RESUMO
We report a case of a woman with a cryptic balanced translocation between chromosomes 5 and 17, suspected during genetic counseling. The woman had a history of previous fetal losses attributed to lissencephaly and intra uterine growth retardation (IUGR) and a daughter with dysmorphic features and mental retardation, previously attributed to a small deletion 5pter, detected years ago by a first generation CGH-array. This peculiar combination of personal and family history suggested the opportunity to carry out a FISH approach, focusing on chromosomes 5 and 17, based on the idea that a malsegregation secondary to a balanced translocation, might have escaped the first CGH array. This approach allowed the identification of a balanced translocation in the mother, FISH in the affected child confirmed the partial 5p deletion predicted by the previous CGH array and identified a new 17p duplication that had not been detected before. The described translocation opens the possibility of alternative imbalances that were probably responsible for previous fetal losses. The imbalances were confirmed by a new high resolution SNP array. We conclude that despite the availability of highly effective and sensitive genomic approaches a careful evaluation of medical history is highly recommended since it can suggest clinical hypothesis that can be confirmed by more classical and molecular cytogenetic based approaches.
RESUMO
The serum- and glucocorticoid-regulated kinase (Sgk1) is essential for hormonal regulation of epithelial sodium channel-mediated sodium transport and is involved in the transduction of growth factor-dependent cell survival and proliferation signals. Growing evidence now points to Sgk1 as a key element in the development and/or progression of human cancer. To gain insight into the mechanisms through which Sgk1 regulates cell proliferation, we adopted a proteomic approach to identify up- or downregulated proteins after Sgk1-specific RNA silencing. Among several proteins, the abundance of which was found to be up- or downregulated upon Sgk1 silencing, we focused our attention of RAN-binding protein 1 (RANBP1), a major effector of the GTPase RAN. We report that Sgk1-dependent regulation of RANBP1 has functional consequences on both mitotic microtubule activity and taxol sensitivity of cancer cells.
Assuntos
Carcinoma/genética , Carcinoma/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Imediatamente Precoces/metabolismo , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transcrição Gênica , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Paclitaxel/farmacologia , Fosforilação , Proteômica , Interferência de RNA , Fator de Transcrição Sp1/metabolismoRESUMO
Mutations in the gene (GJB2) coding for Connexin 26 (Cx26) are responsible for genetic forms of sensorineural hearing loss. This article describes a family characterized by congenital profound hearing loss, inherited in an autosomal dominant fashion and associated to a R75Q substitution in Cx26. Cell transfection and fluorescence imaging, dye transfer experiments and dual patch clamp recording showed that the mutant completely prevents the formation of functional channels despite assembling into junctional plaques, in communication incompetent HeLa cells. The disease is not associated with palmar and plantar keratosis in any of the family members, suggesting that R75Q substitution is not sufficient for the development of the complete syndromic phenotype. The association of palmar and plantar keratosis with profound hearing loss may be dependent on genetic background, requiring a functional interaction between the mutated Cx26 and other epidermally expressed connexins.
Assuntos
Conexinas/genética , Perda Auditiva Neurossensorial/genética , Conexina 26 , Análise Mutacional de DNA , Eletrofisiologia , Genes Dominantes , Células HeLa , Humanos , Ceratodermia Palmar e Plantar/genética , Mutação de Sentido Incorreto , Técnicas de Patch-Clamp , Linhagem , FenótipoRESUMO
The association between angiotensin-converting enzyme (ACE) gene polymorphism and insulin resistance (IR) in hypertensive subjects remains controversial. Thus, we evaluated the possible association between IR and ACE gene polymorphism in a group of hypertensive, never-treated patients compared with that in a normotensive control group. We enrolled 200 (114 men and 86 women; age, 45.5 +/- 4.7 yr) hypertensive patients and 96 (54 men and 42 women; age, 44.0 +/- 4.7 yr) normotensive subjects. A double PCR assay was used to identify ACE genotypes. We determined fasting glucose and insulin by the glucose oxidase method and using a standard RIA technique. IR was estimated using the homeostasis model assessment (HOMA(IR)). Both fasting glucose (5.0 +/- 0.3 vs. 4.7 +/- 0.3 mmol/L; P < 0.0001), insulin levels (12.3 +/- 4.7 vs. 4.9 +/- 1.5 muU/mL; P < 0.0001), and HOMA(IR) (2.7 +/- 1.1 vs. 1.1 +/- 0.3; P < 0.0001) were significantly higher in hypertensive patients than in the normotensive control group. When we subdivided hypertensive patients according to ACE genotype, we observed that fasting insulin and HOMA(IR) were 16.3 +/- 3.3 and 3.6 +/- 0.8 in the DD genotype, 9.4 +/- 3.1 and 2.1 +/- 0.7 in the ID genotype, and 8.3 +/- 2.8 and 1.9 +/- 0.7 muU/mL in the II group (P < 0.0001, by ANOVA). No significant differences were observed in the normotensive control group. In conclusion, we extended previous data regarding the relationship of hypertension and IR by demonstrating a dependence of this relationship upon the ACE gene polymorphism.
