Molecular and cellular outcomes of quercetin actions on healthy and tumor osteoblasts.

There is a global trend in the use of natural bioactive compounds to complement conventional therapies in bone diseases. In this work, we studied the effects of the phytoestrogen quercetin (QUE) in healthy and tumor osteoblasts. We found that QUE (1 µM, 48 h) significantly increased the cell number and the viability of healthy human osteoblasts (hFOB cells) determined by a trypan blue and a MTS assay, respectively, among other concentrations tested. In addition, wound healing and cellular adhesion assays also demonstrated that 1 µM of QUE significantly stimulated both parameters in osteoblasts. Moreover, osteoblast differentiation was also triggered by QUE in an osteogenic medium by measuring alkaline phosphatase activity, calcium deposition, and collagen levels. Herein, a concentration of 0.01 µM of QUE showed an increment in these differentiation markers and an activation of AKT/GSK3ß/ß-catenin pathway, determined by a Western blot analysis. In addition, immunocytochemistry and subcellular fraction studies indicated an increase of ß-catenin localization in the plasma membrane after QUE treatment. Otherwise, QUE (20-100 µM) decreased the cell number and the viability in tumor osteoblasts (ROS 17/2.8 cells) after 48 h. Furthermore, QUE (100 µM) decreased AKT(Ser473) and the pro-apoptotic protein BAD(Ser136) phosphorylation. In addition, the ERK1/2 phosphorylation increased leading to osteosarcoma cell death since pre-treatment with the MEK inhibitor PD98059 had reverted QUE effect. Altogether, these results indicate that low concentrations of QUE stimulate osteoblastogenesis but have no effect on the growth of tumor osteoblast cells, for which only high concentrations are efficient.

Perfil de puérperas assistidas em alojamento conjunto: estudo comparativo entre os serviços público e privado / Profile of postpartum women receiving rooming-in care: a comparative study between public and private services

Introdução: Conhecer as características das puérperas é fundamental para a implantação de estratégias de melhoria dos serviços de saúde. Buscou-se analisar e comparar as características epidemiológicas de puérperas em alojamento conjunto, segundo o local de realização do parto. Métodos: Duzentas puérperas que se encontravam no alojamento conjunto de dois hospitais foram submetidas a questionário estruturado. A amostra foi distribuída igualitariamente entre os dois hospitais, sendo um de caráter particular, e outro de caráter público. Resultados: A análise mostrou, dentre aquelas atendidas no serviço público, menor faixa etária, maior proporção de parto vaginal, maior taxa de tabagismo e menor consumo de álcool durante a gestação. Conclusão: Este estudo permitiu traçar um perfil das puérperas atendidas em alojamento conjunto, possibilitando a construção de novos programas e serviços de saúde em consonância com as características desta população(AU)

Introduction: Knowing the characteristics of mothers is crucial for implementing improvement strategies of health services. Here the authors sought to analyze and compare the epidemiological characteristics of mothers in rooming-in care according to the location of the delivery. Methods: Two hundred mothers who were in rooming-in care at two different hospitals answered a structured questionnaire. The sample was distributed equally between the two hospitals, one being private and the other public. Results: The analysis showed younger age, higher proportion of vaginal delivery, higher rate of smoking, and lower alcohol consumption during pregnancy among mothers receiving public care. Conclusion: This study allowed us to outline a profile of mothers in rooming-in care, allowing the construction of new programs and health services in line with the characteristics of this population(AU)

Avaliação dos níveis séricos de vitamina D em crianças e adolescentes com diabetes mellitus tipo I / Evaluation of vitamin D serum levels in children and adolescents with diabetes mellitus type 1

