RESUMO
UNLABELLED: The recurrence and progression of treated intracranial meningiomas highlights the problem of the type of follow-up that should be used and whether early complementary treatment is indicated. The aim of this study was to evaluate different biochemical markers involved in cell proliferation and transformation to identify new prognostic factors in intracranial meningiomas. Between 1989 and 2003, 120 intracranial meningiomas were studied biochemically. The levels of estrogen receptors (RE), progesterone receptors (RP), cathepsin B (CB), cathepsin L (CL), stefin A (ATA), stefin B (STB), cystatin C (CYSC), urokinase (u-PA), type 1 plasminogen activator inhibitors (PAI-1), cathepsin D (CD) and thymidine kinase activity (TK) were measured in tumor extracts using biochemical assays. RESULTS: Out of 120 meningiomas, 73 were grade I, 39 grade II and eight grade III according to the WHO classification. Of these patients, 17 showed recurrence. The mean follow-up was 47 months. Monofactorial analysis showed that expression of progesterone receptors (RP) had an inverse correlation with recurrence (p=0.0025 %) and that thymidine kinase activity (TK), cathepsin L (CL), the WHO grade and the degree of tumor resection correlated with recurrence (p<0.05). Principal component analysis and linear discriminant analysis confirmed these results. The results of this study confirm the importance of biological parameters (PR, CL, TK) as prognostic factors for the risk of recurrence in intracranial meningiomas.
Assuntos
Neoplasias Meníngeas/cirurgia , Meningioma/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Análise Discriminante , Feminino , Humanos , Masculino , Neoplasias Meníngeas/patologia , Meningioma/patologia , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/epidemiologia , Prognóstico , Estudos Retrospectivos , Adulto JovemRESUMO
Adrenomedullin (AM) is a multifunctional regulatory peptide with important angiogenic and mitogenic properties. Here we identify a region of stable secondary structure in the 5'-untranslated region (5' UTR) of human AM mRNA. Reverse transcriptase-polymerase chain reaction of the 5' UTR consistently resulted, in addition to the product with the expected size of 155 base pair (bp), in a second product with an approximately 65-bp deletion from the central region of the 5' UTR, suggesting the presence of a secondary structure. The presence of a stem-loop structure was confirmed by probing the 5' UTR with RNases with selectivity for single- or double-stranded RNA. We investigated the role of this stem-loop structure in expression of luciferase reporter gene in cultured cell lines. Reporter assays using a chimeric mRNA that combined luciferase and the 5' UTR of AM mRNA demonstrated a dramatic decrease of the reporter activity owing to a decreased translation, whereas the deletion of the stem-loop structure localized between nt +31 and +95 from the cap site led to the recovery of activity. Gel migration shift assays using cytosolic extracts from mammalian cell lines demonstrate a specific binding of a cytosolic protein to riboprobes containing the 5' UTR of AM but not to riboprobes either corresponding to other areas of the message or containing the 5' UTR but lacking the region of secondary structure. Although we conclude that the 5' UTR of the human AM mRNA can modulate the translation of AM mRNA in vivo, and that the predicted stem-loop structure is necessary for this inhibition, the functional consequences of the cis element-binding activity remain to be determined.
Assuntos
Regiões 5' não Traduzidas/química , Peptídeos/química , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/química , Adrenomedulina , Sequência de Bases , Células Cultivadas , DNA Complementar/química , Genes Reporter , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Alinhamento de SequênciaRESUMO
OBJECTIVE: Adrenomedullin (AM), a potent vasodilatator and antioxidative peptide, was shown recently to be expressed by adipose tissue. The aim of our study was to investigate the precise localization of AM within human adipose tissue, and to examine AM regulation in obesity. DESIGN: Subcutaneous (SC) and omental (OM) adipose tissues from 9 lean and 13 obese women were profiled for AM expression changes. Preadipocytes from human adipose tissue were isolated and differentiated under defined adipogenic conditions. METHODS: AM expression was analyzed by immunohistochemistry, in situ hybridization and quantitative RT-PCR. RESULTS: A strong AM expression was observed in vessel walls, stromal cell clusters and isolated stromal cells, some of them being CD 68 positive, whereas mature adipocytes were not labeled. Calcitonin receptor-like receptor and receptor activity-modifying proteins (RAMP) 2 and RAMP 3 were expressed in vessel walls. In vitro, preadipocytes of early differentiation stages spontaneously secreted AM. No difference in AM localization was found between SC and OM adipose tissue. AM levels in SC tissue did not differ between lean and obese subjects. By contrast, AM levels in OM tissue were significantly higher in obese as compared with lean women. Moreover, we found a positive relationship between OM AM and tumor necrosis factor alpha mRNA levels and AM-immunoreactive area in OM tissue followed the features of the metabolic syndrome. CONCLUSION: Stromal cells from human adipose tissue, including macrophages, produce AM. Its synthesis increased in the OM territory during obesity and paralleled the features of the metabolic syndrome. Therefore, AM should be considered as a new member of the adipokine family.
