Biomedical subjects

Daniel Giannella-Neto

Publications and source records attributed to Daniel Giannella-Neto.

13 recordsLinked to original sources

PubMed2007

PTTG expression in different experimental and human prolactinomas in relation to dopaminergic control of lactotropes.

BACKGROUND: Pituitary tumor transforming gene (pttg) is a novel oncogene that is expressed at higher level in most of the tumors analyzed to date compared to normal tissues. Nevertheless, its expression in prolactinomas and its relation with the pituitary dopamine receptor 2 (D2R) are not well defined. We sought to determine the pituitary level of pttg in three different experimental models of prolactinomas with altered dopaminergic control of the pituitary: the dopaminergic D2R knockout female mouse, the estrogen-treated rat, and the senescent female rat. These three models shared the characteristics of increased pituitary weight, hyperprolactinemia, lactotrope hyperplasia and reduced or absent dopaminergic action at the pituitary level. We also studied samples from human macroprolactinomas, which were characterized as responsive or resistant to dopamine agonist therapy. RESULTS: When compared to female wild-type mice, pituitaries from female D2R knockout mice had decreased PTTG concentration, while no difference in pttg mRNA level was found. In senescent rats no difference in pituitary PTTG protein expression was found when compared to young rats. But, in young female rats treated with a synthetic estrogen (Diethylstylbestrol, 20 mg) PTTG protein expression was enhanced (P = 0.029). Therefore, in the three experimental models of prolactinomas, pituitary size was increased and there was hyperprolactinemia, but PTTG levels followed different patterns.Patients with macroprolactinomas were divided in those in which dopaminergic therapy normalized or failed to normalize prolactin levels (responsive and resistant, respectively). When pituitary pttg mRNA level was analyzed in these macroprolactinomas, no differences were found. We next analyzed estrogen action at the pituitary by measuring pituitary estrogen receptor alpha levels. The D2R knockout female mice have low estrogen levels and in accordance, pituitary estrogen receptors were increased (P = 0.047). On the other hand, in senescent rats estrogen levels were slightly though not significantly higher, and estrogen receptors were similar between groups. The estrogen-treated rats had high pharmacological levels of the synthetic estrogen, and estrogen receptors were markedly lower than in controls (P < 0.0001). Finally, in patients with dopamine resistant or responsive prolactinomas no significant differences in estrogen receptor alpha levels were found. Therefore, pituitary PTTG was increased only if estrogen action was increased, which correlated with a decrease in pituitary estrogen receptor level. CONCLUSION: We conclude that PTTG does not correlate with prolactin levels or tumor size in animal models of prolactinoma, and its pituitary content is not related to a decrease in dopaminergic control of the lactotrope, but may be influenced by estrogen action at the pituitary level. Therefore it is increased only in prolactinomas generated by estrogen treatment, and not in prolactinomas arising from deficient dopamine control, or in dopamine resistant compared with dopamine responsive human prolactinomas. These results are important in the search for reliable prognostic indicators for patients with pituitary adenomas which will make tumor-specific therapy possible, and help to elucidate the poorly understood phenomenon of pituitary tumorigenesis.

Adult↗

PubMed2006

Fibronectin and laminin induce expression of islet cell markers in hepatic oval cells in culture.

Hepatic oval cells (OC) are considered to be facultative liver stem cells and, because they may undergo differentiation into a variety of cell lineages, they might have the potential to be used in cellular therapy. Signals delivered by extracellular matrix (ECM) proteins take part in cellular differentiation in cooperation with signals from growth factors; indeed, some ECM proteins, such as laminin (LAM) and fibronectin (FN), have been shown to contribute to beta-cell differentiation and islet development, respectively. Since no previous studies have investigated the effect of ECM proteins on the expression of islet cell markers by cultured OC, the purpose of the present study was to evaluate whether FN and LAM modulate the expression of genes related to the endocrine pancreas in these liver cells. OC proliferation was induced in Wistar rats by prolonged treatment with 2-acetoaminofluorene/allyl alcohol and confirmed by reverse transcription/polymerase chain reaction and hepatic immunocytochemical and histopathological analyses. OC isolation was performed by Ficoll gradient and magnetic-activated cell sorting. OC were cultured for 1 and 2 months under several conditions with specific growth factors, over a FN or LAM substrate or in high glucose, nicotinamide and fetal calf serum. OC cultured on FN substrate expressed Pdx-1, Pax-6, insulin 2 and glucagon. LAM also induced the expression of Pdx-1, insulin 1 and insulin 2, glucagon, somatostatin and GLUT-2. Our results suggest that these ECM proteins can be used in protocols of OC transdifferentiation aimed at reducing the period necessary for complete transdifferentiation.