Introdução: A deficiência de vitamina D é considerada um problema de saúde pública no mundo todo em razão de suas implicações no desenvolvimento de diversas doenças, incluindo a Diabetes Mellitus tipo I (DMI). Os objetivos deste estudo são avaliar os níveis séricos de 25 OH Vitamina D, Cálcio, Fosfatase alcalina, fósforo e hemoglobina glicada em crianças diabéticas tipo I e correlacionar os níveis de vitamina D com a hemoglobina glicada e com a presença de Tireoidite de Hashimoto, microalbuminúria e Doença celíaca. Métodos: Estudo do tipo quantitativo, observacional transversal obtido pela analise de exames laboratoriais de 48 pacientes, maiores de 3 anos, com diagnóstico de Diabetes Mellitus tipo I. Os dados foram obtidos mediante análise dos prontuários. Resultados: Dezenove pacientes (59,4%) apresentaram vitamina D deficiente/insuficiente. A média dos níveis de cálcio, fósforo, fosfatase alcalina e HbA1c foram de 9,24, 281,17, 4,69 e 9,34, respectivamente. Em 31 pacientes (91,17%) o controle da DMI foi considerado ruim ou regular, sendo este avaliado pelo valor da hemoglobina glicosilada. Dez pacientes (29,41%) apresentaram uma ou mais das comorbidades associadas à DMI. Conclusão: Em 59,4 % dos pacientes o nível de vitamina D foi deficiente ou insuficiente. A média dos níveis de cálcio, fósforo e fosfatase alcalina dos pacientes estavam dentro dos valores normais. Em 31 pacientes (91,17%) o controle da DMI, avaliado pela HbA1c, foi considerado ruim ou regular. Não houve associação significativa entre a presença de Tireoidite de Hashimoto, doença celíaca ou microalbuminúria e os níveis de vitamina D.

Introdução: Vitamin D deficiency is considered a public health problem worldwide because of its implications in the development of various diseases, including diabetes mellitus type I (DMI). The aims of this study are to evaluate the serum levels of 25 OH Vitamin D, calcium, alkaline phosphatase, phosphorus and glycated hemoglobin in diabetic children type I and correlate vitamin D levels with glycated hemoglobin and presence of Hashimoto's thyroiditis, microalbuminuria and celiac disease. Methods: A quantitative, observational cross-sectional study performed by analysis of laboratory tests from 48 patients over three years of age and diagnosed with diabetes mellitus type I. The data were obtained by analyzing medical charts. Results: Nineteen patients (59.4%) were vitamin D deficient/insufficient. Calcium, phosphorus, alkaline phosphatase and HbA1c mean levels were 9.24, 281.17, 4.69, and 9.34, respectively. In 31 patients (91.17%), control of DMI was considered poor or fair, as measured by the value of glycosylated hemoglobin. Ten patients (29.41%) had one or more comorbidities associated with DMI. Conclusion: In 59.4% of the patients vitamin D level was deficient or insufficient. Patients’ mean levels of calcium, phosphorus and alkaline phosphatase were within the normal range. In 31 patients (91.17%) the control of DMI, assessed by HbA1c, was considered poor or fair. There was no significant association between presence of Hashimoto's thyroiditis, celiac disease or microalbuminuria and vitamin D levels.

Protein phosphatases: possible bisphosphonate binding sites mediating stimulation of osteoblast proliferation.

We investigated the existence of a bisphosphonate (BP) target site in osteoblasts. Binding assays using [³H]-olpadronate ([³H]OPD) in whole cells showed the presence of specific, saturable and high affinity binding for OPD (K(d)=1.39 ± 0.33 µM) in osteoblasts. [³H]OPD was displaced from its binding site by micromolar concentrations of lidadronate, alendronate and etidronate (K(d)=1.42 ± 0.15 µM, 2.00 ± 0.2 µM and 2.4 ± 0.4 µM, respectively), and by millimolar concentrations of the non-permeant protein phosphatase (PP) substrates p-nitrophenylphosphate and α-naphtylphosphate. PP inhibitors orthovanadate, NaF or vpb(bipy) did not displace [³H]OPD. As expected, specific OPD binding was detected in the plasma membrane of ROS 17/2.8 cells, although significant BP binding was also found intracellularly. Moreover, OPD increased DNA synthesis in these cells with a temporal profile similar to the protein tyrosine phosphatase (PTP) inhibitors, Na3VO4 and vpb(bipy); but different from a general PP inhibitor (NaF). The stimulatory effect of OPD and PTP inhibitors on osteoblast proliferation was inhibited by the protein tyrosine kinase inhibitors genistein and geldanamycin. These results provide new evidence on the existence of a BP target in osteoblastic cells, presumably a PTP, which may be involved in the stimulatory action of BPs on osteoblast proliferation.

