RESUMO
The most prevalent pathogen in bone infections is Staphylococcus aureus; its incidence and severity are partially determined by host factors. Prior studies showed that anti-glucosaminidase (Gmd) antibodies are protective in animals, and 93.3 % of patients with culture-confirmed S. aureus osteomyelitis do not have anti-Gmd levels > 10 ng/mL in serum. Infection in patients with high anti-Gmd remains unexplained. Are anti-Gmd antibodies in osteomyelitis patients of the non-opsonising, non-complement-fixing IgG4 isotype? The relative amounts of IgG4 and total IgG against Gmd and 7 other S. aureus antigens: iron-surface determinants (Isd) IsdA, IsdB, and IsdH, amidase (Amd), α-haemolysin (Hla), chemotaxis inhibitory protein from S. aureus (CHIPS), and staphylococcal-complement inhibitor (SCIN) were determined in sera from healthy controls (Ctrl, n = 92), osteomyelitis patients whose surgical treatment resulted in infection control (IC, n = 95) or an adverse outcome (AD, n = 40), and post-mortem (PM, n = 7) blood samples from S. aureus septic-death patients. Anti-Gmd IgG4 levels were generally lower in infected patients compared to controls; however, levels among the infected were higher in AD than IC patients. Anti-IsdA, IsdB and IsdH IgG4 levels were increased in infected patients versus controls, and Jonckheere-Terpstra tests of levels revealed an increasing order of infection (Ctrl < IC < AD < PM) for anti-Isd IgG4 antibodies and a decreasing order of infection (Ctrl > IC > AD > PM) for anti-autolysin (Atl) IgG4 antibodies. Collectively, this does not support an immunosuppressive role of IgG4 in S. aureus osteomyelitis but is consistent with a paradigm of high anti-Isd and low anti-Atl responses in these patients.
Assuntos
Osteomielite , Infecções Estafilocócicas , Animais , Humanos , Imunoglobulina G , Complicações Pós-Operatórias , Staphylococcus aureusRESUMO
Staphylococcus aureus (S. aureus) osteomyelitis remains a major clinical problem. Anti-glucosaminidase (Gmd) antibodies (1C11) are efficacious in prophylactic and therapeutic murine models. Feasibility, safety and pharmacokinetics of 1C11 passive immunisation in sheep and endogenous anti-Gmd levels were quantified in osteomyelitis patients. 3 sheep received a 500 mg intravenous (i.v.) bolus of 1C11 and its levels in sera were determined by enzyme-linked immunosorbent assay (ELISA) over 52 d. A humanised anti-Gmd monoclonal antibody, made by grafting the antigen-binding fragment (Fab) portion of 1C11 onto the fragment crystallisable region (Fc) of human IgG1, was used to make a standard curve of mean fluorescent intensity versus concentration of anti-Gmd. Anti-Gmd serum levels were determined in 297 patients with culture-confirmed S. aureus osteomyelitis and 40 healthy controls. No complications or adverse events were associated with the sheep 1C11 i.v. infusion and the estimated circulating half-life of 1C11 was 23.7 d. Endogenous anti-Gmd antibody levels in sera of osteomyelitis patients ranged from < 1 ng/mL to 300 µg/mL, with a mean concentration of 21.7 µg/mL. The estimated circulating half-life of endogenous anti-Gmd antibodies in sera of 12 patients with cured osteomyelitis was 120.4 d. A clinically relevant administration of anti-Gmd (500 mg i.v. = 7 mg/kg/70 kg human) was safe in sheep. This dose was 8 times more than the endogenous anti-Gmd levels observed in osteomyelitis patients and was predicted to have a half-life of > 3 weeks. Anti-Gmd passive immunisation has potential to prevent and treat S. aureus osteomyelitis. Further clinical development is warranted.
