RESUMO
CONTEXT/OBJECTIVE: The growth hormone (GH)/insulin-like growth factor-1(IGF-1) axis is the key regulator of somatic growth in humans and its genes are plausible candidates to study the genetics of height variation. Here, we studied polymorphic variation in the GH/IGF-1 axis in the extremely tall Dutch. METHODS: Case-control study of 166 tall cases with height >2 SDS and 206 controls with normally distributed height <2 SDS. Excluded were subjects with endocrine disorders or growth syndromes. We analyzed genomic DNA at 7 common polymorphisms in the GH-1, GH receptor (GHR), IGF-1 and IGFBP-3 genes. RESULTS: The association of the GH-1 1663 SNP with tall stature approached statistical significance, with the T-allele more present in the tall (allele frequency (AF): 0.44 vs. 0.36; p=0.084). Moreover, haplotype frequencies at this locus were significantly different between cases and controls, with the GGT haplotype most commonly seen in cases (p=0.01). Allele frequencies of GHR polymorphisms were not different. For the IGF-1 CA-repeat we observed a higher frequency of homozygous 192-bp carriers among tall males compared to control males (AF: 0.62 vs. 0.55; p=0.02). The IGFBP-3 -202 C-allele occurred more frequently in cases than in controls (AF: 0.58 vs. 0.50; p=0.002). Within cases, those carrying one or two copies of the -202 C-allele were significantly taller than AA genotype carriers (AC, p=0.028 and CC, p=0.009). Serum IGFBP-3 levels were highest in AA genotype carriers, the -202 SNP explained 5.8% of the variation. CONCLUSION: Polymorphic variation in the GH-1, IGF-1 and IGFBP-3 genes is associated with extremely tall stature. In particular, the IGFBP-3 -202 SNP is associated not only with being very tall but also with height variation within the tall.
Assuntos
Estatura/genética , DNA/genética , Hormônio do Crescimento Humano/genética , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Fator de Crescimento Insulin-Like I/genética , Polimorfismo de Nucleotídeo Único/genética , Estudos de Casos e Controles , Criança , Pré-Escolar , DNA/sangue , Feminino , Frequência do Gene , Genótipo , Hormônio do Crescimento Humano/sangue , Humanos , Lactente , Recém-Nascido , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Masculino , Reação em Cadeia da Polimerase , PrognósticoRESUMO
BACKGROUND/AIMS: Genetic variation in several candidate genes has been associated with short stature. Recently, a high-mobility group A2 (HMGA2) gene SNP has been robustly associated with height in the general population. Only few have attempted to study these genes in extremely tall stature. We therefore studied common genetic variation in candidate genes for height in extremely tall Dutch. METHODS: We included 116 constitutionally tall cases with height >2 SD and 103 controls with normally distributed height <2 SD. We genotyped 10 common polymorphisms previously associated with height variation. RESULTS: The HMGA2 gene SNP was significantly associated with tall stature. Using a logistic regression model, we calculated that carrying the HMGA2 (rs1042725) C allele significantly increased the odds of being tall (OR = 1.53, 95% CI 1.02-2.28; p = 0.03). In addition, controls with one or two copies of the C allele were significantly taller than controls carrying the TT genotype [TC: mean (SD) +0.61 (0.21) SDS; p = 0.004, and CC: +0.77 (0.25) SDS; p = 0.003]. CONCLUSION: Our study shows that a common polymorphism in the HMGA2 gene is not only associated with height variation in the general population but also plays an important role in one of the extremes of the height distribution.
Assuntos
Estatura/genética , Variação Genética , Transtornos do Crescimento/genética , Proteína HMGA2/genética , Adolescente , Adulto , Estudos de Casos e Controles , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Proteína HMGA2/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Países Baixos , Adulto JovemRESUMO
We have previously identified a single amino acid mutation (hE149A) in human LCAT that increases its in vitro reactivity with phosphatidylcholine species containing sn-2 arachidonate (Wang et al. 1997. J. Biol. Chem. 272: 280-286). The purpose of the present study was to determine whether in vivo overexpression of hE149A compared with human wild-type LCAT (hLCAT-wt) would be sufficient to enrich the steady state composition of plasma HDL cholesteryl esters (CE) with long chain (>18 carbon) polyunsaturated fatty acyl species. Transgenic lines with 20-fold overexpression of hLCAT were created and studied between 12 and 16 weeks of age while consuming a chow diet. Transgenic overexpression of hE149A compared with hLCAT-wt significantly enriched HDL with CE species containing 20:4 (45%) and 22:6 n-3 (108%), at the expense of those containing 18:2, without a significant change in the plasma HDL concentration, particle size, or phospholipid fatty acyl composition. Removing the contribution of endogenous mouse LCAT by crossing the transgenic mice into the mouse LCAT knockout background resulted in even greater changes in HDL CE composition, with a 2.4-, 5-, and 5-fold increase in 20:4, 20:5 n-3, and 22:6 n-3 cholesteryl esters in the hE149A mice compared with hLCAT-wt Tg mice, respectively. Our results demonstrate that in vivo expression of hE149A significantly enriches HDL cholesteryl esters in 20- and 22-carbon fatty acyl species without affecting HDL concentration or size. Furthermore, the data suggest that endogenous mouse LCAT in hLCAT transgenic mice contributes to the plasma HDL CE pool out of proportion to its mass, presumably because the hLCAT transgene is poorly activated by mouse apolipoprotein A-I.