Assuntos
Hipertensão/fisiopatologia , Resistência à Insulina , Peptidil Dipeptidase A/genética , Polimorfismo Genético , Adulto , Alelos , Glicemia/análise , Feminino , Frequência do Gene , Genótipo , Homeostase , Humanos , Hipertensão/genética , Insulina/sangue , Masculino , Pessoa de Meia-Idade , Valores de ReferênciaRESUMO
Sgk (serum- and glucocorticoid-induced protein kinase) is a serine/threonine-specific protein kinase that is transcriptionally regulated by serum, glucorticoids, and mineralocorticoids. Sgk regulates the amiloride-sensitive sodium channel in kidney principal cells. Insulin and insulin-like growth factor-1 stimulate activity of Sgk by a mechanism mediated by phosphoinositide-dependent kinases (PDK)-1 and -2. In this study, we demonstrate that incubation of transfected cells with 8-(4-chlorophenylthio)-cAMP (8CPT-cAMP; 0.2 mm) led to a 2-fold activation of recombinant Sgk expressed in COS7 cells. Furthermore, the combination of insulin plus 8CPT-cAMP elicited a larger response than either agent alone. The effect of insulin was inhibited by wortmannin (100 nm), but not by the cyclic AMP-dependent protein kinase (PKA) inhibitor, H89 (10 microm). As expected, the effect of 8CPT-cAMP was completely blocked by H89. Surprisingly, the effect of 8CPT-cAMP was also inhibited by wortmannin, suggesting that phosphorylation of Sgk by PDK-1 and/or -2 is required for activation by 8CPT-cAMP. Mutational analysis led to similar conclusions. The Thr(369) --> Ala mutant, lacking the PKA phosphorylation site, was activated by insulin but not 8CPT-cAMP. In contrast, the Ser(422) --> Ala mutant, lacking a PDK-2 phosphorylation site, was inactive and resistant to activation by either insulin or 8CPT-cAMP. In summary, Sgk is subject to complex regulatory mechanisms. In addition to regulation at the level of gene expression, the enzymatic activity of Sgk is regulated by multiple protein kinases, including PKA, PDK-1, and PDK-2. Cross-talk among these signaling pathways may play an important role in the pathogenesis of the hypertension associated with hyperinsulinemia, obesity, and insulin resistance.
Assuntos
AMP Cíclico/farmacologia , Insulina/farmacologia , Proteínas Nucleares , Proteínas Serina-Treonina Quinases/metabolismo , Sulfonamidas , Androstadienos/farmacologia , Animais , Sangue , Células COS , Indução Enzimática , Inibidores Enzimáticos/farmacologia , Glucocorticoides/farmacologia , Proteínas Imediatamente Precoces , Isoquinolinas/farmacologia , Mutagênese , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , WortmaninaRESUMO
The impact of molecular genetics in the diagnosis and management of various forms of heritable cardiac or vascular disorders is continuously increasing thanks to the newly available laboratory tools. Familial hypertrophic cardiomyopathy (FHC), an autosomal dominant inherited disease characterized by unexplained left ventricular hypertrophy and a wide range of clinical symptoms, is the first cardiac disorder whose genetic bases have been elucidated. Linkage analysis studies have shown a statistically significant association between the disease status and at least seven genetic loci, all coding for sarcomeric proteins, in unrelated kindreds. A major challenge for physicians is to make an accurate and early diagnosis, not only on the basis of the traditional tools (i.e. physical examination and electro-echocardiography) but also to focus on the impact of genotype on clinical manifestations of FHC. In this review we present the more recent findings on the genetic basis of FHC and analyze the genotype-phenotype correlations of this disorder, whose expression may be modulated by additional factors (modifier genes, genetic background, environmental factors) other than mutations in any of the sarcometric proteins.