Assuntos
Anticorpos Monoclonais/imunologia , Hexosaminidases/imunologia , Imunização Passiva , Osteomielite/imunologia , Osteomielite/microbiologia , Staphylococcus aureus/fisiologia , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/farmacocinética , Relação Dose-Resposta a Droga , Meia-Vida , Humanos , Camundongos , Padrões de Referência , Ovinos , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologiaRESUMO
The purpose of this study is to identify and chart research literature on safety, efficacy, or effectiveness of exercise prescription following fracture in older adults. We conducted a systematic, research-user-informed, scoping review. The population of interest was adults aged ≥45 years with any fracture. "Exercise prescription" included post-fracture therapeutic exercise, physical activity, or rehabilitation interventions. Eligible designs included knowledge synthesis studies, primary interventional studies, and observational studies. Trained reviewers independently evaluated citations for inclusion. A total of 9,415 citations were reviewed with 134 citations (119 unique studies) identified: 13 knowledge syntheses, 95 randomized or controlled clinical trials, and 11 "other" designs, representing 74 articles on lower extremity fractures, 34 on upper extremity, eight on vertebral, and three on mixed body region fractures. Exercise prescription characteristics were often missing or poorly described. Six general categories emerged describing exercise prescription characteristics: timing post-fracture, person prescribing, program design, functional focus, exercise script parameters, and co-interventions. Upper extremity and ankle fracture studies focused on fracture healing or structural impairment outcomes, whereas hip fracture studies focused more on activity limitation outcomes. The variety of different outcome measures used made pooling or comparison of outcomes difficult. There was insufficient information to identify evidence-informed parameters for safe and effective exercise prescription for older adults following fracture. Key gaps in the literature include limited numbers of studies on exercise prescription following vertebral fracture, poor delineation of effectiveness of different strategies for early post-fracture mobilization following upper extremity fracture, and inconsistent details of exercise prescription characteristics after lower extremity fracture.
Assuntos
Terapia por Exercício/métodos , Fraturas por Osteoporose/reabilitação , Prescrições/estatística & dados numéricos , Idoso , Terapia por Exercício/efeitos adversos , Feminino , Consolidação da Fratura/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Fraturas por Osteoporose/fisiopatologia , Resultado do TratamentoRESUMO
Assessing the body condition of wild animals is necessary to monitor the health of the population and is critical to defining a framework for conservation actions. Body condition indices (BCIs) are a non-invasive and relatively simple means to assess the health of individual animals, useful for addressing a wide variety of ecological, behavioral, and management questions. The Antillean manatee (Trichechus manatus manatus) is an endangered subspecies of the West Indian manatee, facing a wide variety of threats from mostly human-related origins. Our objective was to define specific BCIs for the subspecies that, coupled with additional health, genetic and demographic information, can be valuable to guide management decisions. Biometric measurements of 380 wild Antillean manatees captured in seven different locations within their range of distribution were obtained. From this information, we developed three BCIs (BCI1 = UG/SL, BCI2 = W/SL3, BCI3 = W/(SL*UG2)). Linear models and two-way ANCOVA tests showed significant differences of the BCIs among sexes and locations. Although our three BCIs are suitable for Antillean manatees, BCI1 is more practical as it does not require information about weight, which can be a metric logistically difficult to collect under particular circumstances. BCI1 was significantly different among environments, revealing that the phenotypic plasticity of the subspecies have originated at least two ecotypes-coastal marine and riverine-of Antillean manatees.
Assuntos
Tamanho Corporal , Ecótipo , Trichechus manatus/anatomia & histologia , Animais , Biometria , Feminino , MasculinoRESUMO
