RESUMO
Corticotrophin-releasing factor (CRF) plays a central role in controlling the hypothalamic-pituitary-adrenal axis during stressful periods. CRF neurones are activated in the hypothalamic paraventricular nucleus (PVN) in response to stress, whereas the activated CRF neurones in the PVN are suppressed by glucocorticoids. Glucocorticoids may act directly on CRF neurones because glucocorticoid receptors are expressed highly on these neurones in the PVN. CRF expression levels in the PVN are also increased by adrenalectomy in vivo. The signalling pathways involved in the control of CRF gene transcription in the hypothalamus when negative feedback by glucocorticoids after adrenalectomy is lost remain undetermined. We investigated whether CRF gene transcription is regulated by both glucocorticoids and glucocorticoid withdrawal in hypothalamic cells. The present study demonstrates that CRF gene transcription activity and mRNA levels in the hypothalamic 4B cells were not modulated by incubation with dexamethasone for a short 2-h period, although they were stimulated by incubation for longer than 5 h. CRF gene transcription activity and mRNA levels were increased after 2 h of dexamethasone deprivation. The cAMP-response element (CRE) on the promoter was the main region that is regulated by both glucocorticoids and glucocorticoid withdrawal. We observed that the intracellular cAMP production levels were transiently increased 30 min after the removal of dexamethasone, whereas they were also increased 2.5 h after incubation with dexamethasone without the removal. Phosphorylated-CRE-binding protein (CREB)/CREB protein levels were also increased rapidly after the deprivation of glucocorticoids via an adenylate cyclase pathway. Therefore, the phosphorylation of CREB contributes to the activation of CRF gene transcription after the deprivation of glucocorticoids in hypothalamic cells.
Assuntos
Hormônio Liberador da Corticotropina/genética , Glucocorticoides/deficiência , Glucocorticoides/farmacologia , Hipotálamo/metabolismo , Células Cultivadas , Hormônio Liberador da Corticotropina/metabolismo , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipotálamo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Quinases/metabolismo , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
INTRODUCTION: Corticotropin-releasing factor (CRF) plays a central role in controlling the hypothalamic-pituitary-adrenal (HPA) axis during stressful periods. CRF is synthesized and secreted in the hypothalamic paraventricular nucleus (PVN) in response to stress, and stimulates ACTH in the pituitary corticotrophs. ACTH stimulates the release of glucocorticoids from the adrenal glands, and glucocorticoids sequentially inhibit hypothalamic PVN production of CRF and pituitary production of ACTH. The effects of glucocorticoids on CRF gene regulation, however, are possibly tissue-specific since glucocorticoids stimulate CRF gene expression in the placenta and the bed nucleus of the stria terminalis, while they inhibit it in the hypothalamus. METHODS AND RESULTS: In a hypothalamic cell line, 4B, we found that forskolin-stimulated CRF gene transcription was mediated by a functional cAMP-response element (CRE), which included -220 to -233 bp on the CRF 5'-promoter region. Protein kinase A, protein kinase C, and p38 mitogen-activated protein kinase pathways contributed to forskolin-induced transcriptional activity of CRF in hypothalamic 4B cells. Glucocorticoid-dependent repression of cAMP-stimulated transcriptional activity of CRF was localized to promoter sequences between -278 and -233 bp, which included a glucocorticoid regulatory element and a serum response element. CONCLUSION: Taken together, these findings indicate that the regulatory elements, including CRE, negative glucocorticoid regulatory element, and a serum response element on the promoter, contribute to the regulation of CRF gene transcription in hypothalamic 4B cells.