Assuntos
Cardiomiopatia Hipertrófica/genética , Animais , Galinhas/genética , Genótipo , Humanos , Cadeias Pesadas de Miosina/genética , Cadeias Leves de Miosina/genética , Fenótipo , Proteína C/genética , Troponina T/genéticaRESUMO
The response of the forearm vasculature to acetylcholine (7.5, 15, and 30 microg/min, each for 5 minutes) and sodium nitroprusside (0.8, 1.6, and 3.2 microg/min, each for 5 minutes) was evaluated in 32 never-treated hypertensive outpatients (17 men and 15 women, aged 43+/-7 years) and in 24 normotensive control subjects (14 men and 10 women, aged 42+/-6 years). Drugs were infused into the brachial artery, and forearm blood flow was measured by strain-gauge plethysmography. In both hypertensive and normotensive groups, a deletion (D)/insertion (I) polymorphism in intron 16 of the angiotensin-converting enzyme (ACE) gene was determined by polymerase chain reaction. The response to acetylcholine was significantly reduced in hypertensive patients versus control subjects: at the highest dose (30 microg/min), forearm blood flow was 13.9+/-6.3 mL x 100 mL tissue(-1) x min(-1) in hypertensives versus 27.1+/-9.7 mL x 100 mL tissue(-1) x min(-1) in the controls (P<.001); similarly, vascular resistance was 10.6+/-5.6 U in hypertensive patients and 4.9+/-1.9 U in normotensive subjects. In the hypertensive group, the patients with DD genotype showed significantly less endothelium-dependent vasodilation compared with ID+II genotypes (at the highest dose of acetylcholine, forearm blood flow was 12.1+/-4.2 versus 17.0+/-4.1 mL x 100 mL tissue(-1) x min(-1)) (P<.005). The vasodilator effect of sodium nitroprusside infusions was not statistically different in DD and ID+II hypertensive patients. In conclusion, our data suggest that ACE polymorphism affects endothelium-dependent vasodilation in hypertensive patients and confirm that hypertensive patients had a blunted response to the endothelium-dependent agent acetylcholine.
Assuntos
Endotélio Vascular/fisiologia , Hipertensão/genética , Peptidil Dipeptidase A/genética , Vasodilatação/fisiologia , Adulto , Análise de Variância , Pressão Sanguínea , Feminino , Genótipo , Humanos , Hipertensão/fisiopatologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Polimorfismo GenéticoRESUMO
We report a PCR-based technique for detecting thyroid cancer metastases in small nodes <1.5 cm diameter by the amplification of thyroid specific transcripts TSH-receptor and thyroglobulin. A 100% correspondence with the histopathological diagnosis was observed in the 41/46 nodes (89%) in which an adequate sample was obtained at fine needle aspiration. The genetic analysis resulted more sensitive and accurate than both the cytological analysis (28% inadequate samples, 17% false negative diagnoses) and the thyroglobulin measurement in the aspirates (39% false negatives). The PCR-based genetic analysis may provide a useful tool for diagnosis and follow-up of thyroid cancer.