Assuntos
Ésteres do Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferase/genética , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Mutação Puntual/genética , Substituição de Aminoácidos/genética , Animais , Ésteres do Colesterol/sangue , Ésteres do Colesterol/química , Cromatografia em Gel , Cruzamentos Genéticos , Ácidos Graxos Insaturados/análise , Ácidos Graxos Insaturados/sangue , Feminino , Expressão Gênica , Humanos , Lipídeos/sangue , Lipoproteínas/sangue , Lipoproteínas/química , Lipoproteínas HDL/sangue , Lipoproteínas HDL/química , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Tamanho da Partícula , Fosfatidilcolina-Esterol O-Aciltransferase/sangue , Reação em Cadeia da PolimeraseRESUMO
For many years it was believed that the growth hormone (GH) axis does not play a role in growth before birth or during the first year after birth. More recently, however, studies of genetic disorders of growth have led to developments in our understanding of the onset of GH sensitivity and the relationships between the maternal and fetal GH axes. Many children whose GH deficiency (GHD) is diagnosed in late childhood have a good rate of growth in their first year of life. This can be explained when one considers the differences between pituitary and hypothalamic GHD. Many children in whom GHD is diagnosed later in childhood have hypothalamic GHD, which may be masked in the first year of life by a relative lack of somatostatin secretion. Genetic defects in the GH axis lead to a slight degree of growth retardation at birth, consistent with growth impairment in the third trimester only. Conversely, a boy with a deletion in the insulin-like growth factor I (IGF-I) gene showed severe growth retardation at birth, indicating a role for IGF-I during the second and third trimesters. From these results, there are two possible models for the relationship between IGF-I production and other components of the maternal-fetal GH axis: independence of the IGF-I axis from somatotropic influences during the second trimester or, alternatively, redundancy of somatotropic hormones and their receptors. From reports of individuals with naturally occurring mutations that eliminate contributors to the GH axis, redundancy does indeed appear to be a feature of the maternal-fetal GH axis, with GH sensitivity arising around mid-gestation.
Assuntos
Feto/fisiologia , Hormônio do Crescimento Humano/fisiologia , Desenvolvimento Embrionário e Fetal , Feminino , Hormônio do Crescimento Humano/genética , Humanos , Mutação/fisiologia , Gravidez , Receptores da Prolactina/metabolismo , Receptores da Somatotropina/metabolismoRESUMO
We have previously described a novel pathway for the metabolism of HDL subfractions in which small [2 apolipoprotein (apoA-I) molecules per particle] HDL particles are converted in a unidirectional manner outside the plasma compartment to medium (3 apoA-I molecules per particle) or large (4 apoA-I molecules per particle) HDL particles, which are subsequently removed from the circulation by the liver (Colvin et al. 1999. J. Lipid Res. 40: 1782;-1792; Huggins et al. 2000. J. Lipid Res. 41: 384;-394). The purpose of the present study was to determine whether the reduction in concentration of medium HDL in African green monkeys consuming n-3 polyunsaturated versus saturated fat diets resulted from decreased in vivo production or increased catabolism. Tracer small LpA-I (HDL containing only apoA-I) were isolated, without ultracentrifugation, by gel filtration and immunoaffinity chromatography and radiolabeled. After injection, the specific activity of apoA-I in small, medium, and large HDL was determined, and the kinetic data were analyzed using our previously published multicompartmental model for HDL subfraction metabolism. We found a significant reduction of apoA-I concentration in medium HDL in the animals fed n-3 polyunsaturated fat (31.2 +/- 0.7 mg/dl) compared with animals fed saturated fat (85.4 +/- 11.9 mg/dl; P = 0.002). The production rates of apoA-I in small, medium, and large HDL were similar in both diet groups; however, there was a significant increase in the fractional catabolic rate of apoA-I in medium HDL in the animals fed n-3 polyunsaturated fat (2.188 +/- 0.501 pools/day) compared with animals fed saturated fat (0.714 +/- 0.191 pools/day; P = 0.02). We conclude that n-3 polyunsaturated fat reduces HDL cholesterol concentration by increasing the fractional catabolic rate of medium-sized HDL particles in African green monkeys.
