surgery has been defined as a 'never event'. This article highlights the importance of preventing wrong tooth extraction within orthodontics through an audit spiral over five years investigating the accuracy and clarity of orthodontic extraction letters at the University Dental Hospital of Manchester.Aims To examine compliance with the standards for accuracy and clarity of extraction letters and the incidence of wrong tooth extractions, and to increase awareness of the errors that can occur with extraction letters and of the current guidelines.Method A retrospective audit was conducted examining extraction letters sent to clinicians outside the department.Results It can be seen there has been no occurrence of a wrong site tooth extraction. The initial audit highlighted issues in conformity, with it falling below expected standards. Cycle two generally demonstrated a further reduction in compliance. Cycle three appeared to result in an increase in levels of compliance. Cycles 4 and 5 have demonstrated gradual improvements. However, it is noteworthy that in all cycles the audit standards were still not achieved, with the exception of no incidences of the incorrect tooth being extracted.Conclusion This audit spiral demonstrates the importance of long term re-audit to aim to achieve excellence in clinical care. There has been a gradual increase in standards through each audit.
Assuntos
Erros Médicos/prevenção & controle , Segurança do Paciente , Extração Dentária/normas , Auditoria Odontológica/métodos , Humanos , Erros Médicos/estatística & dados numéricos , Estudos Retrospectivos , Medicina Estatal , Extração Dentária/métodos , Reino UnidoRESUMO
Micro-screws (MSs) have gained rapid popularity among orthodontic specialists over the past decade. Subsequently, as general dental practitioners (GDPs) continue to provide general care for patients undergoing orthodontic treatment they are likely to encounter MSs. This article is aimed at GDPs and provides an overview of MS design, placement, removal and safety. Two examples of treated cases will also be used to demonstrate the use of MSs in contemporary orthodontic practice.
Assuntos
Parafusos Ósseos , Procedimentos de Ancoragem Ortodôntica/instrumentação , Adolescente , Processo Alveolar/diagnóstico por imagem , Processo Alveolar/cirurgia , Parafusos Ósseos/efeitos adversos , Parafusos Ósseos/classificação , Arco Dental/diagnóstico por imagem , Arco Dental/cirurgia , Segurança de Equipamentos , Feminino , Humanos , Consentimento Livre e Esclarecido , Masculino , Má Oclusão Classe I de Angle/terapia , Má Oclusão Classe II de Angle/terapia , Miniaturização , Procedimentos de Ancoragem Ortodôntica/efeitos adversos , Desenho de Aparelho Ortodôntico , Planejamento de Assistência ao Paciente , Radiografia Interproximal , Técnicas de Movimentação Dentária/instrumentação , Dente Impactado/terapia , Adulto JovemRESUMO
The clinical problem of how best to manage an anterior space resulting from a missing central incisor will only be encountered rarely. The goal should be to deliver treatment results that are indistinguishable from normal appearance. This article describes one treatment approach - orthodontic space closure with substitution of the maxillary central incisor by the lateral incisor. Treatment indications, orthodontic and restorative considerations, advantages and disadvantages, as well as the evidence base relating to this treatment modality will be presented and supported by two clinical case examples.
Assuntos
Estética Dentária , Incisivo , Fechamento de Espaço Ortodôntico/métodos , Adolescente , Humanos , Incisivo/anormalidades , Incisivo/lesões , Masculino , Adulto JovemRESUMO
Resorption of lateral incisors caused by impacted maxillary canines is frequently reported. However, resorption of the central incisor is less common and management of such a finding can prove to be a challenge for the clinician. This article reviews the literature of impacted canines and incisor resorption. The management of two cases of severe central incisor resorption caused by an impacted maxillary canine is also described.