Human progesterone receptors (PR) in T47D breast cancer cells are synthesized as two different sized proteins, PR-A [94 kilodaltons (kDa)] and PR-B (120 kDa). Progestin addition to cells (in vivo) causes a 2-fold increase in total phosphorylation of PR and an increase in the apparent mol wt of both PR-A and PR-B on sodium dodecyl sulfate (SDS)-gels. Time-course experiments showed that increased PR phosphorylation that results from hormone addition is a multistep process and involves a rapid increase into total 32P labeling that takes place before the more slowly occurring phosphorylation(s) responsible for the change in electrophoretic mobility of PR on SDS-gels. As an approach to test whether phosphorylation is involved in regulating PR activity, we have examined the effects of cellular modulators of protein phosphorylation on PR-mediated target gene transcription in vivo using a T47D cloned cell line containing a stably transfected mouse mammary tumor virus-chloramphenicol acetyltransferase construct. Treatment with 8-bromo-cAMP (activator of cAMP-dependent protein kinases) or okadaic acid (protein phosphatase-1 and -2A inhibitor) did not stimulate target gene expression in the absence of progestin. When added together with progestin, either compound augmented PR-mediated target gene transcription by 3- to 4-fold. The cyclic nucleotide-dependent protein kinase inhibitor H8 completely blocked target gene responsiveness to hormone. Neither 8-bromo-cAMP, okadaic acid, nor H8 altered the hormone- or DNA-binding activities of PR, as measured in vitro or affected cellular concentrations of PR. These agents, therefore, appeared to selectively modulate PR transcriptional activity. Moreover, none of these compounds altered expression from a control reporter gene, pSV2CAT, indicating that these agents affect PR-mediated processes directly and are not acting through a general effect on transcription. Effects on PR phosphorylation were assessed by measuring 32P labeling of PR in vivo. None of these treatments had a substantial effect on the extent of total 32P labeling of immune isolated PR or on the phosphorylation(s) responsible for PR up-shifts on SDS-gels. This suggests that these agents modulate PR transcriptional activity either through phosphorylation of another protein intimately involved in PR-mediated transcription or through modification of a key site(s) not measurable as a change in total PR phosphorylation or electrophoretic mobility on SDS gels.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Promegestona/farmacologia , Receptores de Progesterona/metabolismo , Transcrição Gênica , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Neoplasias da Mama , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Eletroforese em Gel de Poliacrilamida , Éteres Cíclicos/farmacologia , Feminino , Humanos , Isoquinolinas/farmacologia , Vírus do Tumor Mamário do Camundongo/genética , Peso Molecular , Ácido Okadáico , Fosfatos/metabolismo , Radioisótopos de Fósforo , Fosforilação , Promegestona/metabolismo , Regiões Promotoras Genéticas , Proteína Quinase C/antagonistas & inibidores , Receptores de Progesterona/efeitos dos fármacos , Receptores de Progesterona/isolamento & purificação , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
The human progesterone receptor (PR) is a member of the steroid/thyroid hormone superfamily of nuclear receptors. The receptor is expressed as two forms, PR-B and the shorter PR-A, which lacks the NH2-terminal 164 amino acids of PR-B; whereas PR-B seems to be predominantly a transcriptional activator, PR-A also functions as a repressor. Our previous studies of PR expressed in T47D breast cancer cells have shown that PR is a phosphoprotein whose phosphorylation is enhanced in response to hormone. There is an initial rapid (minutes) increase in phosphorylation followed by a slower, less substantial increase, which results in decreased mobility of the receptor on sodium dodecyl sulfate gels. We now report the identification of three phosphorylation sites, which are predominantly phosphorylated during the later phase of the response to hormone. These sites, Ser102, Ser294, and Ser345, are all found in Ser-Pro consensus sequences. Whereas Ser294 and Ser345 are common to PR-A and PR-B, Ser102 is unique to PR-B. Finally, we demonstrate that phosphorylation of Ser345 is associated with the altered mobility on sodium dodecyl sulfate gels.
Assuntos
Receptores de Progesterona/química , Sequência de Aminoácidos , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Dados de Sequência Molecular , Fosfopeptídeos/química , Fosforilação , Fosfosserina/metabolismo , Congêneres da Progesterona/farmacologia , Promegestona/farmacologia , Receptores de Progesterona/metabolismoRESUMO
We have prepared a monoclonal antibody, C-262, to a synthetic peptide that contains the carboxy-terminal 14 amino acids from progesterone receptors (PR). This sequence is 100% conserved in all species of PRs that have been cloned to date, suggesting that this antibody will recognize all mammalian and avian PR. The C-262 antibody recognizes both native and denatured forms of the receptor. However, it does not recognize PR when they are bound to the hormone agonists progesterone or R5020. Surprisingly the antibody does recognize PR when they are bound to the steroid antagonist RU486. This suggests that progestin agonists induce a conformational change in the receptor that occludes the C-262 epitope in the carboxyl-terminus, whereas unliganded receptors and receptors bound with RU486 assume distinct conformations that leaves the C-terminal tail accessible to the C-262 antibody.