Assuntos
Metástase Linfática/diagnóstico , Metástase Linfática/genética , Neoplasias da Glândula Tireoide/genética , Biópsia por Agulha , Gliceraldeído-3-Fosfato Desidrogenases/análise , Humanos , Linfonodos/metabolismo , Linfonodos/patologia , Metástase Linfática/patologia , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Receptores da Tireotropina/genética , Tireoglobulina/genética , Neoplasias da Glândula Tireoide/patologiaRESUMO
OBJECTIVES: This study sought to evaluate the possible association of polymorphism of the angiotensin-converting enzyme (ACE) gene with blood pressure and left ventricular mass index (LVMI). BACKGROUND: The renin-angiotensin system seems to be involved in the pathogenesis of essential hypertension. Moreover, recent epidemiologic observations demonstrate that many subjects with left ventricular hypertrophy have normal blood pressure levels, suggesting that factors other than hemodynamic overload may contribute to the hypertrophy. METHODS: The study included 140 untreated hypertensive outpatients who underwent ambulatory blood pressure monitoring, echocardiographic evaluation and analysis for insertion (I)/ deletion (D) polymorphism in intron 16 of the ACE gene by polymerase chain reaction. Blood pressure was measured at 24 h, and LVMI was calculated by the Devereux formula, in each patient. RESULTS: Left ventricular mass index values (mean +/- SD) were 137 +/- 28 g/m2 in patients with the DD genotype, 125 +/- 27 g/m2 in those with the ID genotype and 115 +/- 27 g/m2 in those with II genotype. The frequencies of the DD, ID and II genotypes were 45.71% (n = 64), 46.42% (n = 65) and 7.85% (n = 11), respectively, and were in Hardy-Weinberg equilibrium. The strongest association between left ventricular mass and DD genotype in our cohort appeared to be an independent cardiovascular risk factor (DD vs. ID: odds ratio [OR] 2.497, 95% confidence interval [CI] interval 1.158 to 5.412, p < 0.05; DD vs. II: OR 6.577, 95% CI 1.169 to 28.580, p < 0.02). CONCLUSIONS: Our data show that the LVMI was significantly enhanced in patients with the DD genotype.
Assuntos
Deleção de Genes , Hipertensão/genética , Hipertrofia Ventricular Esquerda/genética , Peptidil Dipeptidase A/genética , Polimorfismo Genético , Adulto , Idoso , Ecocardiografia , Feminino , Genótipo , Humanos , Itália , Masculino , Pessoa de Meia-IdadeRESUMO
A previously undescribed de novo insertion-deletion mutation in the beta cardiac myosin heavy chain gene was found in a kindred with familial hypertrophic cardiomyopathy. In the mutated allele there is an inserted-deleted guanine at nucleotides 8823 and 8850 of the beta myosin heavy chain gene, resulting in a dramatic change of the amino acid sequence (AA 395-404). such a mutation, detected in the proband and in his son but not in the proband's parents, is likely to produce major impairment of myosin function.
Assuntos
Cardiomiopatia Hipertrófica/genética , Mutação , Cadeias Pesadas de Miosina/genética , Adulto , Humanos , Masculino , LinhagemRESUMO
The present work describes the distribution of HCV genotypes in Calabria. The data presented suggest that, in the sample of population investigated, genotype 1b is the most prevalent followed by the 2b and the 2a.. In addition it is important to note that in Calabria the prevalence of genotype 1b is strikingly high in respect to the other Italian pullulation. An Association between HCV type 1b and the more severe clinical course of the liver disease has been reported. Although the data presented indicate that in Calabria most of the subjects enrolled in the study are infected by a virulent HCV strain, no association has been found with more severe clinical manifestations.