Assuntos
Dente Canino/diagnóstico por imagem , Incisivo/diagnóstico por imagem , Ortodontia/métodos , Reabsorção da Raiz/diagnóstico , Erupção Ectópica de Dente/complicações , Dente Impactado/terapia , Adolescente , Criança , Tomografia Computadorizada de Feixe Cônico , Dente Canino/crescimento & desenvolvimento , Feminino , Humanos , Incisivo/crescimento & desenvolvimento , Maxila/diagnóstico por imagem , Reabsorção da Raiz/terapia , Erupção Ectópica de Dente/diagnóstico por imagem , Dente Impactado/diagnóstico , Dente Impactado/etiologiaRESUMO
The PR is critical for normal female reproductive function in mammals, including primates, and the PR-knockout mouse is an important model for establishing PR targets. For example, LH secretion is significantly altered both in vivo and in vitro in female PR-knockout mice, but to establish specific mechanisms affected by the absence of the PR in the mouse requires characterization of wild-type mouse cell biology. As steps toward this, the aims were to establish whether altered LH secretion in PR-knockout mice reflects altered mouse gonadotrope cell lineage during development secondary to PR deletion and to test the assumption that PR in wild-type mouse pituitaries has the same exclusive gonadotrope localization and E2 and progesterone regulation as in rat and monkey pituitaries. As an in vitro model, dispersed pituitary cells from 2-wk ovariectomized wild-type or PR-knockout mice were cultured +/- E2 for 3 d. These cells were subjected to dual immunofluorescence staining for PR and LH, PRL, or GH. The proportion of LH-gonadotropes (8-9%) and somatotropes (26-29%) was not different for PR-knockout and wild-type cultures with or without E2. Lactotrope composition (41-42%) was the same in wild-type and PR-knockout, and E2 resulted in a similar and significant increase in the proportion (57-59%) for both mouse types. Nuclear PR immunoreactivity was absent in all PR-knockout pituitary cells. For wild-type, all LH gonadotropes showed nuclear PR immunoreactivity that was up-regulated by E2 (>10-fold increase). Progesterone exposure for 10 h but not 3 h led to a 40% decrease in PR immunoreactivity in LH-gonadotropes. Unexpectedly, PR immunoreactivity also localized to all lactotropes and was up-regulated by E2 and down-regulated by progesterone. In summary, the absence of PR has no effect on the proportion of LH gonadotropes, lactotropes, and somatotropes in ovariectomized PR-knockout mouse pituitary cultures. For ovariectomized wild-type mice, gonadotropes in the in vitro model contain PR that is up-regulated by E2, but the downregulation by progesterone is modest, compared with that previously reported for an in vitro rat model. In contrast to rats and monkeys, E2-dependent PR also is present in lactotropes of ovariectomized wild-type mice. These results underscore the risks in assuming identical cell biology between rats and mice.
Assuntos
Adeno-Hipófise/fisiologia , Receptores de Progesterona/fisiologia , Animais , Células Cultivadas , Feminino , Camundongos , Camundongos Knockout , Ovariectomia , Adeno-Hipófise/citologia , RatosRESUMO
Early C. elegans embryos exhibit protein asymmetries that allow rapid diversification of cells. Establishing these asymmetries requires the novel protein MEX-5. We show that mutations in the efl-1 and dpl-1 genes cause defects in protein localization resembling defects caused by mutations in mex-5. efl-1 and dpl-1 encode homologs of vertebrate E2F and DP proteins that regulate transcription as a heterodimer. efl-1 and dpl-1 mutants have elevated levels of activated Map kinase in oocytes. Their mutant phenotype and that of mex-5 mutants can be suppressed by reducing Ras/Map kinase signaling. We propose this signaling pathway has a role in embryonic asymmetry and that EFL-1/DPL-1 control the level of Map kinase activation.