Assuntos
Hormônio Liberador da Corticotropina/genética , Hipotálamo/metabolismo , Elementos Reguladores de Transcrição/fisiologia , Antracenos/farmacologia , Linhagem Celular , Cromonas/farmacologia , Colforsina/farmacologia , Hormônio Liberador da Corticotropina/metabolismo , Dexametasona/farmacologia , Flavonoides/farmacologia , Genes Reporter/efeitos dos fármacos , Humanos , Hipotálamo/efeitos dos fármacos , Imidazóis/farmacologia , Isoquinolinas/farmacologia , Morfolinas/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Elementos Reguladores de Transcrição/efeitos dos fármacos , Deleção de Sequência , Sulfonamidas/farmacologia , TransfecçãoRESUMO
OBJECTIVE: SKG mice have a point mutation of the zeta-associated protein of 70 kD (ZAP-70) and spontaneously develop a severe polyarthritis in the conventional condition, whereas they are healthy under the specific pathogen free (SPF) condition. The purpose of this study was to investigate the cytokine production from splenocytes in SKG mice developing arthritis under the SPF condition. MATERIAL: SKG and BALB/c mice were intraperitoneally injected with zymosan A under the SPF condition. Spleen was isolated 1, 2 or 8 weeks after the intraperitoneal injection of saline or zymosan A. Splenocytes were cultured with concanavalin A. Cytokine production and proliferation were measured 48 and 72 h after the culture. RESULTS: An intraperitoneal injection of zymosan A induced severe polyarthritis with increased levels of rheumatoid factor and interleukin 6 (IL-6) only in SKG mice. Splenocytes from SKG mice did not proliferate well maybe because of less productivity of IL-2. The IL-4 production from splenocytes of SKG mice was higher, while interferon-gamma production was lower than those of BALB/c mice. An injection of zymosan A reduced the IL-4 production only in SKG mice. CONCLUSIONS: SKG mice do not develop arthritis under the SPF condition possibly because of a low proliferative activity of T cells and Th2-predominance.
Assuntos
Artrite/metabolismo , Citocinas/biossíntese , Baço/metabolismo , Zimosan/farmacologia , Animais , Artrite Experimental/patologia , Modelos Animais de Doenças , Interleucina-2/metabolismo , Interleucina-4/metabolismo , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Especificidade da Espécie , Linfócitos T/metabolismo , Células Th2/metabolismoRESUMO
SETTING: There is sparse epidemiologic information regarding the role of dietary factors in the development of idiopathic pulmonary fibrosis (IPF). OBJECTIVE: To examine the relationship between specific types of fatty acids and selected foods high in fat and IPF in Japan. DESIGN: Included were 104 cases aged > or = 40 years who had been diagnosed in the last 2 years in accordance with the most recent criteria. Controls aged > or = 40 years consisted of 56 hospitalised patients diagnosed as having acute bacterial pneumonia and four out-patients with common cold. RESULTS: Intake of saturated fatty acids, mono-unsaturated fatty acids, n-6 polyunsaturated fatty acids and meat was independently associated with an increased risk of IPF. Specifically, the multivariate OR for comparison of the highest with the lowest quartile of intake of saturated fatty acids was 6.26 (95%CI 1.79-24.96, P for trend = 0.01) and for meat it was 7.19 (95%CI 2.15-27.07, P for trend = 0.02). Intake of cholesterol, n-3 polyunsaturated fatty acids, fish, eggs and dairy products was not related to the risk. CONCLUSION: These findings suggest that consumption of saturated fatty acids and meat may increase the risk of IPF.
Assuntos
Gorduras na Dieta/efeitos adversos , Produtos da Carne/efeitos adversos , Fibrose Pulmonar/etiologia , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Incidência , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Fibrose Pulmonar/epidemiologia , Fatores de Risco , Inquéritos e QuestionáriosRESUMO
Osteocalcin is a noncollagenous protein that is abundant in mineralized bone matrix. Mice have a gene cluster of osteocalcin that consists of OG1, OG2, and ORG. We established a new method to directly analyze the expression levels of OG1, OG2, and ORG mRNAs relative to total osteocalcin mRNA. They were amplified as 371-bp fragments by reverse transcription-polymerase chain reaction (RT-PCR) at the same time using common primers, digested with ApaLI, and separated in a polyacrylamide gel. ApaLI digestion did not affect the mobility of the OG1-derived 371-bp fragment, whereas both 371-bp fragments, derived from OG2 and ORG, were digested into 350 bp. Total RNA prepared from mouse bone was then subjected to RT-PCR followed by ApaLI digestion. OG1 and OG2 mRNAs were found to be expressed at ratios of 80%-86% and 14%-20%, respectively, to the total osteocalcin mRNA in mouse bone. The ratios were almost constant in various bones in vivo, independent of the animal's genetic background, age, or gender, or different parts of bone. RT-PCR using specific primers revealed that mouse bone tissues strongly expressed osteocalcin mRNA derived from OG1 and OG2, but not ORG. In contrast, cells cultured in vitro showed different expression ratios of osteocalcin mRNA: 53%-65% for OG1 and 35%-47% for OG2 to the total osteocalcin mRNA in the osteoblast cell line and primary osteoblasts in culture even though they formed many mineralized bone nodules. Similar results were obtained in both KS483 osteoblasts and C2C12 myoblasts, when they were cultured with bone morphogenetic protein-2 (BMP-2) to induce osteocalcin mRNA. Taken together, these findings indicate that OG1 is the predominant transcript among the three osteocalcin genes in mouse bone in vivo. It is also suggested that the expression of OG1 and OG2 is regulated differently in bone tissues and osteoblast cultures.