Assuntos
Gorduras Insaturadas na Dieta/farmacologia , Ácidos Graxos Ômega-3/farmacologia , Lipoproteínas HDL/sangue , Animais , Apolipoproteína A-I/análise , Apolipoproteína A-I/sangue , Apolipoproteína A-II/sangue , Western Blotting , Chlorocebus aethiops , Colesterol/sangue , HDL-Colesterol/sangue , Gorduras na Dieta/farmacologia , Radioisótopos do Iodo , Cinética , Lipídeos/sangue , Lipoproteínas HDL/análise , Espectroscopia de Ressonância Magnética , Tamanho da Partícula , Triglicerídeos/sangueRESUMO
Pituitary adenomas account for approximately 10% of intracranial tumors, but little is known of the oncogenesis of these tumors. The identification of tumor-specific genes may further elucidate the pathways of tumor formation. We used complementary DNA microarrays to examine gene expression profiles in nonfunctioning, PRL, GH, and ACTH secreting adenomas, compared with normal pituitary. Microarray analysis showed that 128 of 7075 genes examined were differentially expressed. We then analyzed three genes with unique expression patterns and oncogenic importance by RT-real time quantitative PCR in 37 pituitaries. Folate receptor gene was significantly overexpressed in nonfunctioning adenomas but was significantly underexpressed in PRL and GH adenomas, compared with controls and to other tumors. The ornithine decarboxylase gene was significantly overexpressed in GH adenomas, compared with other tumor subtypes but was significantly underexpressed in ACTH adenomas. C-mer proto-oncogene tyrosine kinase gene was significantly overexpressed in ACTH adenomas but was significantly underexpressed in PRL adenomas. We have shown that at least three genes involved in carcinogenesis in other tissues are also aberrantly regulated in the major types of pituitary tumors. The evaluation of candidate genes that emerge from these experiments provides a rational approach to investigate those genes significant in tumorigenesis.
Assuntos
Adenoma/genética , DNA Complementar/análise , Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Hipofisárias/genética , Receptores Proteína Tirosina Quinases , Receptores de Superfície Celular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adenoma/metabolismo , Hormônio Adrenocorticotrópico/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Transporte/genética , Feminino , Receptores de Folato com Âncoras de GPI , Hormônio do Crescimento Humano/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Ornitina Descarboxilase/genética , Neoplasias Hipofisárias/metabolismo , Prolactina/metabolismo , Prolactinoma/genética , Prolactinoma/metabolismo , Proteínas Tirosina Quinases/genética , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , c-Mer Tirosina QuinaseRESUMO
BACKGROUND: Chronic renal failure (CRF) is associated with premature arteriosclerosis, impaired high-density lipoprotein (HDL) maturation, increased pre-beta HDL (a lipid-poor HDL species), reduced HDL/total cholesterol ratio, hypertriglyceridemia, and depressed lipolytic activity. The latter has been, in part, attributed to elevated pre-beta HDL, which is a potent inhibitor of lipoprotein lipase (LPL). Accumulation of cholesterol in the arterial wall is a critical step in atherogenesis, and HDL-mediated cholesterol removal from peripheral tissues mitigates atherosclerosis. Lecithin:cholesterol acyltransferase (LCAT) is essential for maturation of HDL and cholesterol removal by HDL from peripheral tissues. Earlier studies have revealed depressed plasma LCAT enzymatic activity in patients with CRF. This study was conducted to determine whether impaired LCAT activity can be confirmed in CRF animals and if so whether it is due to down-regulation of hepatic LCAT expression. METHODS: Hepatic tissue LCAT mRNA and plasma LCAT enzymatic activity were measured in male Sprague-Dawley rats six weeks after excisional 5/6 nephrectomy or sham operation. RESULTS: Compared with the controls, the CRF group exhibited a significant reduction of hepatic tissue LCAT mRNA abundance. The reduction in hepatic LCAT mRNA was accompanied by a marked reduction of plasma LCAT activity and elevation of serum-free cholesterol in the CRF animals. LCAT activity correlated positively with the HDL/total cholesterol ratio and inversely with free cholesterol and triglyceride concentrations. CONCLUSIONS: CRF leads to a marked down-regulation of hepatic LCAT mRNA expression and plasma LCAT activity. This abnormality can impair HDL-mediated cholesterol uptake from the vascular tissue and contribute to cardiovascular disease. In addition, LCAT deficiency can, in part, account for elevated serum-free cholesterol, reduced HDL/total cholesterol, and elevated pre-beta HDL in CRF. The latter can, in turn, depress lipolytic activity and hinder triglyceride-rich lipoprotein clearance in CRF.