Assuntos
Hepacivirus/genética , Hepatite C/epidemiologia , Hepatite Crônica/epidemiologia , RNA Viral/química , Adulto , Fatores Etários , Idoso , Feminino , Hepacivirus/isolamento & purificação , Hepatite C/virologia , Hepatite Crônica/virologia , Humanos , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Reação em Cadeia da Polimerase , Prevalência , Fatores de RiscoAssuntos
Cardiomiopatia Hipertrófica/genética , Biomarcadores , Feminino , Genótipo , Heterozigoto , Homozigoto , Humanos , Masculino , Mutação , Miosinas/genética , Fenótipo , População , Tropomiosina/genética , Troponina/genética , Troponina TRESUMO
The ACE gene has recently been shown to be associated with myocardial infarction, especially in subjects considered at low risk for coronary heart disease (CHD) according to common classification criteria. The possible relationship between deletion polymorphism in this gene and CHD risk factors, as well as asymptomatic extracoronary atherosclerosis, has been investigated in the present study. One hundred and seventy-four subjects, enrolled in a cardiovascular disease prevention study, underwent clinical and biochemical examination and ACE-I/D polymorphism determination. Subjects > 45 years of age (n = 107) also received echo-Doppler examination of the carotid arteries. Based on the results of ACE-I/D polymorphism, subjects were divided into three groups: homozygous for deletion (D/D), homozygous for insertion (I/I) and heterozygous (I/D). The prevalence of CHD risk factors as well as of extracoronary atherosclerosis was similar in the three genotype groups. Similarly, there was no association between the presence of atherosclerotic lesions and genotype in subjects at low and high CHD risk. Ten subjects with diabetes mellitus had ACE-D/D genotype. Among these subjects seven had hypertension. Eight subjects with diabetes mellitus had ACE-I/D genotype and only one of these was hypertensive. None of the ACE-I/I subjects was diabetic. ACE-I/D polymorphism seems to play a role in the development of hypertension, at least in diabetic subjects. Its determination may help to identify and monitor diabetic subjects prone to hypertension.
Assuntos
Diabetes Mellitus Tipo 2/genética , Hipertensão/genética , Peptidil Dipeptidase A/genética , Polimorfismo Genético , Adulto , Sequência de Bases , Marcadores Genéticos , Humanos , Hipertensão/etiologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência MolecularRESUMO
In the course of studies to elucidate the complex network of interactions controlling FRTL5 cell proliferation, thyroid stimulating hormone (TSH)-independent mutants (M cells), have been obtained from FRTL5 cells by chemical mutagenesis. In the present studies, the role of TSH on the proliferation and on differentiated and metabolic functions in these mutant cells have been investigated and compared to their response to insulin-like growth factor I (IGF-I). The addition of IGF-I to M cells leads to normal stimulation of DNA synthesis. However, inspite of the fact that mutant cells display normal TSH receptors, TSH is unable to stimulate the proliferation of the M cells. Nevertheless, TSH is able to increase intracellular levels of cAMP leading to regulation of TSH function in the M cells. On the other hand, TSH does not influence iodide transport and actin filaments depolimerization in these cells. However, aminoacid transport, stimulated in wild-type FRTL5 cells by both TSH and IGFs, is under the control of IGFs but not of TSH in the mutant cells. Neither TSH or IGF-I modified the expression of c-fos proto-oncogene in the M cells, probably because of high constitutive expression. These data suggest that a crucial signalling step(s) required for TSH induced mitogenesis is impaired in the M cells, and that this signalling step is not required for IGF-I induced mitogenesis.
Assuntos
AMP Cíclico/fisiologia , Transdução de Sinais , Glândula Tireoide/citologia , Ácidos Aminoisobutíricos/metabolismo , Animais , Transporte Biológico , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Replicação do DNA/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Iodetos/metabolismo , Proteínas Proto-Oncogênicas c-fos/biossíntese , Ratos , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/metabolismo , Tireotropina/metabolismo , Tireotropina/farmacologiaRESUMO
Rat thyroid cells (FRTL5), transfected with the sequence coding for rat insulin-like growth factor II (IGF-II) presented mRNA specific for the transfected IGF-II in most of the clones obtained (Tr clones). Tr7 and Tr12 cells maintained their ability to respond to the mitogenic effect of thyrotropin (TSH), while either exogenous IGF-I or IGF-II or insulin failed to stimulate their proliferation. In the absence of exogenous mitogens the Tr7 and Tr12 clones vigorously incorporated [3H]thymidine into DNA. This activity was significantly inhibited by sm1.2, a monoclonal antibody against rat IGF-II. Tr7 and Tr12 clones possess type I IGF receptors, known to mediate the mitogenic effect of IGF-II, with affinity similar to those present on the membrane of the parental cells but with reduced capacity. Finally, media conditioned by Tr7 and Tr12 increase basal thymidine incorporation in quiescent FRTL5 cells and amplify that induced by TSH. Endogenous IGFs may play an important role in the regulation of thyroid cell proliferation by modulating the mitogenic effect of TSH and by supporting TSH-independent growth.