O câncer entrou em meu lar: sentimentos expressos por familiares de clientes / Cancer at home: parents and relatives make a clean breast of their feelings

O objetivo deste estudo foi desvelar as concepções dos familiares ao serem notificados de que um ente querido está com câncer. Utilizamos a abordagem qualitativa fundamentada na fenomenologia existencial, pois busca compreender o ser humano em sua facticidade. Foram realizadas entrevistas com 10 familiares de clientes em seus domicílios, no período de janeiro de 2006 a janeiro de 2007, em uma cidade situada no noroeste do Estado do Paraná. Da linguagem dos familiares, emergiram três categorias: sentimentos avivados nos familiares ante a descoberta do diagnóstico; compartilhar com o ente querido o tratamento; e a importância da espiritualidade para o entendimento da situação. Através do estudo, apreendemos que os sentimentos vivenciados pelos familiares, na confirmação do diagnóstico de neoplasia, expressam angústia, tristeza, incerteza e até mesmo esperança e o desejo de estar com o ente querido.

Parents’ and relatives’ ideas on being notified that one of their members has cancer are provided. A qualitative approach based on existential phenomenology has been used since it is a method for understanding people in their daily experience. Residential interviews with ten relatives were undertaken between January 2006 and January 2007, all of which in a northwestern town in the state of Paraná. Relatives’ discourse may be subdivided into three categories: relatives’ feelings upon acknowledgement of diagnosis; the partaking of feelings with the beloved one during treatment; the importance of spirituality to make sense of the situation. Results showed that feelings experienced by relatives when the diagnosis was confirmed demonstrated concern, sadness, uncertainty, and even hope. The desire to be close to the ill relative was also very strong.

El objetivo de este estudio fue desvelar las concepciones de los familiares al ser notificados de que un ente querido está con cáncer. Utilizamos un enfoque cualitativo fundamentado en la fenomenología existencial, pues ella busca comprender el ser humano en su facticidad. Fueron realizadas entrevistas con 10 familiares de clientes en sus domicilios, en el período de enero de 2006 a enero de 2007; en una ciudad situada en el noroeste del Estado de Paraná - Brasil. Del lenguaje de los familiares, emergieron tres categorías: sentimientos avivados en los familiares ante la descubierta del diagnóstico; compartiendo con el ente querido el tratamiento y la importancia de la espiritualidad para el entendimiento de la situación. Por medio del estudio, aprehendemos que los sentimientos vivenciados por los familiares, en la confirmación del diagnóstico de neoplasia, expresan angustia, tristeza, incertidumbre e incluso esperanza y el deseo de estar con el ente querido.

PTH and phospholipase A2 in the aging process of intestinal cells.