Assuntos
Padronização Corporal , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/embriologia , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Proteínas de Helminto/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas ras/antagonistas & inibidores , Animais , Padronização Corporal/genética , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Divisão Celular , Dimerização , Fatores de Transcrição E2F , Ativação Enzimática , Feminino , Fertilização/genética , Genes de Helmintos/genética , Proteínas de Helminto/genética , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mosaicismo/genética , Mutação/genética , Ovulação/genética , Óvulo/metabolismo , Fenótipo , Transporte Proteico , Fatores de Transcrição/genética , Proteínas ras/metabolismoRESUMO
The progesterone receptor (PR) has a central role in the hypothalamo-pituitary events culminating in the preovulatory LH surge, and mice with genetically ablated PR provide a model for dissecting cellular pathways subserving this role. The aims of this study were to determine 1) whether the GnRH self-priming response and acute progesterone augmentation of secretagogue-stimulated LH secretion are present in cultured wild-type (WT) mouse pituitary cells, and 2) whether the PR is essential for self-priming by comparing the responses in PR knockout (PRKO) cells. Pituitary cells from ovariectomized WT or PRKO mice cultured +/- 17beta-estradiol (E(2)) for 3 days were challenged with hourly pulses of 1 nM GnRH or 54 mM K(+). A background of E(2) had no effect on the initial LH secretory response for either WT or PRKO cells. However, for subsequent GnRH pulses, E(2) was permissive for the GnRH self-priming response in WT cells. PRKO cells exhibited a blunted GnRH self-priming response. Exposure to progesterone for 90 min before secretagogue stimulation resulted in a modest (1.5-fold) augmentation of the LH response to GnRH but not K(+) pulses in WT cells; progesterone had no effect in PRKO cells. Unlike in the rat, the PR antagonists RU486 or ZK98299 failed to prevent potentiation of LH secretory responses to multiple GnRH pulses in WT cells. Although RU486 blocked progesterone augmentation of the initial GnRH pulse, it was ineffective in blocking progesterone's action after multiple GnRH pulses. In WT cells, 8- bromo-cAMP (8-Br-cAMP) was able to substitute for the GnRH priming pulse; 8-Br-cAMP also augmented GnRH-stimulated secretion in PRKO cells but less effectively. 8-Br-cAMP augmented K(+)-stimulated LH secretion in WT and PRKO cells equally. These results suggest that, although mouse gonadotropes show GnRH self-priming, they have adapted strategies different than rat cells for amplifying the GnRH signal as shown by the residual self-priming in PRKO cells, the modest or absent augmentation by acute progesterone of GnRH- or K(+)-stimulated secretion in WT cells, and the reduced ability of PR antagonists to interfere with GnRH self-priming and progesterone augmentation. We speculate that the adaptations could involve, at least in part, differences in the ratio of PR isoforms.
Assuntos
Hormônio Luteinizante/metabolismo , Adeno-Hipófise/citologia , Adeno-Hipófise/metabolismo , Receptores de Progesterona/fisiologia , Animais , Células Cultivadas , AMP Cíclico/farmacologia , Estradiol/farmacologia , Espaço Extracelular/metabolismo , Feminino , Hormônio Liberador de Gonadotropina/farmacologia , Camundongos , Camundongos Knockout/genética , Ovariectomia , Adeno-Hipófise/efeitos dos fármacos , Potássio/metabolismo , Progesterona/farmacologia , Receptores de Progesterona/antagonistas & inibidores , Receptores de Progesterona/genética , Valores de ReferênciaRESUMO
This essay argues that the practice of medicine is not a phronetic activity in the original Aristotelian sense of that term. Jonsen and Toulmin are two philosophers who have conflated the techne of medicine with phronesis. This conflation ignores Aristotle's crucial distinction between techne and phronesis and his use of the medical analogy. It is argued that medical reasoning is similar to phronesis but does not exemplify it. Phronesis will not save the life of medical ethics. The concept could be utilized as a moral prosthetic.