Assuntos
Anticorpos Monoclonais , Mifepristona/metabolismo , Oviductos/metabolismo , Conformação Proteica , Receptores de Progesterona/química , Receptores de Progesterona/metabolismo , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Sítios de Ligação , Western Blotting , Neoplasias da Mama , Galinhas , Citosol/metabolismo , Feminino , Humanos , Ligantes , Dados de Sequência Molecular , Peso Molecular , Peptídeos/síntese química , Peptídeos/imunologia , Receptores de Progesterona/imunologia , Células Tumorais CultivadasRESUMO
The human progesterone receptor (hPR) in T47D breast cancer cells is phosphorylated on at least nine different serine residues. We have previously reported the identification of five sites; three are hormone inducible (Ser102, Ser294 and Ser345), and their phosphorylation correlates with the timing of the change in receptor mobility on gel electrophoresis in response to hormone treatment. The other two sites, Ser81 and Ser162, along with the remaining sites, are basally phosphorylated and exhibit a general increase in phosphorylation in response to hormone. With the exception of Ser81, all of these sites are in Ser-Pro motifs, suggesting that proline-directed kinases are responsible for their phosphorylation. We now report that cyclin A-cyclin-dependent kinase-2 complexes phosphorylate hPR-B in vitro with a high stoichiometry on three sites that are authentic basal sites in vivo. One of these is Ser162, which has been described previously. The other two sites are identified here as Ser190 and Ser400. The specificity and stoichiometry of the in vitro phosphorylation suggest that hPR phosphorylation may be regulated in a cell cycle-dependent manner in vivo.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Progesterona/metabolismo , Serina , Sequência de Aminoácidos , Sítios de Ligação , Quinase 2 Dependente de Ciclina , Humanos , Dados de Sequência Molecular , Peptídeos/metabolismo , Fosforilação , Células Tumorais CultivadasRESUMO
Human progesterone receptors (PR) were overexpressed in Spodoptera frugiperda (Sf9) insect cells using a recombinant baculovirus system. Recombinant viruses were constructed that produced either full-length A (94K) or B (120K) forms of human PR, and each was expressed as a functional protein. Steroid and DNA binding activities were found to be indistinguishable from that of endogenous human PR in T47D breast cancer cells. Moreover, as analyzed by gel-mobility shift, recombinant PR-A and PR-B each bound to specific progesterone response elements in a strictly hormone-dependent manner. Native receptors expressed in Sf9 cells also exhibited structural properties similar to that of endogenous PR. Cytosolic PR (PR-A or PR-B), prepared in low salt buffer, sedimented on density gradients as an 8S oligomeric complex that was converted largely to 4S by treatment with 0.4 M NaCl. Immune isolation of the 8S cytosol PR complex and analysis of protein composition revealed the presence of two specific copurifying proteins of approximately 90K and 70K. The 90-K component was identified immunologically as heat shock protein 90. The 70-K component was not identified but is likely to be the insect equivalent of heat shock protein 70. Immune isolation of PR from Sf9 cells metabolically labeled with [32Pi], revealed that expressed PR was capable of being phosphorylated in insect cells. Hormone addition to Sf9 cells, however, did not stimulate the same increase in PR phosphorylation or upshift in mobility on sodium dodecyl sulfate gels that occurs with endogenous receptors in T47D cells. Thus some, but not all, phosphorylations occur with human PR expressed in Sf9 cells. These phosphorylation data, together with the fact that expressed PR required hormone for DNA binding, indicate that the hormone-dependent phosphorylation step responsible for PR upshifts on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is not required for receptor binding to DNA. The baculovirus expression system, therefore, may prove valuable in dissecting the functional role(s) for both hormone-dependent and hormone-independent PR phosphorylation.
Assuntos
Baculoviridae/genética , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Receptores de Progesterona/metabolismo , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , Cromatografia de Afinidade , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Vetores Genéticos , Humanos , Cinética , Dados de Sequência Molecular , Peso Molecular , Mariposas , Oligodesoxirribonucleotídeos , Fosforilação , Plasmídeos , Promegestona/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Frações Subcelulares/metabolismo , Especificidade por Substrato , TransfecçãoRESUMO
Com o objetivo de promover, por meio de acesso único e com o uso de endoscópio flexível, ampla exploração da cavidade peritoneal de equinos em estação, foi concebida uma cânula laparoscópica para dar sustentação ao endoscópio e possibilitar o acesso sob visualização. O procedimento foi realizado a partir da fossa paralombar. Após pequena incisão cutânea, o endoscópio foi inserido na cânula e os músculos e o peritônio foram divulsionados mediante rotação da cânula. Logo depois da perfuração do peritônio, foi realizada a exploração da cavidade e a identificação das estruturas. Em seguida à exploração do lado ipsilateral ao acesso, realizou-se a transposição do conjunto cânula/endoscópio ventralmente à porção caudal do cólon descendente, seguida de exploração do lado contralateral. Concluída a técnica, foi executado, para fins de comparação, o mesmo procedimento por meio da fossa paralombar contralateral. Foi possível a transposição do conjunto cânula/endoscópio para o lado contralateral ao acesso em todos os procedimentos. Também foi possível a identificação da maioria das estruturas abdominais tanto pelo acesso esquerdo quanto pelo direito. A abordagem por acesso único mostrou-se viável para a exploração ampla da cavidade peritoneal, demonstrando ser uma alternativa à técnica laparoscópica convencional.(AU)
