Serine phosphorylation of human P450c17 increases 17,20-lyase activity: implications for adrenarche and the polycystic ovary syndrome.

Microsomal cytochrome P450c17 catalyzes both steroid 17 alpha-hydroxylase activity and scission of the C17-C20 steroid bond (17,20-lyase) on the same active site. Adrenal 17 alpha-hydroxylase activity is needed to produce cortisol throughout life, but 17,20-lyase activity appears to be controlled independently in a complex, age-dependent pattern. We show that human P450c17 is phosphorylated on serine and threonine residues by a cAMP-dependent protein kinase. Phosphorylation of P450c17 increases 17,20-lyase activity, while dephosphorylation virtually eliminates this activity. Hormonally regulated serine phosphorylation of human P450c17 suggests a possible mechanism for human adrenarche and may be a unifying etiologic link between the hyperandrogenism and insulin resistance that characterize the polycystic ovary syndrome.

Gene conversions and unequal crossovers between CYP21 (steroid 21-hydroxylase gene) and CYP21P involve different mechanisms.

Most cases of congenital adrenal hyperplasia, the inherited inability to synthesize cortisol, are caused by mutations in the steroid 21-hydroxylase gene (CYP21). Steroid 21-hydroxylase deficiency is unusual among genetic diseases in that approximately 95% of the mutant alleles have apparently been generated by recombination between a normally active gene (CYP21) and a linked pseudogene (CYP21P). Approximately 20% of mutant alleles carry DNA deletions of 30 kb that have presumably been generated by unequal meiotic crossing-over, whereas 75% carry one or more mutations in CYP21 that are normally found in the CYP21P pseudogene. These latter mutations are termed "gene conversions," although the mechanism by which they are generated is not well understood. To assess the frequency at which these different recombination events occur, we have used PCR to detect de novo deletions and gene conversions in matched sperm and peripheral blood leukocyte DNA samples from normal individuals. Deletions with breakpoints in a 100-bp region in intron 2 and exon 3 were detected in sperm DNA samples with frequencies of approximately 1 in 10(5)-10(6) genomes but were never detected in the matching leukocyte DNA. Gene conversions in the same region occur in approximately 1 in 10(3)-10(5) genomes in both sperm and leukocyte DNA. These data suggest that whereas deletions occur exclusively in meiosis, gene conversions occur during both meiosis and mitosis, or perhaps only during mitosis. Thus, gene conversions must occur by a mechanism distinct from unequal crossing-over.

Comparative expressed-sequence-tag analysis of differential gene expression profiles in PC-12 cells before and after nerve growth factor treatment.

Nerve growth factor-induced differentiation of adrenal chromaffin PC-12 cells to a neuronal phenotype involves alterations in gene expression and represents a model system to study neuronal differentiation. We have used the expressed-sequence-tag approach to identify approximately 600 differentially expressed mRNAs in untreated and nerve growth factor-treated PC-12 cells that encode proteins with diverse structural and biochemical functions. Many of these mRNAs encode proteins belonging to cellular pathways not previously known to be regulated by nerve growth factor. Comparative expressed-sequence-tag analysis provides a basis for surveying global changes in gene-expression patterns in response to biological signals at an unprecedented scale, is a powerful tool for identifying potential interactions between different cellular pathways, and allows the gene-expression profiles of individual genes belonging to a particular pathway to be followed.

Modulation of pair bonding in female prairie voles (Microtus ochrogaster) by corticosterone.

Glucocorticoid levels in animals may respond to and influence the development of social attachments. This hypothesis was tested in prairie voles (Microtus ochrogaster), monogamous rodents that form long-term heterosexual pair bonds. In socially naive female prairie voles, cohabitation with an unfamiliar male resulted in a dramatic decline in serum corticosterone levels. When corticosterone levels were reduced via adrenalectomy, females developed partner preferences after 1 h of cohabitation, while sham-operated and untreated females required 3 h or more of nonsexual cohabitation to establish a partner preference. In adrenalectomized and intact females, exogenous injections of corticosterone, given prior to social exposure, prevented the development of preferences for the cohabitating male. Although corticosterone inhibited the development of partner preferences, it did not interfere with the expression of previously established social preferences. These results suggest that social stimuli can modulate adrenal activity and that adrenal activity, in turn, is capable of influencing the formation of adult social preferences in female prairie voles. The involvement of the adrenal axis in the formation of partner preferences and the subsequent development of pair bonds provides a mechanism through which environmental and social factors may influence social organization in this species.