Assuntos
Regulação Enzimológica da Expressão Gênica , Falência Renal Crônica/metabolismo , Fígado/enzimologia , Fosfatidilcolina-Esterol O-Aciltransferase/genética , Animais , Northern Blotting , Doenças Cardiovasculares/metabolismo , Colesterol/sangue , Creatinina/sangue , Hipertrigliceridemia/metabolismo , Lipoproteínas HDL/sangue , Masculino , Fosfatidilcolina-Esterol O-Aciltransferase/sangue , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Triglicerídeos/sangueRESUMO
Lecithin-cholesterol acetyltransferase (LCAT) is involved in the synthesis of plasma cholesteryl esters and is pivotal in the maturation of plasma high-density lipoprotein (HDL) and conversion of HDL3 to HDL2. In nephrotic syndrome (NS), the ratio of HDL2 to HDL3 is low even though the total concentration of HDL is generally normal. We hypothesize that the reduced HDL2/HDL3 ratio in NS is due to urinary losses of LCAT, leading to plasma LCAT deficiency. To test this hypothesis, Sprague-Dawley rats were randomized to NS (given 130 mg puromycin aminonucleoside on day 1 and 60 mg ip on day 14) or control groups and were studied on day 30. To dissect the effect of proteinuria from hypoalbuminemia, a group of Nagase rats with inherited hypoalbuminemia was included. Hepatic LCAT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA abundance and plasma and urine LCAT activity were measured. The NS group showed a fourfold rise in serum cholesterol and triglycerides, a fivefold rise in free cholesterol, and a fourfold fall in the HDL-to-total cholesterol ratio. Despite severe hypoalbuminemia, the Nagase rats showed only a mild elevation of serum cholesterol and triglycerides with a normal serum free cholesterol and HDL-to-total cholesterol ratio. The NS group exhibited a normal hepatic LCAT-to-GAPDH mRNA ratio, a marked reduction in plasma LCAT activity, and a significant increase in urinary LCAT excretion. LCAT/GAPDH mRNA and plasma and urine LCAT were normal in Nagase rats. Thus NS led to heavy urinary losses and reduced plasma concentration of LCAT, despite normal hepatic LCAT mRNA abundance. However, hypoalbuminemia, per se, without proteinuria as seen in the Nagase rats had no effect on plasma LCAT or the HDL-to-total cholesterol ratio. Therefore, proteinuria, not hypoalbuminemia, causes LCAT deficiency and a depressed HDL-to-total cholesterol ratio in NS.
Assuntos
Síndrome Nefrótica/enzimologia , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Animais , Northern Blotting , Colesterol/sangue , Testes de Função Renal , Lipídeos/sangue , Fígado/enzimologia , Masculino , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Previous studies demonstrated that structural perturbation of the alpha(1) domain of apolipoprotein B (apoB) blocked the initiation of lipoprotein assembly. We explored the hypothesis that this domain may interact with the inner leaflet of the endoplasmic reticulum membrane in a manner that may nucleate microsomal triglyceride transfer protein-dependent lipid sequestration. ApoB-17 (amino-terminal 17% of apoB), which contains most of the alpha(1) domain, was expressed stably in rat hepatoma cells and recovered from medium in lipid-poor form. On incubation with phospholipid vesicles composed of 1-myristol-2-myristoyl-sn-glycero-3-phosphocholine or 1-palmitoyl-2-oleoyl-sn-gylycero-3-phosphocholine, apoB-17 underwent vesicle binding and was recovered in the d < 1.25 g/ml gradient fraction. To determine whether vesicle binding is disrupted by the same structural perturbations that block lipoprotein assembly in vivo, apoB-17 was subjected to partial and complete chemical reduction. Although normally a soluble peptide, mild reduction of apoB-17 caused its precipitation, suggesting that hydrophobic, solvent-inaccessible domains within the alpha(1) domain of apoB are stabilized by intramolecular disulfide bonds. In contrast to apoB-17 chemically reduced in vitro, forms of apoB-17 bearing pairwise cysteine-to-serine substitutions were recovered in soluble form from transiently transfected COS-1 cell extracts. Although individual disruption of disulfide bond 2 or 4 in apoB-28 and apoB-50 was previously shown to block lipoprotein assembly in vivo, these alterations had no impact on the ability of apoB-17 to bind to phospholipid vesicles in vitro or on its capacity to form recombinant lipoprotein particles. These results suggest that while the vesicle/lipid-binding property of the alpha(1) domain may reflect an essential role required for the initiation of lipoprotein formation, some other aspect of alpha(1) domain function is perturbed by disruption of native disulfide bonds. -- DeLozier, J. A., J. S. Parks, and G. S. Shelness. Vesicle-binding properties of wild-type and cysteine mutant forms of alpha(1) domain of apolipoprotein B. J. Lipid Res. 2001. 42: 399--406.