Assuntos
Osso e Ossos/metabolismo , Osteoblastos/metabolismo , Osteocalcina/genética , Animais , Sequência de Bases , Células Cultivadas , DNA Complementar/química , DNA Complementar/isolamento & purificação , Regulação da Expressão Gênica , Rim/metabolismo , Camundongos , Dados de Sequência Molecular , Família Multigênica , Osteocalcina/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Tíbia/metabolismoRESUMO
Chronic idiopathic intestinal pseudo-obstruction is one of the disorders that is most refractory to medical and surgical treatment. Even when patients are given nutritional support, including total parenteral nutrition, obstructive symptoms seldom disappear. We report a case of chronic idiopathic intestinal pseudo-obstruction, due to myopathy, in which hyperbaric oxygenation therapy was strikingly effective. The presence of myopathy was histologically confirmed on the surgically resected jejunal specimen. Hyperbaric oxygenation resulted not only in relief of the patient's obstructive symptoms but also in a rapid decrease of abnormally accumulated intestinal gas. At last, he could resume oral intake without any critical adverse effects. These observations strongly suggest that hyperbaric oxygenation can be an effective therapy in the management of chronic idiopathic intestinal pseudo-obstruction.
Assuntos
Oxigenoterapia Hiperbárica , Pseudo-Obstrução Intestinal/terapia , Doença Crônica , Humanos , Pseudo-Obstrução Intestinal/diagnóstico por imagem , Pseudo-Obstrução Intestinal/patologia , Masculino , Pessoa de Meia-Idade , RadiografiaRESUMO
It has been reported that central nervous system (CNS) tissue may be more heat labile than other tissues of the body. However, no definite information has been available on how much heat CNS tissue can tolerate without sustaining damage during whole-body hyperthermia, especially in a chronic stage. In this study, whole-body hyperthermia was induced in dogs by extracorporeal heating of blood, to determine the effects 7 days after hyperthermia on the canine brain and spinal cord. The temperatures of both the brain and the spinal cord were raised to 42.0+/-0.1 degrees C and maintained at that level for 60 min. Seven days later, all of the dogs were sacrificed by transcardial perfusion using 10% formaldehyde phosphate buffer for microscopic examination. The thermal dose resulted in neither microscopic damage to the CNS nor neurological symptoms, as determined by comparison of microscopic and neurological findings with those of dogs whose brain and spinal cord temperatures were maintained at 37.0 degrees C for 60 min. The findings suggest that, for medical purposes, whole-body hyperthermia appears promising for application at a thermal dose of up to 42.0 degrees C for 60 min.