In this study we analyzed, for the first time, alterations in phospholipase A2 (PLA2) activity and response to parathyroid hormone (PTH) in rat enterocytes with aging. We found that PTH, rapidly stimulate arachidonic acid (AA) release in rat duodenal cells (+1- to 2-fold), an effect that is greatly potentiated by aging (+4-fold). We also found that hormone-induced AA release in young animals is Ca2+-dependent via cPLA2, while AA released by PTH in cells from aged rats is due to the activation of cPLA2 and the Ca2+-independent PLA2 (iPLA2). In enterocytes from 3 months old rats, PTH induced, in a time and dose-dependent fashion, the phosphorylation of cPLA2 on serine 505, with a maximum at 10 min (+7-fold). Basal levels of cPLA2 serine-phosphorylation were higher in old enterocytes, affecting the hormone response which was greatly diminished (+2-fold at 10 min). cPLA2 phosphorylation impairment in old animals was not related to changes of cPLA2 protein expression and did not explain the substantial increase on PTH-induced AA release with aging, further suggesting the involvement of a different PLA2 isoform. Intracellular Ca2+ chelation (BAPTA-AM, 5 microM) suppressed the serine phosphorylation of cPLA2 in both, young and aged rats, demonstrating that intracellular Ca2+ is required for full activation of cPLA2 in enterocytes stimulated with PTH. Hormone effect on cPLA2 was suppressed to a great extent by the MAP kinases ERK 1 and ERK2 inhibitor, PD 98059 (20 microM), the cAMP antagonist, Rp-cAMP, and the PKC inhibitor Ro31820 both, in young and aged animals. Enterocytes exposure to PTH also resulted in phospho-cPLA2 translocation from cytosol to nuclei and membrane fractions, where phospholipase substrates reside. Hormone-induced enzyme translocation is also modified by aging where, in contrast to young animals, part of phospho-cPLA2 remained cytosolic. Collectively, these data suggest that PTH activates in duodenal cells, a Ca2+-dependent cytosolic PLA2 and attendant AA release and that this activation requires prior stimulation of intracellular ERK1/2, PKA, and PKC. cPLA2 is the major enzyme responsible for AA release in young enterocytes while cPLA2 and the Ca2+-independent iPLA2, potentiate PTH-induced AA release in aged cells. Impairment of PTH activation of PLA2 isoforms upon aging may result in abnormal hormone regulation of membrane fluidity and permeability and thereby affecting intestinal cell membrane function.

1alpha,25(OH)2D3 and parathyroid hormone (PTH) signaling in rat intestinal cells: activation of cytosolic PLA2.

In the current study, we have probed the role of cytosolic phospholipase A2 (cPLA2) activity in the cellular response to the calciotropic hormones, 1alpha,25,dihydroxy-vitamin D(3) [1alpha,25(OH)(2)D(3)] and PTH. Stimulation of rat enterocytes with either hormone, increased release of arachidonic acid (AA) 3H-AA] one-two fold in a concentration and time-dependent manner. The effect of either hormone on enterocytes was totally reduced by preincubation with the intracellular Ca(2+) chelator BAPTA-AM (5 microM), suggesting that the release of AA following cell exposure to the calciotropic hormones occurs mainly through a Ca(2+)-dependent mechanism involving activation of Ca(2+)-dependent cPLA2. Calciotropic homone stimulation of rat intestinal cells increases cPLA2 phosphorylation (three to four fold). This effect was decreased by PD 98059 (20 microM), a MAP kinase inhibitor, indicating that this action is, in part, mediated through activation of the MAP kinases ERK 1 and ERK2. Enterocytes exposure to 1alpha,25(OH)(2)D(3) (1nM) or PTH (10 nM) also resulted in P-cPLA2 translocation from cytosol to nuclei and membrane fractions, where phospholipase subtrates reside. Collectively, these data suggest that PTH and 1alpha,25(OH)(2)D(3) activate in duodenal cells, a Ca(2+)-dependent cytosolic PLA2 and attendant arachidonic acid release and that this activation requieres prior stimulation of intracellular ERK1/2. 1alpha,25(OH)(2)D(3) and PTH modulation of cPLA2 activity may change membrane fluidity and permeability and thereby affecting intestinal cell membrane function.

Characterization of PTH/PTHrP receptor in rat duodenum: effects of ageing.