Assuntos
Apolipoproteínas B/química , Apolipoproteínas B/metabolismo , Cisteína , Lipossomos/metabolismo , Animais , Apolipoproteínas B/genética , Sítios de Ligação , Soluções Tampão , Células COS , Cromatografia de Afinidade , Cisteína/genética , Dimiristoilfosfatidilcolina , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Temperatura Alta , Neoplasias Hepáticas Experimentais/metabolismo , Mutagênese , Desnaturação Proteica , Ratos , Proteínas Recombinantes , Solubilidade , Relação Estrutura-Atividade , Transfecção , Células Tumorais CultivadasAssuntos
Triagem Neonatal/normas , Humanos , Recém-Nascido , Triagem Neonatal/métodos , Estados UnidosRESUMO
OBJECTIVE: To determine the separate and combined effects of the estrogen and progestin components of a modern triphasic oral contraceptive (OC) formulation on extent of coronary artery atherosclerosis. METHODS: Female cynomolgus monkeys (n = 81) were fed atherogenic diets for 32 months. After the first 7 months, they were randomized to four groups and treated triphasically for 21 of each 28 days with ethinyl estradiol (E2) (monkey equivalent of 30-40 microg), levonorgestrel (monkey equivalent of 50-125 microg), a combination of the two steroids, or placebo. RESULTS: Treatment with estrogen alone reduced coronary artery atherosclerosis extent 67% compared with untreated controls (P <.05). Treatment with progestin alone had no effect (P >.20). While atherosclerosis extent in monkeys treated with the combined OC was reduced 28%, this did not differ statistically from the other groups (P >.20). CONCLUSION: In doses used for oral contraception, E2, like all other estrogens studied to date, has a marked inhibitory effect on atherosclerosis progression. Levonorgestrel, at doses used in modern OC formulations, antagonizes this effect. When considered with other experimental evidence, these findings support the concept that progestins used in OCs and hormone replacement therapy can antagonize estrogen's atheroinhibitory effects. Whether this occurs seems to depend on a relative balance between estrogen and progestin with respect to dose, potency, route, and pattern of administration. However, when considered with evidence from previous studies, the findings also indicate a modest atheroinhibitory influence of combination (estrogen-progestin) OCs.
Assuntos
Anticoncepcionais Orais Combinados/uso terapêutico , Doença da Artéria Coronariana/prevenção & controle , Congêneres do Estradiol/uso terapêutico , Etinilestradiol/uso terapêutico , Levanogestrel/uso terapêutico , Congêneres da Progesterona/uso terapêutico , Animais , Anticoncepcionais Orais Combinados/administração & dosagem , Modelos Animais de Doenças , Esquema de Medicação , Congêneres do Estradiol/administração & dosagem , Etinilestradiol/administração & dosagem , Feminino , Levanogestrel/administração & dosagem , Lipoproteínas/sangue , Macaca fascicularis , Congêneres da Progesterona/administração & dosagem , Distribuição AleatóriaRESUMO
The molecular pathogenesis of the majority of sporadic pituitary tumors is largely unknown. Pituitary adenomas can develop sporadically or as a part of multiple endocrine neoplasia type 1 (MEN1). The MEN1 is thought to be a tumor suppressor gene based on loss of heterozygosity (LOH) for polymorphic markers on 11q13 in tumors of the pancreas, parathyroid, and pituitary. Most patients with familial and sporadic MEN1 carry germ-line mutations in the MEN1 gene. Two previous studies and recently a third one have analyzed mutations by sequencing the MEN1 gene in sporadic pituitary tumors but yielded conflicting results. This study was to investigate and clarify the potential role of MEN1 mutations, in sporadic pituitary adenomas. First, we examined 59 sporadic pituitary adenomas by analyzing LOH on 11q13 in the MEN1 minimal interval with microsatellite analysis. We found 3 tumors with LOH in 1 to 4 polymorphic markers in the MEN1 region. Sequencing analysis did not reveal any mutations in the coding region of the MEN1 gene. However, we found 3 polymorphisms, one of which was a novel CAC to CAT transition encoding His433His, in exon 9. The data show that while LOH occurs in some sporadic pituitary tumors, inactivating mutations of the tumor suppressor gene MEN1 are rare. These results also suggest there may be another additional tumor suppressor gene at this locus which is involved in the pathogenesis of sporadic pituitary neoplasms.