A transgene coding for a human insulin analog has a mitogenic effect on murine embryonic beta cells.

We have investigated the mitogenic effect of three mutant forms of human insulin on insulin-producing beta cells of the developing pancreas. We examined transgenic embryonic and adult mice expressing (i) human [AspB10]-proinsulin/insulin ([AspB10]ProIN/IN), produced by replacement of histidine by aspartic acid at position 10 of the B chain and characterized by an increased affinity for the insulin receptor; (ii) human [LeuA3]insulin, produced by the substitution of leucine for valine in position 3 of the A chain, which exhibits decreased receptor binding affinity; and (iii) human [LeuA3, AspB10]insulin "double" mutation. During development, beta cells of AspB10 embryos were twice as abundant and had a 3 times higher rate of proliferation compared with beta cells of littermate controls. The mitogenic effect of [AspB10]ProIN/IN was specific for embryonic beta cells because the rate of proliferation of beta cells of adults and of glucagon (alpha) cells and adrenal chromaffin cells of embryos was similar in AspB10 mice and controls. In contrast to AspB10 embryos, the number of beta cells in the LeuA3 and "double" mutant lines was similar to the number in controls. These findings indicate that the [AspB10]ProIN/IN analog increased the rate of fetal beta-cell proliferation. The mechanism or mechanisms that mediate this mitogenic effect remain to be determined.

A wortmannin-sensitive phosphatidylinositol 4-kinase that regulates hormone-sensitive pools of inositolphospholipids.

The synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], the immediate precursor of intracellular signals generated by calcium-mobilizing hormones and growth factors, is initiated by the conversion of phosphatidylinositol to phosphatidylinositol 4-phosphate [PtdIns(4)P] by phosphatidylinositol 4-kinase (PtdIns 4-kinase). Although cells contain several PtdIns 4-kinases, the enzyme responsible for regulating the synthesis of hormone-sensitive PtdIns(4,5)P2 pools has not been identified. In this report we describe the inhibitory effect of micromolar concentrations of wortmannin (WT) on the synthesis of hormone-sensitive PtdIns(4)P and PtdIns(4,5)P2 pools in intact adrenal glomerulosa cells, and the presence of a WT-sensitive PtdIns 4-kinase in adrenocortical extracts. In addition to its sensitivity to the PtdIns 3-kinase inhibitor WT, this enzyme is distinguished from the recognized membrane-bound PtdIns 4-kinases by its molecular size and weak membrane association. Inhibition of this PtdIns 4-kinase by WT results in rapid loss of the hormone-sensitive PtdIns(4,5)P2 pool in angiotensin II-stimulated glomerulosa cells. Consequently, WT treatment inhibits the sustained but not the initial increases in inositol 1,4,5-trisphosphate and cytoplasmic [Ca2+] in a variety of agonist-stimulated cells, including adrenal glomerulosa cells, NIH 3T3 fibroblasts, and Jurkat lymphoblasts. These results indicate that a specific WT-sensitive PtdIns 4-kinase is critical for the maintenance of the agonist-sensitive polyphosphoinositide pool in several cell types.

Human steroidogenic acute regulatory protein: functional activity in COS-1 cells, tissue-specific expression, and mapping of the structural gene to 8p11.2 and a pseudogene to chromosome 13.