A laparoscopic cannula was designed to support a single access approach with a flexible endoscope for the wide exploration of the peritoneal cavity of standing horses. It provides support to the endoscope and allows access to the peritoneal cavity with a visual aid. This procedure was performed through the paralumbar fossa. After a small cutaneous incision, the endoscope was inserted into the cannula, and the muscles and peritoneum were divulsed through the rotation of the cannula. After the peritoneal perforation, cavity exploration and identification of structures were performed. After the exploration of the ipsilateral side of the access, the cannula/endoscope was transposed ventrally to the caudal portion of the descending colon; this was followed by the exploration of the contralateral side. Once this process was completed, the same procedure was performed through the contralateral paralumbar fossa for comparison. It was possible to transpose the cannula/endoscope set to the contralateral access side in all procedures. Further, it was possible to identify most of the abdominal structures in both the left and right access. This single access approach proved to be feasible for the extensive exploration of the peritoneal cavity, thereby indicating it can be an alternative to the conventional laparoscopic technique.(AU)
Assuntos
Animais , Peritônio/diagnóstico por imagem , Endoscópios/veterinária , Cirurgia Vídeoassistida/veterinária , Cânula , Cavalos , Procedimentos Cirúrgicos Minimamente Invasivos/métodosRESUMO
Progesterone (P4) can alter the synthesis and secretion of FSH from pituitary gonadotropes of sheep. In this study, the 5'-flanking region (4.7 kilobases) of the ovine FSH beta gene was tested for binding by human progesterone receptors (hPR), using an immunoprecipitation technique. Three fragments were bound by hPR. Competition experiments using homologous and heterologous DNA fragments revealed this binding to be specific and of high affinity (Kd = 1.2-47 nM). The fragment sequences were screened for potential P4 response elements (PREs). Six PRE-like elements were found among the three immunoprecipitated fragments. Band shift experiments discerned that each of these PRE-like sequences could be bound by hPR. In functional studies, each of the PRE-like elements could enhance the expression of a reporter gene driven by a heterologous promoter in a hormone-dependent manner. The 5'-flanking region of the ovine FSH beta gene was tested for P4 responsiveness using a luciferase reporter. In the presence of P4, there was a 2- to 3-fold increase in luciferase activity when the entire 4.7 kilobases of the 5'-flanking sequence were present, whereas no increase was seen in a construct that contained only 84 basepairs 5' to the transcription start site. This effect on transcription was dose dependent for P4. Deletion studies revealed that the three PRE-like elements closest to the transcription start site (-250 to -137) were sufficient to create the hormone-dependent enhancement. These results indicate that the 5'-flanking sequence of the ovine FSH beta gene contains sequences capable of being bound by hPR and may be responsible for the effects of P4 on FSH beta synthesis and secretion. This study is the first to show binding and function of PR for a gonadotropin gene.
Assuntos
Hormônio Foliculoestimulante/genética , Genes , Progesterona/farmacologia , Transcrição Gênica , Animais , Quimera , Feminino , Genes Reguladores , Humanos , Luciferases/genética , Luciferases/metabolismo , Testes de Precipitina , Progesterona/genética , Receptores de Progesterona/metabolismo , OvinosRESUMO
To determine whether the steroid antagonist RU486 mediates its antiglucocorticoid and antiprogestin activities by the same or different receptor mechanisms, a direct comparison of RU486 interaction with glucocorticoid (GR) and progesterone (PR) receptors was made. The effects of RU486 on transformation of GR and PR 8-10S complexes in the intact cell and in vitro were analyzed by sucrose density gradient centrifugation, and the in vitro stability of receptor-heat shock protein-90 interactions was analyzed by coimmunoprecipitation. Compared to agonist, RU486 binding produced a reduction in the amount of GR converted from 8S to 4S and stabilized the GR-heat shock protein-90 complex. By contrast, PR-RU486 complexes were transformed both in vitro and in the intact cell to the same extent as receptor-agonist complexes. PR-RU486 complexes sedimented at 5-6S, whereas PR-R5020, GR-RU486, and GR-agonist complexes sedimented at 4S. The portion of GR that undergoes nuclear transformation when bound to RU486 was examined for binding to the glucocorticoid-progesterone response element of the mouse mammary tumor virus by an immunoprecipitation assay. The nuclear-transformed GR-RU486 complex bound the glucocorticoid-progesterone response element with the same affinity as the nuclear-transformed GR-triamcinolone acetonide complex. The electrophoretic mobilities of GR-RU486 complexes and GR-agonist complexes were the same, as determined by gel retardation assay. These results suggest that RU486 exerts its antiglucocorticoid activity at two levels of receptor action: prevention of complete GR transformation and alteration of a step subsequent to GR-DNA binding. As an antiprogestin, RU486 action is exerted predominantly at a post-DNA-binding step.