Assuntos
Adenoma/genética , Genes Supressores de Tumor , Neoplasia Endócrina Múltipla Tipo 1/genética , Mutação , Neoplasias Hipofisárias/genética , Adulto , Idoso , Autorradiografia , Sequência de Bases , Cromossomos Humanos Par 11 , DNA/química , Feminino , Humanos , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo Genético , Análise de Sequência de DNARESUMO
The purpose of this study was to test the hypothesis that lipid fluidity regulates lecithin:cholesterol acyltransferase (LCAT) activity. Phosphatidylcholine (PC) species were synthesized that varied in fluidity by changing the number, type (cis vs. trans), or position of the double bonds in 18 or 20 carbon sn-2 fatty acyl chains and recombined with [(3)H]cholesterol and apolipoprotein A-I to form recombinant high density lipoprotein (rHDL) substrate particles. The activity of purified human plasma LCAT decreased with PC sn-2 fatty acyl chains containing trans versus cis double bonds and as double bonds were moved towards the methyl terminus of the sn-2 fatty acyl chain. The decrease in LCAT activity was significantly correlated with a decrease in rHDL fluidity (measured by diphenylhexatriene fluorescence polarization) for PC species containing 18 carbon (r(2) = 0.61, n = 18) and 20 carbon (r(2) = 0.93, n = 5) sn-2 fatty acyl chains. rHDL were also made containing 10% of the 18 carbon sn-2 fatty acyl chain PC species and 90% of an inert PC ether matrix (sn-1 18:1, sn-2 16:0 PC ether) to normalize rHDL fluidity. Even though fluidity was similar among the PC ether-containing rHDL, the order of PC reactivity with LCAT was significantly correlated (r(2) = 0.71) with that of 100% PC rHDL containing the same 18 carbon sn-2 fatty acyl chain species, suggesting that PC structure in the active site of LCAT determines reactivity in the absence of measurable differences in bilayer fluidity. We conclude that PC fluidity and structure are major regulators of LCAT activity when fatty acyl chain length is constant.
Assuntos
Lipoproteínas HDL/metabolismo , Fluidez de Membrana , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Apolipoproteína A-I/metabolismo , Humanos , Isomerismo , Espectrometria de FluorescênciaRESUMO
Isolated growth hormone deficiency (IGHD) IB is an autosomal recessive disorder characterized by a good response to exogenous growth hormone (GH) treatment without development of anti-GH antibodies. Patients with IGHD IB were found to be compound heterozygotes for deletion and frameshift mutations as well as homozygotes for splicing mutations in the GH-1 gene. Recently, a novel splicing mutation in the GH-1 gene was identified in an extended, consanguineous Arab-Bedouin family from Israel with IGHD IB. Prior to the identification of this mutation, a considerable number of children with short stature in this family were found normal on pharmacological stimulation for GH release. This observation prompted a genotype/phenotype correlation of potential heterozygotes in the family. Carriers of the mutant GH-1 allele were found as a group to have a significantly shorter stature than normal homozygote (mean standard deviation scores, 1.67 and -0.40, respectively, P<0.05). Moreover, 11 of 33 (33%) heterozygotes, but only 1 of 17 (5.9%) normal homozygotes, had their height at 2 or more SD below the mean. Overall, 48.5% of studied heterozygotes were found to be of appreciably short stature with height at or lower than the 5th centile (> or = -1.7 SD), whereas only 5.9% of the normal homozygotes did (P<0.004). This phenomenon of heterozygotes for a recessive mutation in the GH-1 gene manifesting short stature, might imply that some such mutations may account for non-GH deficiency reduced height in the general population.