Steroidogenic acute regulatory protein (StAR) appears to mediate the rapid increase in pregnenolone synthesis stimulated by tropic hormones. cDNAs encoding StAR were isolated from a human adrenal cortex library. Human StAR, coexpressed in COS-1 cells with cytochrome P450scc and adrenodoxin, increased pregnenolone synthesis > 4-fold. A major StAR transcript of 1.6 kb and less abundant transcripts of 4.4 and 7.5 kb were detected in ovary and testis. Kidney had a lower amount of the 1.6-kb message. StAR mRNA was not detected in other tissues including placenta. Treatment of granulosa cells with 8-bromo-adenosine 3',5'-cyclic monophosphate for 24 hr increased StAR mRNA 3-fold or more. The structural gene encoding StAR was mapped using somatic cell hybrid mapping panels to chromosome 8p. Fluorescence in situ hybridization placed the StAR locus in the region 8p11.2. A StAR pseudogene was mapped to chromosome 13. We conclude that StAR expression is restricted to tissues that carry out mitochondrial sterol oxidations subject to acute regulation by cAMP and that StAR mRNA levels are regulated by cAMP.

Isolation and characterization of a MAGE gene family in the Xp21.3 region.

A human gene with strong homology to the MAGE gene family located in Xq27-qter has been isolated by using exon-trapping of cosmids in the Xp21.3 region. We have mapped and sequenced cDNA and genomic clones corresponding to this gene, MAGE-Xp, and shown that the last exon contains the open reading frame and is present in a minimum of five copies in a 30-kb interval. MAGE-Xp is expressed only in testis and, unlike the Xq27-qter MAGE genes, it is not expressed in any of 12 different tumor tissues tested. However, the gene and predicted protein structure are conserved, suggesting a similar function. MAGE-Xp is located in the 160-kb critical interval defined for the locus involved in sex determination within Xp21 and is 50 kb distal to the DAX-1 gene, which is responsible for X-chromosome-linked adrenal hypoplasia congenita.

Otimização dos valores de referência da 17OH-progesterona neonatal de acordo com o peso ao nascimento no programa de triagem neonatal da APAE DE SÃO PAULO

Introdução: A Hiperplasia Adrenal Congênita por deficiência da 21-hidroxilase (HAC) é uma doença com mortalidade neonatal elevada sendo elegível para programas públicos de Triagem Neonatal (TN). A HAC é causada por mutações no gene CYP21A2, as quais acarretam diferentes comprometimentos da atividade enzimática e resultam em espectro amplo de manifestações clínicas. Apesar da eficiência da TN para diagnosticar os casos graves, a taxa elevada de resultados falso-positivos (RFP), principalmente relacionados à prematuridade, é um dos maiores problemas. Porém, resultados falso-negativos também podem ocorrer em coletas antes de 24 horas de vida. No Brasil, a coleta da amostra neonatal difere entre os municípios, podendo ser no terceiro dia de vida como após. Objetivo: Avaliar se os valores da 17OH-progesterona neonatal (N17OHP) das coletas no terceiro dia de vida diferem significativamente das coletas a partir do quarto dia. Determinar qual percentil (99,5 ou 99,8) pode ser utilizado como valor de corte para a N17OHP, de acordo com o peso ao nascimento e tempo de vida na coleta, a fim de que proporcione taxa menor de RFP. Métodos: Foi avaliada, retrospectivamente, a N17OHP de 271.810 recém-nascidos (Rns) de acordo com o tempo de vida na coleta (G1: 48 - = 72h) e peso ao nascimento (P1: <= 1.500g, P2: 1.501-2.000g, P3: 2.001-2.500g e P4: >= 2.500g), pelo método imunofluorimétrico. Testes com resultados alterados foram confirmados no soro por Espectrometria de Massas em Tandem - LC-MS/MS. Rns afetados e/ou assintomáticos e com valores persistentemente elevados de 17OHP sérica foram submetidos ao estudo molécular, sequenciamento do gene CYP21A2. Resultados: os valores da N17OHP no grupo G1 foram significativamente menores do que em G2 em todos os grupos de peso (p < 0.001). A taxa de RFP em G1 e G2 foi de 0,2% para o percentil 99,8 e de 0,5% para o percentil 99,5 em ambos os grupos. O percentil 99,8 da N17OHP foi o melhor valor de corte para distinguir os Rns não afetados dos afetados, cujos valores são: G1 (P1: 120; P2: 71; P3: 39 e P4: 20 ng /mL) e em G2 (P1: 173; P2: 90; P3: 66 e P4: 25 ng/mL). Vinte e seis Rns do grupo G1 apresentaram a forma perdedora de sal (PS) (13H e 13M), nestes a N17OHP variou de 31 a 524 ng/mL e vinte Rns no grupo G2 (8H e 12M), nestes a N17OHP variou de 53 a 736 ng/mL. Para ambos os grupos foram encontrados três Rns com a forma virilizante simples (1H e 2M) e os valores da N17OHP variaram de 36 a 51 ng/mL. Resultados falso-negativos não foram relatados. O valor preditivo positivo (VPP) no teste do papel filtro foi de 5,6% e 14,1% nos grupos G1 e G2, respectivamente, ao se utilizar o percentil 99,8, e de 2,3% e 7% nos grupos G1 e G2 ao se utilizar o percentil 99,5. Dentre os casos com TN alterada (RFP), 29 deles também apresentaram 17OHP sérica elevada quando dosada por LC-MS/MS. Os casos assintomáticos foram acompanhados até normalização da 17OHP sérica e/ou submetidos ao estudo molecular, que identificou dois Rns com genótipo que prediz a forma não clássica. Conclusão: a melhor estratégia para otimização do diagnóstico da HAC na triagem neonatal é se padronizar valores de corte da N17OHP em dois grupos de acordo com o tempo de vida na coleta (antes e depois de 72 horas), subdivididos em quatro grupos de peso. A utilização dos valores de corte do percentil 99,8 se mantém eficaz no diagnóstico da HAC-21OH na triagem neonatal, reduzindo de forma significativa a taxa de RFP, sem perda do diagnóstico da forma PS