Assuntos
Sistema Nervoso Central/lesões , Hipertermia Induzida/efeitos adversos , Animais , Temperatura Corporal , Lesões Encefálicas/etiologia , Lesões Encefálicas/patologia , Cães , Feminino , Masculino , Segurança , Traumatismos da Medula Espinal/etiologia , Traumatismos da Medula Espinal/patologia , Fatores de TempoRESUMO
Recent evidence suggests that nitric oxide (NO), a free radical gas, plays an important role in regulating the function of a variety of neuroendocrine systems. With respect to the hypothalamo-pituitary-gonadal axis, a stimulatory effect of NO on the release of gonadotropin-releasing hormone (GnRH) from rat hypothalamus has been demonstrated in vitro. However, no previous study has reported NO-stimulated secretion of GnRH from in vivo hypothalamus, and also the precise cellular site of action of NO within the GnRH neuronal system remains to be elucidated. In the present study, utilizing the push-pull perfusion technique of rat hypothalamus, we examined the effect of L-arginine (L-Arg), an NO donor, on the release of GnRH, neuropeptide Y and cyclic GMP (c-GMP), which is a pivotal second messenger molecule of the NO system. For comparison, we also examined the effect of carbon monoxide (CO), another putative gaseous neurotransmitter, using hematin, a CO donor. During the period of 11.00-18.00 h, we collected blood and hypothalamic perfusates from ovariectomized adult rats that had been implanted with an estradiol capsule 2 days before. During the entire period of observation, L-Arg (1.0 or 10 mM), hematin (10 or 100 microM) or artificial cerebrospinal fluid alone (as the control) was infused into the medial preoptic area (MPOA) where there are cell bodies of GnRH neurons, or the median eminence-arcuate nucleus complex (ME-ARC) where axon terminals of GnRH neurons are localized. Although 10 mM of L-Arg significantly stimulated GnRH and c-GMP, but not neuropeptide Y, levels in both the MPOA and ME-ARC, GnRH and c-GMP in the ME-ARC were already increased by 1.0 mM of L-Arg. By contrast, both concentrations of hematin were without effect at either site of the hypothalamus. This study is the first to demonstrate that NO is capable of stimulating GnRH release from rat hypothalamus in vivo. Our data also suggests that both cell bodies and axon terminals of GnRH neurons may be sites of action of NO. Our data do not support a previous study by other investigators that reported a stimulatory effect of CO on the GnRH release.
Assuntos
Monóxido de Carbono/farmacologia , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/metabolismo , Hormônio Luteinizante/metabolismo , Neuropeptídeo Y/metabolismo , Óxido Nítrico/farmacologia , Animais , Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Núcleo Arqueado do Hipotálamo/metabolismo , Arginina/administração & dosagem , Arginina/farmacologia , Monóxido de Carbono/administração & dosagem , Cateterismo , Ritmo Circadiano , GMP Cíclico/metabolismo , Estradiol/farmacologia , Feminino , Hipotálamo/efeitos dos fármacos , Eminência Mediana/efeitos dos fármacos , Eminência Mediana/metabolismo , Óxido Nítrico/administração & dosagem , Área Pré-Óptica/efeitos dos fármacos , Área Pré-Óptica/metabolismo , Ratos , Ratos WistarRESUMO
Genistein, an isoflavone abundantly present in soybeans, has structural similarity to estrogen, suggesting that genistein may act as a phytoestrogen. To examine the possible role of genistein in hemopoiesis and bone metabolism, female mice were either sham-operated or ovariectomized (OVX), and selected OVX mice were administered genistein for 2-4 weeks (0.1-0.7 mg/day) or 17beta-estradiol (E2; 0.01-0.1 microg/day) s.c., using a miniosmotic pump (Alza Corp., Palo Alto, CA). In OVX mice, uterine weight declined but was completely restored by E2 administration. In contrast, genistein did not demonstrate a reversal of the OVX-induced uterine atrophy. The number of bone marrow cells markedly increased, 2-4 weeks after OVX, and most of these were B220-weakly positive pre-B cells. The increased B-lymphopoiesis was completely restored, not only by E2 but also by genistein administration. In OVX mice, the trabecular bone volume of the femoral distal metaphysis, measured by microcomputed tomography scanning and dual-energy x-ray absorptiometry, was markedly reduced; and genistein restored this, as did E2. These results indicate that genistein exhibits estrogenic action in bone and bone marrow, to regulate B-lymphopoiesis and prevent bone loss, without exhibiting estrogenic action in the uterus. Phytoestrogens may be useful for preventing bone loss caused by estrogen deficiency in females.