2-Acetylaminofluorene↗

PubMed2006

Hepatic gene expression profile associated with non-alcoholic steatohepatitis protection by S-nitroso-N-acetylcysteine in ob/ob mice.

BACKGROUND/AIMS: To understand the molecular mechanisms underlying non-alcoholic steatohepatitis (NASH) prevention by S-nitroso-N-acetylcysteine (SNAC), an NO donor that inhibits lipid peroxidation, we examined hepatic differentially expressed genes between ob/ob mice receiving or not SNAC treatment concomitantly with a methionine-choline deficient (MCD) diet. METHODS: Ob/ob mice were assigned to receive oral SNAC fed solution (MCD+SNAC group) or vehicle (MCD group) by gavage. After four weeks, histopathological analysis and microarray hybridizations were conducted in liver tissues from groups. GeneSifter system was used to identify differentially expressed genes and pathways according to Gene Ontology. RESULTS: NASH was absent in the MCD+SNAC group and no significant changes in food intake or body weight were observed in comparison to MCD group. After SNAC treatment, several genes belonging to oxidative phosphorylation, fatty acid biosynthesis, fatty acid metabolism and glutathione metabolism pathways were down-regulated in comparison to the MCD group. CONCLUSIONS: SNAC treatment promotes down regulation of several genes from fatty acid (FA) metabolism related pathways, possibly through abrogation of the cytotoxic effects of reactive oxygen species and lipid peroxides with consequent prevention of mitochondrial overload. Further studies are required to investigate the clinical implications of these findings, in attempt to develop novel therapeutic strategies for NAFLD treatment.

Acetylcysteine↗

PubMed2006

Increased hepatic expression of insulin-like growth factor-I receptor in chronic hepatitis C.

AIM: Although increased insulin-like growth factor-I receptor (IGF-IR) gene expression has been reported in hepatocellular carcinoma, studies assessing IGF-IR in chronic hepatitis C (CHC) and cirrhosis are scarce. We therefore aimed to evaluate IGF-IR and IGF-I mRNA expression in liver from patient with CHC. METHODS: IGF-IR and IGF-I mRNA content were determined by semi-quantitative RT-PCR and IGF-IR protein expression was determined by immunohisto-chemistry in hepatic tissue obtained from patients with CHC before (34 patients) and after (10 patients) therapy with interferon-alpha and ribavirin. RESULTS: An increase of IGF-IR mRNA content was observed in hepatic tissue obtained from all CHC patients as well as from 6 cadaveric liver donors following orthopic transplantation (an attempt to evaluate normal livers) in comparison to normal liver, while no relevant modifications were detected in IGF-I mRNA content. The immunohistochemical results showed that the raise in IGF-IR mRNA content was related both to ductular reaction and to increased IGF-IR expression in hepatocytes. A decrease in IGF-IR mRNA content was observed in patients who achieved sustained virological response after therapy, suggesting an improvement in hepatic damage. CONCLUSION: The up-regulation of IGF-IR expression in hepatocytes of patients with CHC could constitute an attempt to stimulate hepatocyte regeneration. Considering that liver is the organ with the highest levels of IGF-I, our finding of increased IGF-IR expression after both acute and chronic hepatic damage highlights the need for additional studies to elucidate the role of IGF-I in liver regeneration.

Antiviral Agents↗

PubMed2006

Hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) and guanylate kinase 1 (GUK1) are differentially expressed in GH-secreting adenomas.