Assuntos
Ética Médica , Filosofia , Humanos , Princípios MoraisRESUMO
The basic helix-loop-helix transcription factor NeuroD (Neurod1) has been implicated in neuronal fate determination, differentiation and survival. Here we report the expression and functional analysis of cnd-1, a C. elegans NeuroD homolog. cnd-1 expression was first detected in neuroblasts of the AB lineage in 14 cell embryos and maintained in many neuronal descendants of the AB lineage during embryogenesis, diminishing in most terminally differentiated neurons prior to hatching. Specifically, cnd-1 reporter genes were expressed in the precursors of the embryonic ventral cord motor neurons and their progeny. A loss-of-function mutant, cnd-1(ju29), exhibited multiple defects in the ventral cord motor neurons. First, the number of motor neurons was reduced, possibly caused by the premature withdrawal of the precursors from mitotic cycles. Second, the strict correlation between the fate of a motor neuron with respect to its lineage and position in the ventral cord was disrupted, as manifested by the variable expression pattern of motor neuron fate specific markers. Third, motor neurons also exhibited defects in terminal differentiation characteristics including axonal morphology and synaptic connectivity. Finally, the expression patterns of three neuronal type-specific transcription factors, unc-3, unc-4 and unc-30, were altered. Our data suggest that cnd-1 may specify the identity of ventral cord motor neurons both by maintaining the mitotic competence of their precursors and by modulating the expression of neuronal type-specific determination factors. cnd-1 appears to have combined the functions of several vertebrate neurogenic bHLH proteins and may represent an ancestral form of this protein family.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/embriologia , Genes de Helmintos , Neurônios Motores/citologia , Proteínas do Tecido Nervoso/genética , Sistema Nervoso/embriologia , Proteínas Nucleares , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Caenorhabditis elegans/genética , Diferenciação Celular , Linhagem da Célula , Sequências Hélice-Alça-Hélice/genética , Proteínas de Helminto/isolamento & purificação , Proteínas de Homeodomínio/isolamento & purificação , Dados de Sequência Molecular , Proteínas Musculares/isolamento & purificação , Mutação , Homologia de Sequência de Aminoácidos , Células-Tronco , Fatores de Transcrição/isolamento & purificaçãoRESUMO
For rat pituitary cells, progesterone receptor (PR) protein localizes to gonadotropes and PR messenger RNA is induced by E2 and rapidly but transiently down-regulated by progesterone. Here we quantitatively establish the down-regulatory effect of progesterone on PR protein and evaluate possible mechanisms. Nuclear PR-immunoreactivity (PR-IR) in gonadotropes, identified by dual immunofluorescence, was analyzed by quantitative confocal microscopy. Pituitary cells from female rats were cultured +/- 0.2 nM E2 for 3 days. We confirmed the E2 requirement for PR induction in gonadotropes and determined that the increase in PR-IR required about 24 h. After removal of E2, PR-IR decreases were not found until 24-36 h. Addition of progesterone (40 nM) to E2-treated cells led to a dramatic loss in PR-IR by 9 h (26% of control); by 24 h, PR-IR was barely detectable. Reappearance of nuclear PR-IR required progesterone removal (8-fold increase by 12 h after progesterone removal) and protein synthesis (cycloheximide inhibited the reappearance of PR-IR). Although progesterone decreased PR-IR whether or not E2 was present concurrent with progesterone, the recovery of PR-IR required E2. RU486 completely blocked progesterone-induced PR down-regulation. Because the sustained progesterone-induced loss of PR protein did not correlate with previously reported temporal changes in PR messenger RNA levels, we examined a role for protein degradation. When cells were coincubated with progesterone and the proteasome inhibitor, MG132 (1 microM), the expected decrease in PR protein was abrogated. In summary, progesterone leads to a rapid and extensive reduction in nuclear PR protein in gonadotropes. The progesterone-dependent down-regulation of PR occurs, at least in part, by a proteasome-mediated pathway. Recovery of PR protein requires removal of progesterone, the presence of E2, and protein synthesis. These dynamic changes in nuclear PR levels coincide with the temporal extent of the preovulatory LH surge in rats and could provide a basis for progesterone's biphasic action on LH secretion.