Assuntos
Estatura/genética , Mutação da Fase de Leitura , Genes Recessivos , Triagem de Portadores Genéticos , Hormônio do Crescimento/deficiência , Feminino , Homozigoto , Humanos , Masculino , Linhagem , FenótipoRESUMO
In vivo multicompartmental modeling of the turnover of HDL subfractions has suggested that HDL containing four molecules of apoA-I per particle and no other apolipoproteins (large LpA-I) are terminal particles in plasma. We hypothesized that these terminal particles were the end product of HDL metabolism and, as such, would be cleared preferentially by the liver. Thus, the purpose of this study was to determine: 1) the tissue sites of catabolism of large LpA-I in African green monkeys, and 2) whether saturated versus n;-6 polyunsaturated dietary fat affected tissue accumulation. Large LpA-I were isolated, without ultracentrifugation, by size exclusion and immunoaffinity chromatography and radiolabeled with either the residualizing compound, (125)I-labeled tyramine cellobiose (TC), or with (131)I. After injection into recipient animals, the plasma die-away of the radiolabels was followed for 12 or 24 h, after which the animals were killed and tissues were collected for determining radiolabel sites of catabolism. The plasma die-away of the (125)I-labeled TC-LpA-I and (131)I-labeled LpA-I doses was similar suggesting that the TC radiolabeling did not modify the metabolism of the large LpA-I dose. The liver, adrenal, kidney, and spleen had the greatest accumulation of large LpA-I degradation products on a per gram tissue basis. On a whole organ basis, the liver was the major site of large LpA-I degradation in both the 12-h (15.4 +/- 0.3% of injected dose) and 24-h (9.1 +/- 0.6% of injected dose) catabolic studies. The kidney, compared to the liver, had less uptake of large LpA-I radioactivity in either study (1.3 +/- 0.4% and 1.2 +/- 0.3% of injected dose). There was no apparent influence of dietary fat type on the tissue accumulation of large LpA-I. We conclude that the liver is the primary site of catabolism of large LpA-I in the African green monkey.
Assuntos
Apolipoproteína A-I/metabolismo , Animais , Apolipoproteína A-I/isolamento & purificação , Chlorocebus aethiops , Cromatografia de Afinidade/métodos , Cromatografia em Gel , Gorduras na Dieta/administração & dosagem , MasculinoRESUMO
We have found a novel mutation in intron 4 of the GH-1 gene in a Bedouin kindred with isolated growth hormone deficiency type IB (IGHD IB). RFLP analysis suggested linkage between the GH-1 gene and IGHD. Nested PCR amplification followed by single stranded conformation polymorphism (SSCP) analysis indicated sequence variation between introns 2 and 4. Sequencing showed a G-->C transversion at the fifth base in the splice donor region of intron 4. Affected individuals were homozygous for the mutation, which creates a new Mae III restriction site. Reverse transcription and PCR of GH-1 transcripts in EBV transformed lymphocytes indicated predominance of a species lacking 73 bp of exon 4. Amplification with a bridging primer showed that the same mRNA species is present in lymphocytes from normal individuals. The first 102 amino acids of the predicted protein are identical to wild-type GH, but the next 94 amino acids are completely divergent.
Assuntos
Processamento Alternativo , Hormônio do Crescimento Humano/deficiência , Hormônio do Crescimento Humano/genética , Árabes , Sequência de Bases , Pré-Escolar , Consanguinidade , Feminino , Humanos , Íntrons , Linhagem , Polimorfismo de Fragmento de Restrição , Polimorfismo Conformacional de Fita Simples , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Basic and translational research achievements over the past 2 decades have disclosed the molecular mechanisms underlying several genetic forms of hypopituitarism. Disorders that are limited to the hypothalamic, pituitary, GH axis are caused by mutations in individual components of that axis. Disorders involving GH and one or more additional pituitary hormones are caused by mutations in the homeodomain transcription factors that direct embryological development of the anterior pituitary gland. Pit-1 has a POU-specific and a POU-homeo DNA-binding domain. The phenotype produced by mutations in the PIT1 gene involves deficiencies of GH, PRL, and TSH. Pituitary glands are either small or normally sized. The PROP1 gene encodes a transcription factor with a single paired-like DNA-binding domain. Persons with inactivating mutations in PROP1 have deficiencies of LH and FSH, as well as GH, PRL, and TSH. Their pituitary glands may be small, normally sized, or extremely large and show suprasellar extension. Pituitary degeneration may produce acquired deficiency of ACTH. Expression of the HESX1 gene precedes expression of PROP1 and PIT1, and it is much more widespread. The protein has a paired-like domain, and it competes with the product of PROP1 for DNA-binding. Homozygosity for inactivating mutations of HESX1 produces a complex phenotype that resembles septo-optic dysplasia. Much more needs to be learned about the role of HESX1 mutations in other forms of hypopituitarism.