Pituitary tumors, adenomas in their vast majority, represent around 10-15% of the intracranial neoplasms. Pituitary carcinomas are exceedingly rare. Clinically, these neoplasms cause hormonal dysfunctions, and mass effect symptoms as headache and visual disorders in the case of macroadenomas. Pituitary tumorigenesis is still poorly understood. In order to investigate the expression of cancer-related genes in pituitary tumors, we employed a human cancer cDNA macroarray membrane with 1176 well-characterized human genes related to cancer and tumor biology. We were able to identify several differentially expressed genes, among them hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) and guanylate kinase 1 (GUK1) which were over expressed in a pool of clinically nonfunctioning pituitary adenomas, compared with a spinal cord metastasis of a nonfunctioning pituitary carcinoma. HGS and GUK1 mRNA expression were chosen to be validated by quantitative RT-qPCR, however, only GUK1 had the differential expression confirmed between the adenomas and the metastasis of a pituitary carcinoma. We have also investigated HGS and GUK1 mRNA expressions in a series of 46 pituitary adenomas (18 nonfunctioning, 12 GH-secreting, nine PRL-secreting, and seven ACTH-secreting adenomas). HGS and GUK1 were significantly over expressed in GH-secreting adenomas, compared with ACTH-secreting adenomas and nonfunctioning tumors, and with PRL-secreting adenomas, respectively. We have shown that these genes, involved in tumorigenesis in other tissues, are as well over expressed in the pituitary tumors, however, their role in the oncogenesis of these tumors need to be further investigated.

ACTH-Secreting Pituitary Adenoma↗

PubMed2006

Effect of gastric bypass on spontaneous growth hormone and ghrelin release profiles.

OBJECTIVE: The purpose of this study was to analyze growth hormone (GH) concentrations in obese women before and after Roux-en-Y gastric bypass (RYGBP) and how resulting changes in weight, fat mass, ghrelin levels, and insulin sensitivity affect GH secretion. RESEARCH METHODS AND PROCEDURES: Blood was sampled at 20-minute intervals for 24 hours in 10 non-diabetic premenopausal severely obese women before and 6 months after RYGBP. GH concentrations were measured in all samples, and serum ghrelin was collected at five time-points. RESULTS: After a 27% BMI drop (55.9 +/- 6.2 to 40.7 +/- 5.8 kg/m2), blunted GH profiles underwent partial recovery. Basal, postprandial, and mean ghrelin concentrations were not changed. A negative correlation was found between mean GH levels and insulin and homeostasis model assessment (p < 0.01). BMI accounted for 54% of GH variation. DISCUSSION: Partial recovery of GH secretion after RYGBP-induced weight loss suggests that a blunted secretion is not a causal factor of obesity but a consequence of the obese state and does not seem to be ghrelin-level dependent.

Adult↗

PubMed2006

Somatostatin receptor subtype 5 (SSTR5) mRNA expression is related to histopathological features of cell proliferation in insulinomas.

Insulinomas are rare endocrine neoplasias that constitute the most frequent islet cell tumours. Somatostatin (SST) analogs are tentatively used to inhibit insulin secretion and control tumour growth in patients with local invasion or inoperative metastasis, but variable responses have been reported. Data regarding somatostatin receptor (SSTR) subtypes expression in insulinomas are conflicting. In this study, we evaluated 16 cases of primary insulinomas (including four primary plurihormonal tumours) and two hepatic metastases. Histopathological and immunohistochemical analysis for some features associated with tumour aggressiveness and semi-quantitative RT-PCR for SSTR1-5 and real-time qPCR for SSTR5 were performed. SSTR subtypes 1, 3, and 5 were expressed in 100%, SSTR2 in 89%, and SSTR4 only in 22% of the insulinomas. SSTR5 mRNA was positively correlated with histopathological features related to tumour aggressiveness (large tumour diameter, well-differentiated endocrine tumour with uncertain behaviour and higher number of cells with nuclear atypia). SSTR5 mRNA expression in primary insulinomas was lower than in primary plurihormonal tumours (P < 0.05). The observed positive correlation between SSTR5 expression and tumour size suggests that the use of SST analogues more specific to SSTR5 in the treatment of insulinomas deserves attention.

Adenoma, Islet Cell↗

PubMed2005

Human macroprolactin displays low biological activity via its homologous receptor in a new sensitive bioassay.