Assuntos
Fator de Crescimento Insulin-Like II/farmacologia , Glândula Tireoide/efeitos dos fármacos , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura/farmacologia , Replicação do DNA/efeitos dos fármacos , Sinergismo Farmacológico , Insulina/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Insulin-Like II/genética , Mitose/efeitos dos fármacos , Ratos , Proteínas Recombinantes/farmacologia , Glândula Tireoide/citologia , Tireotropina/farmacologia , TransfecçãoRESUMO
A tumor surface protein (TSP-180) has been identified on murine lung carcinomas using two monoclonal antibodies (MoAbs) (135-13C and 346-11A). Quantitative analysis of TSP-180 on 3LL variants maintained either in vitro or in vivo indicates that TSP-180 is highly expressed in highly malignant metastatic cells. In reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns of TSP-180 obtained with MoAb 135-13C from cell lysates of 3LL metastatic cells show three proteins migrating to Mr 204,000, 134,000, and 116,000. In the same experimental conditions MoAb 135-13C precipitates from low metastasizing ones only one band, corresponding to the lower molecular weight (Mr 116,000). All bands of TSP-180 observed in 3LL variants are labeled by lactoperoxidase-catalyzed radioiodination of viable cells, incorporate 32PO4, and contain carbohydrates, as judged by binding to wheat germ agglutinin. These results indicate that all proteins have external exposure on the cell surface and that at least some of TSP-180 proteins could be differentially regulated in different tumor cells (highly metastatic versus low metastatic). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns and immunoblots obtained from cell lysates of 3LL variants by using a monoclonal antibody to phosphotyrosine (IG-2) indicate that this MoAb recognizes proteins migrating with molecular weights identical to those reported for TSP-180. Moreover, the immunoblots of solubilized immunocomplex, obtained from cell lysates of 3LL variants by using MoAb 135-13C, demonstrate that MoAb IG-2 specifically reacts with TSP-180 proteins. Experiments undertaken in order to assess if some or all of TSP-180 proteins have tyrosine kinase activity demonstrate that MoAb 135-13C binding to the cell surface induces specific phosphorylation of the Mr 204,000 protein of TSP-180. Phosphoaminoacid analysis of the ligand-induced phosphorylated protein (pp204) demonstrates that this protein is phosphorylated at serine and tyrosine. Results reported lead us to hypothesize that TSP-180 is involved in growth-regulation mechanisms and that its high expression on cells with more malignant phenotype could be responsible for a proliferative advantage of such tumor clones.
Assuntos
Antígenos de Neoplasias/metabolismo , Antígenos de Superfície/metabolismo , Metástase Neoplásica , Aminoácidos/análise , Animais , Anticorpos Monoclonais , Antígenos de Neoplasias/análise , Antígenos de Neoplasias/imunologia , Integrina alfa6beta4 , Ligantes , Neoplasias Pulmonares/análise , Masculino , Camundongos , Peso Molecular , Neoplasias Experimentais/análise , Fenótipo , FosforilaçãoRESUMO
Receptors for Insulin, Epidermal Growth Factor, Platelet-Derived Growth Factor and Insulin-like Growth Factor type 1 are tyrosine-specific protein kinases. This enzymatic activity may play a role in mediating the biological actions of these peptides. It has recently been identified a Mr 120 KDa glycoprotein in rat liver plasma membranes which can be phosphorylated by the insulin receptor and by the EGF receptor in a cell-free system and by the insulin receptor in intact cultured H-35 hepatoma cells. In the present report it is shown that the solubilized Insulin-like Growth Factor type 1 receptor can phosphorylate tyrosine residues in the same 120 KDa glycoprotein from the AS-30D rat hepatoma cells.