In rat enterocytes, signaling through the parathyroid hormone (PTH)/PTH-related peptide receptor type 1(PTHR1) includes stimulation of adenylyl cyclase, increases of intracellular calcium, activation of phospholipase C, and the MAP kinase pathway, mechanisms that suffer alterations with ageing. The purpose of this study was to evaluate whether an alteration at the level of the PTH receptor (PTHR1) is the basis for impaired PTH signaling in aged rat enterocytes. Western Blot analysis with a specific monoclonal anti-PTHR1 antibody revealed that a 85 kDa PTH binding component, the size expected for the mature PTH/PTHrP receptor, localizes in the basolateral (BLM) and brush border (BBM) membranes of the enterocyte, being the protein expression about 7-fold higher in the BLM. Two other bands of 105 kDa (corresponding to highly glycosylated, incompletely processed receptor form) and 65 kDa (proteolytic fragment) were also seen. BLM PTHR1 protein expression significantly decreases with ageing, while no substantial decrease was observed in the BBM from old rats. PTHR1 immunoreactivity was also present in the nucleus where PTHR1 protein levels were similar in enterocytes from young and aged rats. Immunohistochemical analysis of rat duodenal sections showed localization of PTHR1 in epithelial cells all along the villus with intense staining of BBM, BLM, and cytoplasm. The nuclei of these cells were reactive to the PTHR1 antiserum, but not all cells showed the same nuclear staining. The receptor was also detected in the mucosae lamina propria cells, but was absent in globets cells from epithelia. In aged rats, PTHR1 immunoreactivity was diffused in both membranes and cytoplasm and again, PTH receptor expression was lower than in young animals, while the cell nuclei showed a similar staining pattern than in young rats. Ligand binding to PTHR1 was performed in purified BLM. rPTH(1-34) displaced [I(125)]PTH(1-34) binding to PTHR1 in a concentration-dependent fashion. In both, aged (24 months) and young (3 months) rats, binding of [I(125)]PTH was characterized by a single class of high-affinity binding sites. The affinity of the receptor for PTH was not affected by age. The maximum number of specific PTHR1 binding sites was decreased by 30% in old animals. The results of this study suggest that age-related declines in PTH regulation of signal transduction pathways in rat enterocytes may be due, in part, to the loss of hormone receptors.

Effect of ageing in the early biochemical signals elicited by PTH in intestinal cells.

In previous work, we have demonstrated that rPTH(1-34) increases cytoplasmic calcium concentration ([Ca(2+)](i)) in isolated rat enterocytes. In the present study, we have identified the sources of PTH-mediated increase in [Ca(2+)](I) and the implication of Ca(2+) on hormone early signals in enterocytes isolated from young (3-month-old) and aged (24-month-old) rats. In young enterocytes, PTH raised [Ca(2+)](i) in a dose-dependent manner (1 pM-100 nM). In cells from aged rats, hormone concentrations higher than physiological (>/=1 nM) were required to observe significant increases in [Ca(2+)](i). Phospholipase C (PLC) inhibitors blocked the initial acute elevation of the [Ca(2+)](i) biphasic response to PTH of young enterocytes while in old cells, no effects were observed. The voltage-dependent calcium-channel blocker (VDCC), nitrendipine, suppressed PTH-dependent changes of the sustained [Ca(2+)](i) phase in young and aged animals. In this study, we analysed, for the first time, alterations in phosphatidylinositol 3-kinase (PI3K) activity and response to PTH in rat enterocytes with ageing. Basal PI3K activity was significantly modified by ageing. Acute treatment with 10(-8) M PTH increased enzyme activity, with a maximun at 2 min (+3-fold) in young rats and only elevated by less than 1-fold basal PI3K activity in aged animals. Hormone-induced tyrosine phosphorylation of p85alpha, the regulatory subunit of PI3K, as well as the phosphorylation on Thr(308) of its downstream effector Akt/PKB was evident in enterocytes from 3-month-old rats, whereas it was greatly reduced in the cells from 24-month-old animals. Intracellular Ca(2+) chelation (BAPTA-AM, 5 microM) affected the tyrosine phosphorylation of p85alpha and inhibited PTH-dependent PI3K activation by 75% in young rats and completely abolished the enzyme activity in aged animals, demonstrating that Ca(2+) is required for full activation of PI3K in enterocytes stimulated with PTH. The Thr phosphorylation of PI3K downeffector, Akt/PKB, was also fully dependent on Ca(2+). Taken together, these results suggest that PTH regulation of enterocyte [Ca(2+)](i) involves Ca(2+) mobilization from IP(3)-sensitive stores and the influx of the cation from the extracellular milieu, the former pathway being blunted during ageing. The data also indicates a positive role for intracellular calcium in one of the early signals of PTH in rat enterocytes, the activation of PI3K, and that hormone regulation of PI3K activity and Akt/PKB phosphorylation on Thr(308) is impaired with ageing.