Assuntos
Mifepristona/farmacologia , Receptores de Glucocorticoides/efeitos dos fármacos , Receptores de Progesterona/efeitos dos fármacos , Animais , Sequência de Bases , Western Blotting , Neoplasias da Mama/metabolismo , Núcleo Celular/metabolismo , Centrifugação com Gradiente de Concentração , Citosol/metabolismo , DNA/metabolismo , Proteínas de Choque Térmico/metabolismo , Hormônios/farmacologia , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Mifepristona/metabolismo , Dados de Sequência Molecular , Concentração Osmolar , Ratos , Receptores de Glucocorticoides/metabolismo , Receptores de Progesterona/metabolismo , Triancinolona Acetonida/metabolismo , Células Tumorais CultivadasRESUMO
Replicative DNA synthesis, as measured by thymidine incorporation, has been measured in rat uterine cells in primary culture in response to growth factors. Insulin, insulin-like growth factor-I (IGF-I), multiplication-stimulating activity (MSA) and platelet-derived growth factor (PDGF) stimulated DNA synthesis, while estradiol, epidermal growth factor (EGF), transforming growth factor-beta (TGF-beta), basic fibroblast growth factor (bFGF) and relaxin did not stimulate or did so weakly and only at very high concentrations. Uterine acid extracts also stimulated DNA synthesis. IGF-I stimulated at concentrations consistent with its acting through the IGF-I receptor; however, insulin stimulated at concentrations higher than expected for its acting through its receptor and this its action may be mediated through the IGF-I receptor. IGF-I was found in uterine tissue by radioimmunoassay (RIA). There was a 5- to 10-fold increase in IGF-I in the uteri from ovariectomized rats that had been treated with estradiol 24 h earlier. This is analogous to the increase in growth factor activity found previously in rat uterus after 24-h estradiol treatment (Beck, C.A. and Garner, C.W. (1989) Mol. Cell. Endocrinol. 63, 93-101). These data are consistent with the hypothesis that estradiol effects in the uterus are in part mediated through IGF-I.
Assuntos
Replicação do DNA/efeitos dos fármacos , Substâncias de Crescimento/farmacologia , Útero/metabolismo , Animais , Extratos Celulares/farmacologia , Relação Dose-Resposta a Droga , Estradiol/farmacologia , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Técnicas In Vitro , Insulina/farmacologia , Fator de Crescimento Insulin-Like I/biossíntese , Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Insulin-Like II/farmacologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Radioimunoensaio , Ratos , Ratos Endogâmicos , Receptores de Estrogênio/análise , Relaxina/farmacologia , Timidina/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Acid-stable uterine-derived growth factor activity, extracted from uteri of several species (rat, rabbit and bovine), stimulates DNA synthesis as measured by [3H]thymidine incorporation into hamster uterine carcinosarcoma (UCS) cells. A time course of [3H]thymidine incorporation demonstrates maximum incorporation at 24 h. These extracts also stimulate [3H]thymidine incorporation in a variety of other cell types from 17 beta-estradiol (E2) target tissues and non-target tissues. Uterine extracts from E2-treated ovariectomized rats show a 3-fold increase in growth factor activity above control values. Activity is elevated within 18-24 h after estradiol injection and remains elevated wtih subsequent injections. Growth factor activity is acid-stable, heat-labile, reduced by trypsin but not reduced by treatment with dextran-coated charcoal. Gel filtration shows molecular weight (Mr) heterogeneity with activity eluting at Mr of 10,000-30,000. Since uterine extracts can restore in vitro the estrogen-regulated properties of uterine growth observed previously in vivo, it is possible that the substances found in these extracts may be mediators of E2 actions.