Assuntos
Hipopituitarismo/genética , Proteínas de Membrana , Hipófise/crescimento & desenvolvimento , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteínas de Transporte/genética , Proteínas de Homeodomínio/genética , Humanos , Mutação , Proteínas de Transferência de Fosfolipídeos , Fatores de Transcrição HES-1 , Fatores de Transcrição/genéticaRESUMO
This review will address contributions of nuclear transcription factors to the embryologic development and definitive function of the anterior pituitary gland. The HESX1, PITX1, PITX2, PROP1 and POU1F1 genes are of particular interest because of their recognized or potential associations with human disease. Mutations of any of the first three genes produce complex disease phenotypes such as septo-optic dysplasia, Treacher Collins Franceschetti syndrome or Rieger syndrome that may include deficiency of one or more pituitary hormones. Mutations in PROP1 or POU1F1, or their mouse homologous, result in severe hypopituitarism as well as morphological abnormalities of the pituitary gland.
Assuntos
Proteínas Nucleares , Hipófise/embriologia , Fatores de Transcrição/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Modelos Animais de Doenças , Genes Homeobox , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Humanos , Hipopituitarismo/embriologia , Hipopituitarismo/genética , Hipopituitarismo/fisiopatologia , Camundongos , Camundongos Mutantes , Fatores de Transcrição Box Pareados , Fenótipo , Hipófise/fisiologia , Proteínas Repressoras , Fatores de Transcrição HES-1 , Fatores de Transcrição/genética , Proteína Homeobox PITX2RESUMO
Mechanisms producing the divergent phenotypes, Wolman disease (WD) and cholesterol ester storage disease (CESD), associated with the genetic deficiency of human lysosomal acid lipase/cholesterol ester hydrolase (hLAL) function were investigated with the determination of HLAL activity levels, mRNA and protein expression, and defects in structural gene sequences in cells from three WD and five CESD patients. Measured with natural substrates, HLAL activities were all below 2% of normal, regardless of phenotype. Immunoblotting showed a lack of detectable hLAL protein in all mutant fibroblasts. Four CESD, but no WD genomes contained at least one allele with a specific exon 8 splice junction mutation, c.894 G>A, that encodes a shortened form of hLAL mRNA. Other CESD mutations were identical in type to the WD defects: nucleotide deletions (positions 397, 684, 980), insertions (594), or substitutions (193, 347) that result in premature terminations precluding any function. The only exception was a substitution at nucleotide 866 in the CESD case without an exon 8 splicing mutation; expression of the predicted S289C change in a transfection assay produced a low, but clearly measurable, level of acid esterase activity. Although it is not easily demonstrated in conventional assays, CESD is distinct from WD in that at least one mutant allele has the potential to produce enough residual enzymatic function to ameliorate the phenotype; in the majority of CESD cases this may come from a single, easily detected, splicing mutation in one allele.
Assuntos
Doença do Armazenamento de Colesterol Éster/genética , Lipase/genética , Mutação , Doença de Wolman/genética , Adulto , Substituição de Aminoácidos , Animais , Sequência de Bases , Células COS , Células Cultivadas , Criança , Doença do Armazenamento de Colesterol Éster/enzimologia , Éxons , Feminino , Fibroblastos/enzimologia , Humanos , Lactente , Lipase/metabolismo , Masculino , Mutagênese Sítio-Dirigida , Fenótipo , Mutação Puntual , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Deleção de Sequência , Transfecção , Doença de Wolman/enzimologiaRESUMO
We hypothesized that small HDL particles, containing two apoA-I molecules but no apoA-II (LpAI), may be converted in vivo into medium and large HDL particles, containing three or four apoA-I molecules, respectively, and that more conversion will occur in animals with higher HDL concentrations. To test this possibility, kinetic studies of small LpAI were performed in African green monkeys with either high plasma HDL cholesterol concentrations (120 +/- 36 mg/dl, mean +/- SD, n = 3) or low plasma HDL cholesterol concentrations (40 +/- 13 mg/dl, n = 3). Tracer small LpAI was purified, without ultracentrifugation, by immunoaffinity and gel filtration. After injection, the specific activity of apoA-I in small, medium, and large HDL, consisting of both LpAI and LpAI:AII particles, was followed. A multicompartmental model was developed with the simultaneous analysis of urine and plasma turnover data for the kinetics of apoA-I in small, medium, and large HDL. These analyses indicated that small HDL is converted to either medium or large HDL with little or no interconversion of medium HDL and large HDL. Much of the metabolic conversion of small HDL occurs in a sequestered pool, effectively outside the circulating plasma, in a unidirectional manner before reentering the circulating plasma as medium or large HDL. The mean fractional catabolic rate of apoA-I in small, medium, and large HDL was not different comparing the high and low HDL group. In contrast, the mean production rate of apoA-I was greater in the high HDL group compared with the low HDL group. These data support the hypothesis that the plasma concentration of HDL is primarily a function of the rate of appearance of apoA-I in medium and large HDL.