Assuntos
Linfócitos B/citologia , Estrogênios não Esteroides/farmacologia , Estrogênios/deficiência , Genisteína/farmacologia , Hematopoese/efeitos dos fármacos , Isoflavonas , Osteoporose Pós-Menopausa/prevenção & controle , Animais , Densidade Óssea/efeitos dos fármacos , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Estradiol/farmacologia , Estrogênios não Esteroides/uso terapêutico , Feminino , Genisteína/uso terapêutico , Humanos , Camundongos , Tamanho do Órgão , Osteoporose Pós-Menopausa/etiologia , Ovariectomia , Fitoestrógenos , Preparações de Plantas , Glycine max , Útero/anatomia & histologiaRESUMO
To identify genes expressed in myeloid differentiation, we isolated a cDNA fragment by differential display using RNA prepared from HT93A cells, a human cell line capable of differentiating into neutrophil and eosinophil lineages in response to retinoic acid (RA). Evaluation of the full-length clone isolated from an HT93A cDNA library showed that it encoded a 24 kDa protein comprised of several domains conserved in the Ras superfamily. Comparison of the deduced amino acid sequence of this clone with Rab proteins revealed that it had highest homology to a small GTP-binding protein, murine Rab3D. The mRNA expression of human Rab3D was upregulated in the course of myeloid differentiation, and it was preferentially expressed in granulocytes. These results suggest that human Rab3D may play a specific role in granulocytes, for example in exocytosis of neutrophil-specific granules or in degranulation of both eosinophils and basophils.
Assuntos
DNA Complementar/biossíntese , Proteínas de Ligação ao GTP/genética , Sequência de Aminoácidos , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular/efeitos dos fármacos , Clonagem Molecular , Sequência Conservada , DNA Complementar/isolamento & purificação , Proteínas de Ligação ao GTP/biossíntese , Regulação da Expressão Gênica , Biblioteca Gênica , Granulócitos/metabolismo , Humanos , Dados de Sequência Molecular , Tretinoína/farmacologia , Regulação para Cima , Proteínas rab3 de Ligação ao GTPRESUMO
Sequential changes in hormone levels were assessed for one year in postmenopausal breast cancer patients administered the aromatase inhibitor fodrozole hydrochloride hydrate (Afema) in order to evaluate its efficacy and safety in long-term treatment. Forty patients received Afema alone as postoperative adjuvant therapy, while 30 received it with 5-FU. Plasma estrone and estradiol levels decreased significantly in both groups (p < 0.001) and were not affected by body mass index (BMI). Plasma androstenedione and testosterone levels increased within the normal range. Aldosterone levels hit bottom 3 months after treatment was started, and tended to return to the pre-treatment baseline thereafter. Plasma cortisol levels increased significantly within the normal range. FDP and alpha 2-PIPC, parameters related to blood coagulation and fibrinolysis, showed no significant change. Adverse reactions, mainly nausea and elevation of LDH levels, were all slight. Thus, long-term administration of Afema resulted in significant decreases in estrogen levels with few adverse reactions regardless of the BMI, suggesting that it can be safely used as effective postoperative adjuvant therapy.
Assuntos
Androgênios/sangue , Antineoplásicos Hormonais/administração & dosagem , Neoplasias da Mama/sangue , Neoplasias da Mama/tratamento farmacológico , Inibidores Enzimáticos/administração & dosagem , Estrogênios/sangue , Fadrozol/administração & dosagem , Pós-Menopausa , Idoso , Androstenodiona/sangue , Androsterona/sangue , Neoplasias da Mama/cirurgia , Quimioterapia Adjuvante , Estradiol/sangue , Feminino , Humanos , Hidrocortisona/sangue , Pessoa de Meia-Idade , Testosterona/sangueRESUMO
Saireito, a saiko agent (a Chinese herbal drug), increases the synthesis and secretion of ACTH by stimulating hypothalamic CRH release. In the present study, we examined the effect of food containing saireito (1.5%) on the recovery of the hypothalamic-pituitary-adrenal axis after treating male rats with prednisolone (PSL, 200 microM) in drinking water for 14 days. Saireito was administered during and after PSL administration. The rats were decapitated at various times after PSL administration. Tail-pinch stress had been applied to some rats. The plasma ACTH response to tail-pinch stress in the PSL + saireito group recovered to the control level on day 1, but that in the group given PSL alone recovered on day 3. The ACTH level in the anterior pituitary and the CRH level in the median eminence of the PSL + saireito group returned to the control level on day 3, and that in the group given PSL alone returned to it on day 5. These results indicate that the administration of saireito reduces the negative feedback effect of PSL on the hypothalamus and pituitary and accelerates the recovery of the hypothalamic CRH and pituitary ACTH level after glucocorticoid treatment.