Assuntos
Estrogênios/fisiologia , Substâncias de Crescimento/metabolismo , Útero/metabolismo , Animais , Carcinossarcoma/metabolismo , Carcinossarcoma/patologia , Bovinos , Cromatografia em Gel , Feminino , Especificidade de Órgãos , Coelhos , Timidina/metabolismo , Extratos de Tecidos/análise , Células Tumorais Cultivadas , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologia , Útero/análiseRESUMO
Estradiol-17 beta was previously shown to stimulate glucose transport (as measured by phosphorylation of 2-deoxyglucose) in rat uterine tissue in vivo (Meier, D.A. and Garner, C.W. (1987) Endocrinology 121, 1366-1374) but attempts to demonstrate this in uterine organ strips in vitro, in uterine tumor cell lines or in uterine cells in primary culture have been unsuccessful. However, aqueous uterine extracts and uterine luminal fluid did stimulate glucose transport in uterine tumor cells and uterine cells in primary culture. Estradiol in vivo and uterine extracts in vitro each increased the initial rate of glucose transport 1.5- to 3-fold. In each case, 2-3 h were required for the stimulation to be fully expressed. The stimulation was not inhibited by cycloheximide suggesting that protein synthesis was not required. Uteri from ovariectomized rats injected daily for 4 days with 10 micrograms estradiol contained 4-fold more activity than uteri from saline-injected control animals. The activity was acid- and heat-stable, inactivated by trypsin treatment but not removed by dextran-coated charcoal treatment, suggesting that the activity is (or is associated with) a protein. The activity eluted in the 6-12 kDa range upon chromatography on Sephadex G-50. Insulin (1-1000 ng/ml) and epidermal growth factor (1-100 ng/ml) stimulated glucose transport, but only less than 50% of the stimulation by extracts. The substance(s) present in the extracts, possibly a known growth factor, may be involved in the estradiol stimulation of glucose transport and other estradiol actions in vivo.
Assuntos
Glucose/metabolismo , Extratos de Tecidos/farmacologia , Útero/citologia , Animais , Transporte Biológico , Proteínas de Transporte/análise , Células Cultivadas , Cicloeximida/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Estradiol/fisiologia , Feminino , Glucose/fisiologia , Insulina/farmacologia , Coelhos , Ratos , Ratos Endogâmicos , Útero/efeitos dos fármacosRESUMO
The narcotic antagonists naloxone and naltrexone were used as respiratory stimulants in two patients failing traditional medical therapy for COPD. Both patients demonstrated improvement while receiving the drugs, but developed respiratory failure when they were discontinued abruptly. In selected patients with COPD, narcotic antagonists may offer an additional mode of therapy.
Assuntos
Pneumopatias Obstrutivas/tratamento farmacológico , Naloxona/uso terapêutico , Naltrexona/uso terapêutico , Idoso , Dióxido de Carbono/sangue , Feminino , Humanos , Pneumopatias Obstrutivas/sangue , Masculino , Pessoa de Meia-Idade , Oxigênio/sangueRESUMO
Currently available progesterone antagonists have been suggested to fall into two categories based on differences in how they interact with and inactivate the progesterone receptor (PR). The anti-progestin ZK98299 (Type I) impairs PR association with DNA, while Type II compounds (RU486, ZK112993, ZK98734) promote PR binding to DNA. Type II agents, therefore, appear to inhibit receptor activity at a step downstream of DNA binding, presumably failing to induce conformational changes in PR structure requird for enhancement of transcription. This paper discusses both published and unpublished data supporting the concept of two types of progestin antagonists. Using PR-mediated induction of reporter genes in breast cancer cells as an assay for biological response, both types of anti-progestins, after correction for difference in steroid binding affinity, inhibit progestin induction substoichiometrically. However, Type II anti-progestins are more potent, inhibiting at lower ratios of antagonist to agonist than ZK98299. This suggests that in addition to behaving by classical competitive mechanisms these compounds (in particular Type II) may exhibit additional activity as transrepressors of PR in the same cell bound to hormone agonist. Transrepression may occur by the combined mechanisms of heterodimerization and competition for binding to DNA. In support of this, mixed ligand dimers form readily in solution between a PR subunit bound to agonist and another bound to either type of anti-progestin, whereas these mixed ligand dimers bind poorly, if at all, to specific progesterone response elements (PREs) in vitro. Additionally, when added as a single ligand, Type II agents increase PR dimerization in solution and PR affinity for PREs as compared with single ligand dimers formed by progestin agonist. This contrasts with ZK98299, when given as a single ligand, which reduces PR affinity for PREs without disrupting solution dimerization. Thus the higher affinity of PR for PREs may account for the greater biological potency of Type II compounds as compared with ZK98299. As a further distinction between types of antiprogestins, ZK98299 minimally stimulates phosphorylation of PR whereas RU486 increases site-specific phosphorylation of PR in a manner indistinguishable from that of hormone agonist. Additionally, ZK98299 is not susceptible in vivo to functional switching to a partial agonist by cross talk with cAMP signal transduction pathways, as occurs with Type II compounds. Thus, ZK98299 under certain conditions may be a more pure antagonist than Type II compounds.