Involvement of PI3-kinase and its association with c-Src in PTH-stimulated rat enterocytes.

Phosphoinositide-3-kinase (PI3K) is a lipid kinase, which phosphorylates the D3 position of phosphoinositides, and is known to be activated by a host of protein tyrosine kinases. PI3K plays an important role in mitogenesis in several cell systems. However, whether parathyroid hormone (PTH) affects the activity and functional roles of PI3K in intestinal cells remain to be determined. The objective of this study was to identify and characterize the PI3K pathway, and its relation to other non-receptor tyrosine kinases in mediating PTH signal transduction in rat enterocytes. PTH dose- and time-dependently increased PI3K activity with a peak occurring at 2 min. The tyrosine kinase inhibitor genistein, c-Src inhibitor PP1 and two structurally different inhibitors of PI3K, LY294002 and wortmannin, suppressed PI3K activity dependent on PTH. Co-immunoprecipitation analysis showed a constitutive association between c-Src and PI3K, which was enhanced by PTH treatment, suggesting that the cytosolic tyrosine kinase forms an immunocomplex with PI3K probably via the N-SH2 domain of the p85alpha regulatory subunit. In response to PTH, tyrosine phosphorylation of p85alpha was enhanced, effect that was abolished by PP1, the inhibitor of c-Src kinase. PTH causes a rapid (0.5-5 min) phosphorylation of Akt/PKB, effect that was abrogated by PI3K inhibitors, indicating that in rat enterocytes, PI3K is an upstream mediator of Akt/PKB activation by PTH. We report here that PI3K is also required for PTH activation of the mitogen-activated protein kinases ERK1 and ERK2. Taken together, the present study demonstrate, for the first time, that PTH rapidly and transiently stimulates PI3K activity and its down effector Akt/PKB in rat enterocytes playing c-Src kinase a central role in PTH-dependent PI3K activation and that PI3K signaling pathway contributes to PTH-mediated MAPK activation.

Non-genomic stimulation of tyrosine phosphorylation cascades by 1,25(OH)(2)D(3) by VDR-dependent and -independent mechanisms in muscle cells.

Studies with different cell types have shown that modulation of various of the fast as well as long-term responses to 1,25(OH)(2)D(3) depends on the activation of tyrosine kinase pathways. Recent investigations of our laboratory have demonstrated that 1,25(OH)(2)D(3) rapidly stimulates in muscle cells tyrosine phosphorylation of PLC-gamma and the growth-related proteins MAPK and c-myc. We have now obtained evidence using antisense technology indicating that VDR-dependent activation of Src mediates the fast stimulation of tyrosine phosphorylation of c-myc elicited by the hormone. This non-genomic action of 1,25(OH)(2)D(3) requires tyrosine phosphorylation of the VDR. Immunoprecipitation under native conditions coupled to Western blot analysis revealed 1,25(OH)(2)D(3)-dependent formation of complexes between Src and the VDR and c-myc. However, the activation of MAPK by the hormone was only partially mediated by the VDR and required in addition increased PKC and intracellular Ca(2+). Following its phosphorylation, MAPK translocates into the nucleus where it regulates c-myc transcription. Altogether these results indicate that tyrosine phosphorylation plays a role in the stimulation of muscle cell growth by 1,25(OH)(2)D(3). Data were also obtained involving tyrosine kinases and the VDR in hormone regulation of the Ca(2+) messenger system by mediating the stimulation of store-operated calcium (SOC; TRP) channels. Congruent with this action, 1,25(OH)(2)D(3) induces a rapid translocation of the VDR to the plasma cell membrane which can be blocked by tyrosine kinase inhibitors. Of mechanistic relevance, an association between the VDR and TRP proteins with the participation of the scaffold protein INAD was shown.