Assuntos
Doenças das Glândulas Suprarrenais/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Doenças Hipotalâmicas/tratamento farmacológico , Doenças da Hipófise/tratamento farmacológico , Prednisolona/toxicidade , Doenças das Glândulas Suprarrenais/induzido quimicamente , Hormônio Adrenocorticotrópico/sangue , Hormônio Adrenocorticotrópico/metabolismo , Animais , Peso Corporal , Hormônio Liberador da Corticotropina/metabolismo , Doenças Hipotalâmicas/induzido quimicamente , Cinética , Masculino , Eminência Mediana/metabolismo , Doenças da Hipófise/induzido quimicamente , Adeno-Hipófise/metabolismo , Ratos , Ratos Wistar , Estresse Fisiológico/sangueRESUMO
A full-length cDNA for the rat kidney mitochondrial cytochrome P450 mixed function oxidase, 25-hydroxyvitamin D3-1alpha-hydroxylase (P4501alpha), was cloned from a vitamin D-deficient rat kidney cDNA library and subcloned into the mammalian expression vector pcDNA 3.1(+). When P4501alpha cDNA was transfected into COS-7 transformed monkey kidney cells, they expressed 25-hydroxyvitamin D3-1alpha-hydroxylase activity. The sequence analysis showed that P4501alpha was of 2,469 bp long and contained an ORF encoding 501 amino acids. The deduced amino acid sequence showed a 53% similarity and 44% identity to the vitamin D3-25-hydroxylase (CYP27), whereas it has 42.6% similarity and 34% identity with the 25-hydroxyvitamin D3-24-hydroxylase (CYP24). Thus, it composes a new subfamily of the CYP27 family. Further, it is more closely related to the CYP27 than to the CYP24. The expression of P4501alpha mRNA was greatly increased in the kidney of vitamin D-deficient rats. In rats with the enhanced renal production of 1alpha,25-dihydroxyvitamin D3 (rats fed a low Ca diet), P4501alpha mRNA was greatly increased in the renal proximal convoluted tubules.
Assuntos
Esteroide Hidroxilases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Cálcio/administração & dosagem , Colestanotriol 26-Mono-Oxigenase , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , Primers do DNA , DNA Complementar , Regulação da Expressão Gênica , Rim/enzimologia , Masculino , Dados de Sequência Molecular , Fósforo/administração & dosagem , Ratos , Ratos Sprague-Dawley , Homologia de Sequência de Aminoácidos , Vitamina D3 24-HidroxilaseRESUMO
We analyzed the effects of cold oxygen plasma ashing of neurobiological specimens on different elements with synchrotron spectromicroscopy. Our results demonstrate that while carbon is almost completely removed, phosphorus, calcium, potassium, sulfur, and, to some extent, nitrogen are retained and their relative concentration is enhanced.
Assuntos
Microscopia Eletrônica/métodos , Fixação de Tecidos/métodos , Animais , Cálcio/química , Carbono/química , Cerebelo/ultraestrutura , Nitrogênio/química , Oxigênio/química , Fósforo/química , Potássio/química , Ratos , Espectrometria por Raios X/métodos , Enxofre/químicaRESUMO
The 25-hydroxyvitamin D3 1 alpha-hydroxylase (1 alpha-hydroxylase) is a cytochrome P450 enzyme that catalyzes the conversion of 25-hydroxyvitamin D3 to 1 alpha,25-dihydroxyvitamin D3. This enzyme plays an important role in calcium homeostasis. Here we report the molecular cloning of cDNA and gene for human 1 alpha-hydroxylase. The cDNA clone was obtained from a human kidney cDNA library by cross-hybridization with a previously cloned rat cDNA probe. The cDNA consists of 2469 bp and encodes a protein of 508 amino acids that shows 82.5% sequence identity with the rat enzyme. A computer-aided homology search revealed that 1 alpha-hydroxylase shares a relatively high homology with vitamin D3 25-hydroxylase (about 40% amino acid identity). Northern blot analysis showed that the 2.5-kb mRNA is most abundant in kidney. The gene for human 1 alpha-hydroxylase spans approximately 6 kb, is composed of nine exons, and is present as a single copy. This molecular cloning makes it possible to investigate the genetic mechanism of diseases related to calcium metabolism, including vitamin D-dependency rickets type I.
Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , DNA Complementar/isolamento & purificação , DNA/isolamento & purificação , Genes , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Éxons , Biblioteca Genômica , Humanos , Íntrons , Masculino , Dados de Sequência Molecular , Especificidade de Órgãos/efeitos dos fármacos , RNA Mensageiro/metabolismo , RatosRESUMO
The sequential oxidation and cleavage of the side chain of 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) initiated by the hydroxylation at C-24 is considered to be the major pathway of this hormone in the target cell metabolism. In this study, we examined renal metabolism of a synthetic analog of 1alpha,25(OH)2D3, 24, 24-difluoro-1alpha,25-dihydroxyvitamin D3 (F2-1alpha,25(OH)2D3), C-24 of which was designed to resist metabolic hydroxylation. When kidney homogenates prepared from 1alpha,25(OH)2D3-supplemented rats were incubated with F2-1alpha,25(OH)2D3, it was mainly converted to a more polar metabolite. We isolated and unequivocally identified the metabolite as 24,24-difluoro-1alpha,25,26-trihydroxyvitamin D3 (F2-1alpha,25,26(OH)3D3) by ultraviolet absorption spectrometry, frit-fast atom bombardment liquid chromatography/mass spectroscopy analysis, and direct comparison with chemically synthesized F2-1alpha,25,26(OH)3D3. Metabolism of F2-1alpha,25(OH)2D3 into F2-1alpha,25,26(OH)3D3 by kidney homogenates was induced by the prior administration of 1alpha,25(OH)2D3 into rats. The C-24 oxidation of 1alpha,25(OH)2D3 in renal homogenates was inhibited by F2-1alpha,25(OH)2D3 in a concentration-dependent manner. Moreover, F2-1alpha,25,26(OH)3D3 was formed in ROS17/2.8 cells transfected with a plasmid expressing 1alpha,25(OH)2D3-24-hydroxylase (CYP24) but not in the cells transfected with that expressing vitamin D3-25-hydroxylase (CYP27) or containing inverted CYP27 cDNA. These results show that CYP24 catalyzes not only hydroxylation at C-24 and C-23 of 1alpha,25(OH)2D3 but also at C-26 of F2-1alpha,25(OH)2D3, indicating that this enzyme has a broader substrate specificity of the hydroxylation sites than previously considered.
Assuntos
Calcifediol/análogos & derivados , Calcitriol/análogos & derivados , Sistema Enzimático do Citocromo P-450/metabolismo , Rim/metabolismo , Esteroide Hidroxilases/metabolismo , Animais , Calcifediol/metabolismo , Calcitriol/metabolismo , Carbono/metabolismo , Hidrólise , Ratos , Vitamina D3 24-HidroxilaseRESUMO
Three distinct osteoblastic cell lines (KS418, KS460, and KS483) were subcloned from the mouse osteoblastic KS-4 cells, which possessed the abilities not only to differentiate into mature osteoblasts, but also to support osteoclast differentiation in coculture with spleen cells. The order of the magnitude of the basal alkaline phosphatase (ALP) activity was KS483 > KS418 > KS460. KS483 cells were also more differentiated than KS418 and KS460 in terms of ALP activity and osteocalcin production, when cultured in growth medium containing 10% fetal bovine serum. In long-term culture, KS418 and KS483 apparently differentiated into mature osteoblasts and formed calcified nodules without addition of beta-glycerophosphate. Electron microscopic analysis demonstrated that calcification occurring in the nodules was initiated in the matrix vesicles as observed in bone formation in vivo. Nodule formation and mineral deposition occurred simultaneously in the presence of beta-glycerophosphate, but the former always preceded the latter without addition of beta-glycerophosphate. In contrast, KS460 cells did not show time-dependent increases of ALP activity, type I collagen expression and osteocalcin production, which were induced by treatment with recombinant osteogenic protein-1 (OP-1). The three cell lines similarly supported osteoclast differentiation in coculture with spleen cells in response to 1,25-dihydroxyvitamin D3. These results indicate that the three cell lines subcloned from the original KS-4 cells represent phenotypically distinct osteoblasts during osteoblast differentiation, but are equipped similarly with the capacity to support osteoclast differentiation. The subcloned cells of the KS-4 series may provide useful systems in which to study osteoblast differentiation and function.