Assuntos
Progesterona/fisiologia , Receptores de Progesterona/fisiologia , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Células Cultivadas , Regulação para Baixo/genética , Estradiol/farmacologia , Feminino , Imunofluorescência , Imuno-Histoquímica , Microscopia Confocal , Mifepristona/farmacologia , Ovariectomia , Adeno-Hipófise/citologia , Adeno-Hipófise/efeitos dos fármacos , Adeno-Hipófise/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Progesterona/efeitos dos fármacosRESUMO
During the preovulatory period, the pituitary action of progesterone is biphasic, moving from a severalfold augmentation of the gonadotropin release action of GnRH to a suppression of GnRH efficacy, which occurs in rats over a period of about 12 h, but the extent to which these biphasic effects are dependent on alterations in progesterone receptor (PR) expression is not known. To address this, as well as the localization of PR in cultured rat pituitary cells, we used cells from ovariectomized rats cultured +/- 0.2 nM E2 with acute progesterone treatment on day 3. Northern blot of poly(A+) RNA extracts showed multiple PR messenger RNA (mRNA) transcripts between 4.8-10.2 kb; E2 treatment led to a 5- to 6-fold increase in the predominant PR mRNA transcripts (5.1 and 10.1 kb). In the presence of E2, 200 nM progesterone resulted in a decrease in steady-state PR mRNA levels by 3 h of exposure, with the greatest decrease around 6 h (50% of E2 control) and recovery by 12 h. Similarly treated pituitary cultures were subjected to dual immunofluorescence staining for LH and PR. In the absence of E2, PR was undetectable. In the presence of E2, essentially all LH-positive cells were positive for PR and only 1-2% of PR-immunopositive cells were negative for LH, possibly reflecting FSH-exclusive gonadotropes. PR staining was predominantly nuclear, but 20 nM progesterone led to a gradual increase in cytoplasmic staining, with the nuclear-to-cytoplasmic ratio decreasing to near unity by 9-12 h of exposure. In summary, we show for the first time, that PR colocalizes with LH in cultured female rat pituitary cells and that E2 induces expression of PR mRNA, as well as PR protein, in rat gonadotropes. In the presence of E2, progesterone causes a rapid but transient down-regulation of PR message; recovery of PR mRNA is accompanied by an increase in cytoplasmic PR, suggestive of an increase in synthesis. These dynamic changes implicate the gonadotrope PR as having a significant role within the temporal context of the rat preovulatory period.
Assuntos
Estradiol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Gonadotropinas Hipofisárias/análise , Adeno-Hipófise/metabolismo , Progesterona/farmacologia , Receptores de Progesterona/genética , Animais , Células Cultivadas , Feminino , Ovariectomia , Adeno-Hipófise/química , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Receptores de Progesterona/análise , Distribuição TecidualRESUMO
In the female, androgens can have negative and positive actions in the regulation of LH, but it is not clear how they may function during the reproductive cycle. Toward resolving these potentially conflicting roles for androgen, we used an in vitro model of preovulatory gonadotropes to examine the effect of proestrous levels of testosterone (1.7 nM) or dihydrotestosterone (DHT; 0.7 nM) on LH secretion in response to pulsatile GnRH (1 nM) or elevated extracellular K+ (54 mM). For female rat pituitary cells cultured in 17beta-estradiol (E2)-containing medium, androgen treatment for 16 h, but not for 4 h, inhibited the LH secretory response to a pulse of either GnRH or K+ by about 60% and suppressed the acute augmentation action of 20 nM progesterone on GnRH- or K+-induced LH secretion. In the absence of E2, DHT also decreased LH secretion induced by a pulse of GnRH. DHT's suppressive effect on progesterone could be partially overcome with increased progesterone (200 nM) or by removal of DHT during progesterone exposure. For pituitary cells transfected with a reporter plasmid containing three progesterone response elements, DHT only partially suppressed progesterone-stimulated transcriptional activity. The positive action of androgen (16 h) on LH secretion was elicited by multiple GnRH pulses with a latency of about 2 h after the first pulse; this facilitatory action of androgen did not require an E2 background and, therefore, is distinct from GnRH self priming. In summary, these data demonstrate both facilitatory and inhibitory actions of androgen on LH secretion function in female gonadotropes in vitro in the absence or presence of E2; these actions occur with a time course suggestive of a role for androgen in shaping the preovulatory LH surge. Androgen also markedly suppresses progesterone augmentation of stimulated LH secretion, which could be due in part to interference with the trans-activation function of the progesterone receptor.