CONTEXT: Macroprolactinemia is a frequent finding in hyperprolactinemic individuals, usually without clinical impact. Data on biological activity of macroprolactin (bbPRL) are controversial and mostly based on a heterologous rat Nb2 cell bioassay. Biological activity of bbPRL observed in vitro but not in vivo may be due to its high molecular weight, preventing its passage through capillary barrier. Alternatively, bbPRL bioactivity may differ depending on the prolactin (PRL) receptor (PRLR) species specificity. OBJECTIVE: The objective of the study was to characterize the bioactivity of bbPRL in a homologous bioassay: Ba/F-3 cells stably expressing the human PRLR. DESIGN/SETTING/PATIENTS: Chromatography-purified bbPRL from macroprolactinemic individuals (group I, n = 18) and monomeric PRL from hyperprolactinemic patients without macroprolactinemia (group II, n = 5) were tested in Nb2 and Ba/F-LLP bioassays. Both groups were followed up at the neuroendocrinology outpatients' clinic. MAIN OUTCOME MEASURE: Biological activity of bbPRL presented in the two bioassays was measured. RESULTS: In group I, no patient had hypogonadism. Mean ratio bioactivity to immunoactivity of bbPRL in the Nb2 assay was 0.69. There was no dose-response in 15 of the 18 samples tested in Ba/F-LLP assay. In group II, three patients had galactorrhea and all five had hypogonadism. Mean ratio bioactivity to immunoactivity of monomeric PRL samples was 1.35 in Nb2 and 0.91 in Ba/F-LLP assay. CONCLUSION: Whereas both bioassays achieve similar results with respect to monomeric PRL activity, our results indicate that the activity displayed by bbPRL toward the rat receptor may be inappropriate because it is not observed in the human PRLR-mediated assay, consistent with the apparent absence of bioactivity in vivo.

Adult↗

PubMed2004

Identification and complete sequencing of novel human transcripts through the use of mouse orthologs and testis cDNA sequences.

The correct identification of all human genes, and their derived transcripts, has not yet been achieved, and it remains one of the major aims of the worldwide genomics community. Computational programs suggest the existence of 30,000 to 40,000 human genes. However, definitive gene identification can only be achieved by experimental approaches. We used two distinct methodologies, one based on the alignment of mouse orthologous sequences to the human genome, and another based on the construction of a high-quality human testis cDNA library, in an attempt to identify new human transcripts within the human genome sequence. We generated 47 complete human transcript sequences, comprising 27 unannotated and 20 annotated sequences. Eight of these transcripts are variants of previously known genes. These transcripts were characterized according to size, number of exons, and chromosomal localization, and a search for protein domains was undertaken based on their putative open reading frames. In silico expression analysis suggests that some of these transcripts are expressed at low levels and in a restricted set of tissues.

Amino Acid Sequence↗

PubMed2004

A transcript finishing initiative for closing gaps in the human transcriptome.

We report the results of a transcript finishing initiative, undertaken for the purpose of identifying and characterizing novel human transcripts, in which RT-PCR was used to bridge gaps between paired EST clusters, mapped against the genomic sequence. Each pair of EST clusters selected for experimental validation was designated a transcript finishing unit (TFU). A total of 489 TFUs were selected for validation, and an overall efficiency of 43.1% was achieved. We generated a total of 59,975 bp of transcribed sequences organized into 432 exons, contributing to the definition of the structure of 211 human transcripts. The structure of several transcripts reported here was confirmed during the course of this project, through the generation of their corresponding full-length cDNA sequences. Nevertheless, for 21% of the validated TFUs, a full-length cDNA sequence is not yet available in public databases, and the structure of 69.2% of these TFUs was not correctly predicted by computer programs. The TF strategy provides a significant contribution to the definition of the complete catalog of human genes and transcripts, because it appears to be particularly useful for identification of low abundance transcripts expressed in a restricted set of tissues as well as for the delineation of gene boundaries and alternatively spliced isoforms.

Alternative Splicing↗

PubMed2003

The generation and utilization of a cancer-oriented representation of the human transcriptome by using expressed sequence tags.