Assuntos
Gonanos/química , Progesterona/antagonistas & inibidores , Receptores de Progesterona/antagonistas & inibidores , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Estrenos/química , Humanos , Técnicas In Vitro , Mifepristona/análogos & derivados , Mifepristona/química , Progesterona/química , Promegestona/química , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Transcrição Gênica/efeitos dos fármacosRESUMO
Four phosphorylation sites have been identified in the chicken progesterone receptor. Two of these sites exhibit basal phosphorylation which is enhanced upon treatment with hormone and two of the sites are phosphorylated in response to hormone. Mutation of one of these hormone dependent sites, Ser530 to Ala530, causes a decrease in transcriptional activation at low concentrations of hormone, but the activity is unaffected at high concentrations. However, the hormone binding of the mutant is unaffected suggesting that phosphorylation of Ser530 plays a role in facilitating the response of the receptor to low concentrations of hormone. The chicken progesterone receptor can be activated by modulators of kinases in the absence of hormone. The finding that signals initiated by tyrosine phosphorylation (through treatment with EGF) or through the dopamine receptor suggests that there are multiple means of activating chicken progesterone receptor. In contrast, the human progesterone receptor does not exhibit ligand independent activation; however, its activity in the presence of the agonist R5020 is enhanced by treatment with 8-Br-cAMP, an activator of protein kinase A, and treatment with 8-Br-cAMP causes the antagonist, RU486, to act as an agonist.
Assuntos
Receptores de Progesterona/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Ligantes , Dados de Sequência Molecular , Fosforilação , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Mammalian progesterone receptors activated by hormone binding in nuclei of intact cells exhibit substantially higher binding activity for specific DNA sequences than receptors bound with hormone and activated in cell-free cytosol. Differences in DNA-binding activity occur despite the fact that both activated receptor forms sediment at 4S on sucrose gradients and are apparently dissociated from the heat shock protein 90. This suggests that hormone-induced release of heat shock protein 90 from receptors is necessary, but not sufficient for maximal activation of DNA binding. This report is a review of studies from our laboratories that have examined the role of receptor interaction with other nuclear protein factor(s), and receptor dimerization in solution, as additional regulatory steps involved in the process of receptor activation and binding to specific gene sequences.
Assuntos
DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Receptores de Progesterona/metabolismo , Sequência de Bases , Western Blotting , Eletroforese em Gel de Poliacrilamida , Hormônios/metabolismo , Humanos , Vírus do Tumor Mamário do Camundongo/genética , Dados de Sequência Molecular , Proteínas Nucleares/fisiologia , Receptores de Progesterona/isolamento & purificação , Sequências Reguladoras de Ácido Nucleico , Células Tumorais CultivadasRESUMO
Activation of protein kinase A potentiates the transcriptional response mediated by the glucocorticoid receptor in responsive fibroblasts and in mammary carcinoma cells. This potentiation is ligand-dependent and occurs in responsive fibroblasts and in mammary carcinoma cells. This potentiation is ligand-dependent and occurs without detectable change in the phosphorylation of receptor. The transcriptional response to glucocorticoid or progestin agonists can be blocked by potent antagonists like RU 486. However, upon activation of protein kinase A, the antagonist action of RU 486 on both receptors is blunted. Indeed, RU 486 can itself activate transcription of a hormone-responsive promoter. The conditional agonist activity is observed with type II antagonists, those which recapitulate many of the early steps of ligand-dependent receptor activation, but not type I antagonists, which do not. These studies have now been extended to antimineralocorticoids. In COS-1 cells transfected with a mineralocorticoid receptor expression vector, treatment with 8-BromocAMP potentiates the response to the agonist aldosterone and elicits additional agonist activity in mineralocorticoid antagonists. A model is proposed wherein type II antagonist-receptor complexes occupy receptor binding sites on the genome. The antagonist, however, fails to promote a receptor conformation that can interact productively with a coactivator mediating the communication between receptor and the basal transcription apparatus. Activation of protein kinase A results in the recruitment or activation of a coactivator that permits recovery of receptor-mediated activation function.(ABSTRACT TRUNCATED AT 250 WORDS)