Assuntos
Di-Hidrotestosterona/farmacologia , Hormônio Luteinizante/metabolismo , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Testosterona/farmacologia , Animais , Células Cultivadas , Di-Hidrotestosterona/administração & dosagem , Estradiol/farmacologia , Feminino , Hormônio Liberador de Gonadotropina/administração & dosagem , Hormônio Liberador de Gonadotropina/farmacologia , Ovariectomia , Periodicidade , Potássio/farmacologia , Progesterona/farmacologia , Ratos , Ratos Sprague-Dawley , Elementos de Resposta/genética , Testosterona/administração & dosagem , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
1. Exocytosis and intracellular [Ca2+] were determined simultaneously in single anterior pituitary gonadotrophs from ovariectomized female rats. Dispersed cells were cultured for 2-4 days with or without 0.2 nM oestradiol-17 beta (E2) before use. Cells were stimulated with either gonadotrophin releasing hormone (GnRH) or by membrane depolarization. Exocytosis was determined from the change in membrane capacitance (Cm) using the perforated-patch whole-cell recording technique. Intracellular [Ca2+] was measured using fura-2 fluorescence. 2. The exocytotic response to 1 nM GnRH was characterized by a wide spectrum of responses, ranging from exocytotic bursts to relatively slow, graded increases that were dependent on the evoked intracellular Ca2+ pattern. A kinetic model is presented that incorporates the observed steep dependence of exocytosis on measured intracellular [Ca2+]; simulated exocytosis reasonably approximated observed exocytotic responses, both kinetically and quantitatively. The model also suggests that the modulatory effects of E2 are brought about either by a change in the Ca2+ sensitivity of exocytosis or by a preferential clustering of docked-secretory granules close to sites of Ca2+ release. The results suggest that in gonadotrophs an oscillatory Ca2+ signal is sensed by the exocytotic apparatus in a modified form of digital encoding. 3. Exocytosis in E2-treated cells was 3-fold greater than in non-treated cells for GnRH-evoked secretion, and 38% greater for depolarization; however, there was no effect of E2 on the intracellular Ca2+ response to either stimulus. The results show that maximum expression of the effect of E2 on exocytosis requires activation of GnRH-dependent pathways.
Assuntos
Adeno-Hipófise/citologia , Animais , Cálcio/fisiologia , Estimulação Elétrica , Eletrofisiologia , Endocitose/fisiologia , Estradiol/farmacologia , Exocitose/fisiologia , Feminino , Cinética , Potenciais da Membrana/fisiologia , Modelos Biológicos , Ovariectomia , Técnicas de Patch-Clamp , Adeno-Hipófise/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyAssuntos
Padronização Corporal , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Genes Homeobox , Animais , Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Células Epidérmicas , Epiderme/embriologia , Feminino , Masculino , Neurônios/citologia , Neurônios/fisiologiaRESUMO
We have used single gonadotropes from the newly derived line, LbetaT2, to investigate the modulation of Ca2+ signaling and exocytosis by the steroid hormone environment. This cell line, derived by targeted oncogenesis in transgenic mice, has recently been shown to secrete LH in response to GnRH. We have characterized the effects of both GnRH and membrane depolarization on exocytosis and intracellular [Ca2+] ([Ca2+]i) in individual LbetaT2 cells. GnRH (1-100 nM) evoked concentration-dependent increases in [Ca2+]i and secretion, as monitored by measurement of plasma membrane capacitance (Cm) using the whole-cell perforated-patch technique, and the extent of these changes were dependent upon steroid hormone background. GnRH treatment of cells cultured in medium containing charcoal-treated FBS (ct-FBS) showed smaller changes in [Ca2+]i than cells cultured in untreated FBS. However, when estradiol (E2) and dexamethasone (Dex) were added to the ct-FBS medium (E2/Dex-ct-FBS), the elevations in [Ca2+]i stimulated by GnRH increased almost 2-fold. Additionally, the rates of secretion in the E2/Dex-ct-FBS-cultured cells were greater than in either ct-FBS- or FBS-cultured cells. The increase in secretory response observed with E2/Dex-ct-FBS appeared to be due to both an increase in the peak [Ca2+]i stimulated by GnRH and a shift toward increased sensitivity of the Ca2+ dependency of exocytosis. In contrast to GnRH-evoked responses, the increases in [Ca2+]i elicited by depolarization were greater in cells cultured in ct-FBS than in E2/Dex-ct-FBS; however, the secretory rates were no different in the two groups. Likewise, there was no apparent effect of steroid treatment on the Ca2+ dependency of depolarization-evoked exocytosis. In summary, these results 1) clearly demonstrate the utility of this cell line for single-cell studies of both agonist- and depolarization-evoked secretion; 2) reveal that steroid hormone background has profound effects on LbetaT2 cells, both on stimulus-induced calcium mobilization and on the apparent Ca2+ -sensitivity of exocytosis; and 3) show that expression of the steroid hormone effect on Ca2+ -sensitivity is dependent upon receptor occupation by GnRH.