Assuntos
Calcificação Fisiológica/genética , Osteoblastos/citologia , Osteoclastos/citologia , Actinas/biossíntese , Actinas/genética , Fosfatase Alcalina/metabolismo , Animais , Proteína Morfogenética Óssea 7 , Proteínas Morfogenéticas Ósseas/farmacologia , Calcificação Fisiológica/fisiologia , Calcitriol/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Clonagem Molecular , Colágeno/biossíntese , Colágeno/genética , Meios de Cultura , DNA Complementar/metabolismo , Glicerofosfatos/farmacologia , Camundongos , Microscopia Eletrônica , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Osteoblastos/ultraestrutura , Osteocalcina/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/enzimologia , Osteoclastos/ultraestrutura , Fenótipo , RNA/metabolismo , Proteínas Recombinantes/farmacologia , Baço/citologia , Baço/enzimologia , Fator de Crescimento Transformador beta/farmacologiaRESUMO
HTK is a receptor tyrosine kinase that belongs to the Eph subfamily. An extensive screening using BIAcore system revealed that a colon cancer cell line, C-1, expressed the ligand for HTK. From the conditioned medium of C-1 cells, a soluble form of ligand was purified by receptor affinity chromatography, and the isolation of full-length cDNA revealed that this ligand is identical to the human HTK ligand (HTKL) previously reported. HTK receptor tyrosine phosphorylation was induced by membrane-bound or clustered soluble HTKL but not by unclustered soluble HTKL, indicating that HTKL requires cell-to-cell interaction for receptor activation. Binding analysis demonstrated that HTKL binds to HTK with a much higher affinity (Kd: 1.23 nM) than the other transmembrane-type ligand for Eph family, LERK-2/ELKL (Kd: 135 nM). The expression of HTK in cord blood cells was upregulated after the culture in the presence of stem cell factor. Clustered soluble HTKL stimulated the proliferation of sorted HTK+ cord blood cells and a hematopoietic cell line, UT-7/EPO from which HTK was isolated. These findings suggest the involvement of HTK-HTKL system in the proliferation of HTK+ hematopoietic progenitor cells in the hematopoietic environment.
Assuntos
Sangue Fetal/metabolismo , Proteínas de Membrana/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Primers do DNA , DNA Complementar , Efrina-B2 , Sangue Fetal/citologia , Humanos , Ligantes , Proteínas de Membrana/genética , Dados de Sequência Molecular , Receptor EphB4 , Proteínas Recombinantes/metabolismoRESUMO
To assess whether the cAMP-dependent protein kinase-A and/or the diacylglycerol-dependent protein kinase C (PKC) pathways play important roles in the activation of CRF neurons in vivo under physiological conditions, we tested the effect of microinjection of 8-bromo-cAMP (8-Br-cAMP) or 12-O-tetradecanoyl phorbol 13-acetate (TPA) into both paraventricular nuclei (PVN) of the hypothalamus in conscious rats. Both 8-Br-cAMP and TPA increased plasma ACTH concentrations and the POMC messenger RNA (mRNA) concentrations in the anterior pituitary. While injection of 8-Br-cAMP also increased CRF mRNA concentrations in hypothalamic tissue containing the PVN, TPA injection had no effect on CRF mRNA concentrations there. During insulin-induced hypoglycemia, which stimulates CRF gene expression and release, c-fos and c-jun mRNA increases in the hypothalamic tissue preceded the increase in the CRF mRNA level after insulin-induced hypoglycemia. Antisense oligodeoxyribonucleotides (oligos) directed against c-fos, c-jun, or the cAMP response element binding protein (CREB) mRNA were injected into both PVN before insulin-induced hypoglycemia to assess whether activator protein-1 or CREB mediates transcriptional activation of CRF during hypoglycemia. Only antisense oligo against CREB mRNA reduced the CRF mRNA level after insulin-induced hypoglycemia. These results suggest that protein kinase A may transduce intracellular signals in CRF neurons under physiological conditions and raises the possibility that CREB may be involved in stress-induced CRF gene expression.