Whereas genome sequencing defines the genetic potential of an organism, transcript sequencing defines the utilization of this potential and links the genome with most areas of biology. To exploit the information within the human genome in the fight against cancer, we have deposited some two million expressed sequence tags (ESTs) from human tumors and their corresponding normal tissues in the public databases. The data currently define approximately 23,500 genes, of which only approximately 1,250 are still represented only by ESTs. Examination of the EST coverage of known cancer-related (CR) genes reveals that <1% do not have corresponding ESTs, indicating that the representation of genes associated with commonly studied tumors is high. The careful recording of the origin of all ESTs we have produced has enabled detailed definition of where the genes they represent are expressed in the human body. More than 100,000 ESTs are available for seven tissues, indicating a surprising variability of gene usage that has led to the discovery of a significant number of genes with restricted expression, and that may thus be therapeutically useful. The ESTs also reveal novel nonsynonymous germline variants (although the one-pass nature of the data necessitates careful validation) and many alternatively spliced transcripts. Although widely exploited by the scientific community, vindicating our totally open source policy, the EST data generated still provide extensive information that remains to be systematically explored, and that may further facilitate progress toward both the understanding and treatment of human cancers.

Chromosome Mapping↗

PubMed2002

Genotype-phenotype correlation in inherited severe insulin resistance.

The insulin receptor is a ligand-activated tyrosine kinase. Mutations in the corresponding gene cause the rare inherited insulin-resistant disorders leprechaunism and Rabson-Mendenhall syndrome. Patients with the most severe syndrome, leprechaunism, have growth restriction, altered glucose homeostasis and early death (usually before 1 year of age). Rabson-Mendenhall syndrome is less severe, with survival up to 5-15 years of age. These disorders are transmitted as autosomal recessive traits. Here we report six new patients and correlate mutations in the insulin receptor gene with survival. Patients with leprechaunism were homozygous or compound heterozygous for mutations in the extracellular domain of the insulin receptor and their cells had markedly impaired insulin binding (<10% of controls). Mutations in their insulin receptor gene inserted premature stop codons (E124X, R372X, G650X, E665X and C682X), resulting in decreased levels of mature mRNA, or affected the extracellular domain of the receptor (R86P, A92V, DeltaN281, I898T and R899W). Three patients with Rabson-Mendenhall syndrome had at least one missense mutation in the intracellular domain of the insulin receptor (P970T, I1116T, R1131W and R1174W). Expression studies in CHO cells indicated that the R86P, A92V, DeltaN281, I898T, R899W and R1131W mutations markedly impaired insulin binding (<5% of control), while the P970T, I1116T and R1174W mutant receptors retained significant insulin-binding activity. These results indicate that mutations in the insulin receptor retaining residual insulin-binding correlate with prolonged survival in our series of patients with extreme insulin resistance.

Amino Acid Substitution↗

PubMedDate not supplied

Correlation between genotype and phenotype in primary open angle glaucoma of Brazilian families with mutations in exon 3 of the TIGR/MYOC gene.

PURPOSE: To investigate the phenotype of primary open-angle glaucoma (POAG) in Brazilian families with mutation in exon 3 of TIGR/MYOC. METHODS: Seventy-eight POAG patients with a positive family history and eighteen unrelated patients with POAG were screened by automated DNA sequencing for mutations in exon 3 of the TIGR/MYOC gene. The pedigrees of POAG patients with mutations that lead to amino acid change were built. All available relatives of the index cases were also examined and genotyped by sequencing. RESULTS: Four sequence variants were identified in exon 3 of the TIGR/MYOC gene (Tyr347Tyr, Pro370Pro, Lys398Arg and Cys433Arg) from the 96 initially screened patients. The Lys398Arg mutation was previously described as a polymorphism and in our study did not segregate with POAG. The most prevalent mutation was Cys433Arg, affecting 3 index cases (3.1% or 3/96). In two different families, 8/56 subjects presented Cys433Arg mutation and had POAG, 5/56 had ocular hypertension and 8/56 had no disease manifestation. POAG patients had a median age at diagnosis of 43.25 yr (17-58 yr) and intraocular pressure (IOP) with a mean of 36.3 +/- 3.8mmHg for the right eye and 37.6 +/- 9.75 mmHg for the left eye. The group of patients with Cys433Arg mutation had significantly higher IOP (p<0.0007) and vertical cup/disc ratio when compared to the patients without mutation (p<0.023). CONCLUSIONS: Cys433Arg mutation in exon 3 of the TIGR/MYOC gene is related to juvenile-onset POAG (J-POAG) in Brazilian families and autosomal dominant inheritance. The phenotype of this mutation is characterized by varied ages at diagnosis, causing J-POAG and late-onset POAG, associated with high IOP.

Adolescent↗