Assuntos
Membrana Celular/fisiologia , Estradiol/farmacologia , Hormônio Liberador de Gonadotropina/farmacologia , Hormônio Luteinizante/metabolismo , Adeno-Hipófise/metabolismo , Animais , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Células Clonais , Meios de Cultura , Dexametasona/farmacologia , Exocitose , Cinética , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Técnicas de Patch-Clamp , Adeno-Hipófise/efeitos dos fármacosRESUMO
We describe a genetic mosaic analysis procedure in which Caenorhabditis elegans mosaics are generated by spontaneous loss of an extrachromosomal array. This technique allows almost any C. elegans gene that can be used in germline transformation experiments to be used in mosaic analysis experiments. We identified a cosmid clone that rescues the mutant phenotype of ncl-1, so that this cell-autonomous marker could be used to analyze mosaic animals. To determine the sites of action for unc-29 and lin-31, an extrachromosomal array was constructed containing the ncl-1(+) cosmid linked to lin-31(+) and unc-29(+) cosmids. This array is mitotically unstable and can be lost to produce a clone of mutant cells. The specific cell division at which the extrachromosomal array had been lost was deduced by scoring the Ncl phenotypes of individual cells in genetic mosaics. The Unc-29 and Lin-31 phenotypes were then scored in these animals to determine in which cells these genes are required. This analysis showed that unc-29, which encodes a subunit of the acetylcholine receptor, acts in the body muscle cells. Furthermore, lin-31, which specifies cell fates during vulval induction and encodes a putative transcription factor similar to HNF-3/fork head, acts in the Pn.p cells.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Mosaicismo , Fatores de Transcrição/genética , Animais , Herança Extracromossômica , Mutação , Fenótipo , Receptores Colinérgicos/genética , Transformação GenéticaRESUMO
Properties of a pituitary gonadotrope include the capacity to regulate gonadotropin synthesis and secretion in response to a GnRH signal. Progress in identifying the steps involved in these processes has been impeded by the lack of a homogeneous in vitro model of gonadotropes. This paper presents functional characterization of a L beta T2 gonadotrope cell line generated by tumorigenesis in transgenic mice carrying the rat LH beta-subunit regulatory region linked to the SV40 T-antigen oncogene. This cell line expresses LH beta, alpha-subunit, and GnRH-receptor (GnRH-R) mRNAs (though not FSH beta), responds to glucocorticoid treatment with a reversible dampening of proliferation, and responds to pulsatile, concentration-dependent GnRH administration with LH secretion. L beta T2 cells presented with four GnRH pulses (10 nM, 90-min interpulse interval) on each of 4 days respond with incremental increases in LH secretion on successive days. This increase was greatest (15-fold) in the presence of estradiol and dexamethasone. Part of the enhanced responsiveness is apparently due to an increase in GnRH-R; pulsatile GnRH treatment alone as well as steroid treatment alone led to an increase in GnRH-R mRNA levels. When secretion was stimulated on day 4 with 54 mM [K+] pulses, bypassing the GnRH-R, the LH-secretory response indicated that the GnRH pulse history as well as estradiol and dexamethasone have actions on L beta T2-secretory capacity distinct from changes in the GnRH-R. This increase can be explained in part by the marked up-regulation of LH beta, but not alpha-subunit, mRNA observed in GnRH-pulsed cells. In summary, L beta T2 clonal gonadotropes exhibit functional characteristics consistent with those of normal pituitary gonadotropes such as LH secretion via a regulated pathway and changes in GnRH-R and LH beta gene expression in response to signaling by GnRH and steroid hormones and therefore should be a useful tool for dissecting the cellular and molecular events involved in these fundamental gonadotrope properties.