Evolução de lesão neoplásica da suprarrenal : caso clínico

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

A cateterização dos seios petrosos inferiores no diagnóstico da doença de Cushing

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Avaliação dos internamentos em endocrinologia

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Doença celíaca num doente com síndrome poliglandular autoimune tipo III : caso clínico

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Doença de Addison no adulto

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Pulmonary hypoplasia and anasarca syndrome in Cika cattle

BACKGROUND: Hydrops foetalis is defined as excessive fluid accumulation within the foetal extravascular compartments and body cavities. It has been described in human and veterinary medicine, but despite several descriptive studies its aetiology is still not fully clarified. Pulmonary hypoplasia and anasarca (PHA) syndrome is a rare congenital abnormality in cattle that is characterised by hydrops foetalis including extreme subcutaneous oedema (anasarca) and undeveloped or poorly formed lungs (pulmonary hypoplasia). Until now, sporadic cases of PHA were reported in cattle breeds like Australian Dexter, Belted Galloway, Maine-Anjou, and Shorthorn. This report describes the first known cases of PHA syndrome in Slovenian Cika cattle. CASE PRESENTATION: A 13-year-old cow aborted a male calf in the seventh month of pregnancy, while a male calf was delivered by caesarean section on the due date from a 14-year-old cow. The pedigree analysis showed that the calves were sired by the same bull, the dams were paternal half-sisters and the second calf was the product of a dam-son mating. Gross lesions were similar in both cases and characterized by severe anasarca, hydrothorax, hydropericardium, ascites, hypoplastic lungs, absence of lymph nodes, and an enlarged heart. The first calf was also athymic. Histopathology of the second affected calf confirmed severe oedema of the subcutis and interstitium of the organs, and pulmonary hypoplasia. The lymph vessels in the subcutis and other organs were severely dilated. Histopathology of the second calf revealed also lack of bronchus associated lymphoid tissue and adrenal gland hypoplasia. CONCLUSIONS: The findings were consistent with known forms of the bovine PHA syndrome. This is the first report of the PHA syndrome occurring in the local endangered breed of Cika cattle. Observed inbreeding practice supports that this lethal defect most likely follows an autosomal recessive mode of inheritance. In the light of the disease phenotype it is assumed that a mutation causing an impaired development of lymph vessels is responsible for the hydrops foetalis associated malformations in bovine PHA.