Assuntos
Neoplasias Hepáticas Experimentais/enzimologia , Glicoproteínas de Membrana/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptor de Insulina/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Feminino , Técnicas de Imunoadsorção , Insulina/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Microssomos Hepáticos/análise , Peso Molecular , Fosforilação , Fosfotirosina , Ratos , Ratos Endogâmicos , Receptores de Somatomedina , Células Tumorais Cultivadas , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
A tumor surface protein (TSP-180) that is highly expressed on highly malignant metastatic cells has been identified on murine lung carcinomas. On SDS-PAGE under reducing conditions, TSP-180 shows a complex banding pattern corresponding to 204, 183, 150, 135, and 116 kDa. All bands of the TSP-180 complex are glycosylated and are labeled by lactoperoxidase-catalyzed radioiodination of viable cells. The mouse TSP-180 complex described here is homologous to the human integrin alpha 6 beta 4 complex, and in particular it has been demonstrated that protein corresponding to 204 kDa is homologous to the beta 4 subunit of the integrin complex. It has been shown recently that monoclonal antibody to TSP-180 (MoAb 135-13C) stimulates cell growth in vitro and induces phosphorylation of the 204-kDa protein. We now report that insulin increases the phosphorylation of the 204-kDa protein 30-fold in intact carcinoma cells and epidermal growth factor (EGF) causes a threefold increase. Insulin-like growth factor (IGF-I) and platelet-derived growth factor have no effect. The effect of insulin and of IGF-I on phosphorylation of their own receptors was studied using solubilized cell membranes. Insulin and IGF-I each induced a fivefold increase in the phosphorylation of their respective receptor beta subunits. In order to test if phosphorylation of the 204-kDa protein was induced by direct binding of growth factors to TSP-180 and to identify growth factor receptors on line 1 cells, affinity cross-linking studies were performed. Affinity labeling of receptors demonstrated that insulin and IGF-I both bind to a 135-kDa protein that corresponds to the insulin and IGF-I receptor alpha subunits. Affinity labeling of EGF receptors failed to demonstrate EGF receptor molecules (175-kDa protein) on line 1 cells. Further investigations by using a different approach confirmed the very low amount of EGF receptors on line 1 cells. Direct phosphoamino acid analysis of the 204-kDa protein purified from insulin-stimulated cells demonstrated that this beta 4 integrin subunit is phosphorylated on serine and tyrosine. We conclude that beta 4 integrin molecule is a target for phosphorylation through an indirect receptor-mediated mechanism.
Assuntos
Antígenos de Neoplasias/metabolismo , Carcinoma/metabolismo , Insulina/farmacologia , Integrinas/metabolismo , Aminoácidos/análise , Animais , Antígenos de Neoplasias/análise , Eletroforese em Gel de Poliacrilamida , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/análise , Feminino , Integrina alfa6beta4 , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , FosforilaçãoRESUMO
FRTL-5 rat thyroid cells have receptors for both insulin and IGF-I which can be distinguished in binding studies. The ability of TSH to regulate each in an antiparallel manner is atypical. If these receptors are shown to have independent as well as coordinate activities, studies of the mechanisms of their receptor cross-talk in these cells will be relevant to understanding IGF-I and insulin receptors in other tissues.
Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Insulina/metabolismo , Receptor de Insulina/metabolismo , Receptores de Superfície Celular/metabolismo , Somatomedinas/metabolismo , Glândula Tireoide/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Reagentes de Ligações Cruzadas/farmacologia , Interações Medicamentosas , Insulina/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Ligação Proteica , Ratos , Receptores de Somatomedina , Glândula Tireoide/citologia , Tireotropina/farmacologiaRESUMO
The aim of this study was to compare the metabolic effects of a high-carbohydrate (CHO), high-fiber diet with only moderate protein restriction with those of a low-CHO, low-fiber diet with a low protein content in six diabetic patients with moderate chronic renal failure. The high-CHO, high-fiber diet induced a significant improvement in blood glucose control, a significant decrease in serum cholesterol, and a significant increase in fecal nitrogen losses. Other variables evaluated were not significantly different between the two diets, except for a significant increase in serum phosphorus during the high-CHO, high-fiber diet. N balance was not significantly different from 0 at the end of either dietary period and was very similar for both diets. The high-CHO, high-fiber diet presents many beneficial metabolic effects in diabetic patients with chronic renal failure.