Assuntos
Regulação Neoplásica da Expressão Gênica , Hormônio Liberador de Gonadotropina/farmacologia , Hormônio Luteinizante/biossíntese , Hormônio Luteinizante/metabolismo , Neoplasias Hipofisárias/fisiopatologia , Receptores LHRH/biossíntese , Análise de Variância , Animais , Antígenos Virais de Tumores/biossíntese , Antígenos Virais de Tumores/genética , Sequência de Bases , Divisão Celular/efeitos dos fármacos , Primers do DNA , Dexametasona/farmacologia , Estradiol/farmacologia , Feminino , Hormônio Foliculoestimulante , Subunidade beta do Hormônio Folículoestimulante , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Subunidade alfa de Hormônios Glicoproteicos/biossíntese , Cinética , Hormônio Luteinizante/genética , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Oncogenes , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Ratos , Vírus 40 dos Símios/genética , Fatores de Tempo , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Signal amplification is fundamental to the normal operation of the preovulatory LH surge and is achieved through processes such as GnRH self-priming and augmentation of stimulated LH secretion by progesterone. We have proposed a model for GnRH self-priming that requires cross-communication between a GnRH receptor-activated protein kinase A pathway and the progesterone receptor (PR) to achieve amplification of the GnRH signal. We found that a pulse of GnRH administered to gonadotrope-enriched pituitary cells cultured in medium containing charcoal-treated serum plus estradiol (E2) potentiated the LH secretory response to subsequent GnRH pulses, and this potentiation could be blocked by a PR antagonist, RU486, in the absence of progesterone. Similarly, exposure of gonadotrope-enriched cultures to forskolin augmented the response to a pulse of GnRH, and the augmentation due to cAMP elevation could be reduced by RU486 in the absence of progesterone. To directly test whether stimulation with either GnRH or a cAMP analog results in transactivation of the endogenous PR, we used rat anterior pituitary cells cultured in the presence of E2 and transfected with reporter plasmids containing progesterone-responsive elements (PRE) and either a E1b or a thymidine kinase (tk) promoter linked to the chloramphenicol acetyltransferase (CAT) gene. For pituitary cells transfected with the PRE-E1b-CAT plasmid, exposure to either progesterone, GnRH, or 8-bromo-cAMP (8BrcAMP) for 6 h resulted in an induction of CAT activity which could be suppressed by coincubation with RU486. RU486 by itself had no effect on CAT activity. Similar results were obtained when a plasmid containing a different promoter (PRE-tk-CAT) was used. For cells transfected with a construct lacking a PRE (pSV2CAT), 8BrcAMP was without effect on CAT expression. When cells were made PR-deficient by omission of E2 from the incubation medium and transfected with PRE-E1b-CAT, neither progesterone, GnRH, nor 8BrcAMP was able to induce CAT activity. In summary we found that either GnRH or 8BrcAMP is able to stimulate transcription of reporter genes linked to two different PRE-containing promoters in anterior pituitary cells that contain endogenous PR; this occurred in the absence of progesterone and was suppressed by a PR antagonist. A simple interpretation of these data is that a GnRH-triggered signaling cascade can result in progesterone-independent transactivation of the PR. We propose that, in the normal operation of the preovulatory LH surge, the pathways for GnRH self-priming and progesterone augmentation converge at the PR and that the pathways serve as physiological redundancies to ensure the LH surge.
Assuntos
Hormônio Liberador de Gonadotropina/fisiologia , Receptores de Progesterona/fisiologia , Transdução de Sinais/fisiologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Estradiol/farmacologia , Feminino , Hormônio Luteinizante/metabolismo , Mifepristona/farmacologia , Adeno-Hipófise/citologia , Adeno-Hipófise/metabolismo , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Taxa Secretória/efeitos dos fármacos , TransfecçãoRESUMO
Communication between a cell surface peptide hormone receptor and an intracellular steroid hormone receptor can take various routes, as dictated by the physiology of a particular cell type. There is increasing evidence for a novel route which requires that a peptide hormone receptor pathway converge on a steroid hormone receptor, leading to its activation. One consequence of such a process can be signal amplification for the peptide hormone receptor agonist. This is exemplified by the self-potentiating action of GnRH, which is a critical component in events leading to a surge in LH secretion and ovulation. One signaling pathway stimulated by the GnRH receptor may entail a phosphorylation cascade resulting in progesterone-independent modulation of progesterone receptor activity.