Assuntos
Tecido Adiposo/metabolismo , Obesidade/metabolismo , Peptídeos/metabolismo , Adrenomedulina , Adulto , Antropometria , Análise Química do Sangue , Peso Corporal/fisiologia , Diferenciação Celular/fisiologia , Feminino , Hemodinâmica/fisiologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Pessoa de Meia-Idade , Peptídeos/genética , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismo , Células Estromais/metabolismo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
There is an increasing demand for the evaluation of HER2 status in breast cancer. In this study, sections from fixed tissues and triton extracts of tissue homogenates were obtained from 163 malignant breast tumors and analyzed in parallel using immunohistochemistry combined with fluorescence in situ hybridization, as gold standard tests, and an ELISA test (c-erbB2/c-neu Rapid Format ELISA, Oncogene Research Products, USA). Tumor DNA was employed to evaluate two quantitative PCR methods: the HER2/neu DNA Quantification Kit (Roche Diagnostics GmbH, Germany), which uses the gastrin chromosome 17 reference gene, and our recently developed Oncolab qPCR assay, where both a chromosome 17 gene (somatostatin receptor type II (SSTR2)) and a non-chromosome 17 reference gene (glyceraldehyde-3-phosphate deshydrogenase (GAPDH)) were used to detect an increase in HER2 gene copy number and to evaluate the aneusomy of chromosome 17, respectively. By IHC/FISH and ELISA, HER2 was overexpressed in 27 (16.6%) and 24 (14.7%) samples, respectively. With the Roche and Oncolab qPCR assays, 29 (17.8%) samples showed a ratio of HER2/gastrin > or = 2.0 and 26 (16.0%) showed a ratio of HER2/SSTR2 > or = 2.0, respectively. In samples presenting HER2/SSTR2 <2.0 and HER2/GAPDH > or = 2.0, which was indicative of a chromosome 17 polysomy, we observed a modest increase in HER2 protein expression. Complete agreement between the four methods for HER2 status determination was obtained for 154 (94.5%) samples. Overall, these results demonstrate that quantitative PCR is a reliable method for analyzing HER2 status and chromosome 17 polysomy.
Assuntos
Neoplasias da Mama/genética , Aberrações Cromossômicas , Cromossomos Humanos Par 17/genética , Genes erbB-2 , Reação em Cadeia da Polimerase/métodos , Biomarcadores Tumorais/análise , Neoplasias da Mama/patologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Estudos Prospectivos , Receptor ErbB-2/biossíntese , Receptores de Estrogênio/análise , Sensibilidade e EspecificidadeRESUMO
After castration or therapeutic hormone deprivation, most cancer of the prostate (CaP) cells develop androgen-independent (AI) growth. In this work, we studied the effect of androgen depletion (castration) on the growth of experimental model LuCaP 23.1 xenograft. A total of 101 nude mice were implanted and analysed for their growth profile before experimental period 1 (11 weeks) and after castration experimental period 2 (15 weeks). For specific periods, tumors were harvested and assessed for molecular marker expression specific for CaP. Taking into account tumor dynamic growth, prior to castration we found 37 fast growing (FG) tumors (948.9+/-76.9 mm3) and 63 slow growing (SG) tumors (229.6+/-18.4 mm3). Real-time quantitative RT-PCR showed that in comparison to SGs, FGs contained elevated expression of epidermal growth factor receptor type 1 (HER1), urokinase plasminogen activator (uPA), thymidine phosphorylase (TP) and thymidilate synthase (TS) mRNAs expression and low levels of 5alpha-reductase 2 (5alpha-R2) mRNA. After castration all FG tumors progressed rapidly (by 5 weeks) to AI growth (FG-P). In SG castrated tumors, 66% of tumors showed retarded progression (by 12 weeks) to AI (SG-P), whereas 34% responded to castration (SG-R). Molecular analysis demonstrated distinct molecular profiles integrating different pathways associated with AI progression. The progressive tumors FG-P, and some tumors of SG-P subgroup, presented significantly high levels of HER1, epidermal growth factor receptor type 2 (HER2), TS, uPA, TP, tumor necrosis factor superfamily member 6 (FAS) and peptidylglycine alpha-amidating mono-oxygenase (PAM) mRNA all of which correlated with androgen receptor (AR) mRNA. The second subgroup of SG-P tumors showed a high expression of the anti-apoptotic gene Bcl-2. A third subgroup of SG-P tumors showed significant expression of hypoxia-related genes such as adrenomedullin (AM) after castration. LuCaP 23.1 xenograft represent a useful dynamic model to study pre-clinically new therapeutic molecules and evaluate non-randomized therapeutics protocols combining different target inhibition specific to each AI pathways.
Assuntos
Androgênios/fisiologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Neoplasias da Próstata/metabolismo , Transplante Heterólogo , Adrenomedulina , Animais , Castração , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Peptídeos/metabolismo , Antígeno Prostático Específico/metabolismo , Timidina Fosforilase/metabolismo , Fatores de Necrose Tumoral/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM) catalyzes the first step of the reaction and is dependent on copper, ascorbate, and molecular oxygen. Peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalyzes the second step of the reaction. Previous studies demonstrated that alternative splicing results in the production of bifunctional PAM proteins that are integral membrane or soluble proteins as well as soluble monofunctional PHM proteins. Rat PAM is encoded by a complex single copy gene that consists of 27 exons and encompasses more than 160 kilobases (kb) of genomic DNA. The 12 exons comprising PHM are distributed over at least 76 kb genomic DNA and range in size from 49-185 base pairs; four of the introns within the PHM domain are over 10 kb in length. Alternative splicing in the PHM region can result in a truncated, inactive PHM protein (rPAM-5), or a soluble, monofunctional PHM protein (rPAM-4) instead of a bifunctional protein. The eight exons comprising PAL are distributed over at least 19 kb genomic DNA. The exons encoding PAL range in size from 54-209 base pairs and have not been found to undergo alternative splicing. The PHM and PAL domains are separated by a single alternatively spliced exon surrounded by lengthy introns; inclusion of this exon results in the production of a form of PAM (rPAM-1) in which endoproteolytic cleavage at a paired basic site can separate the two catalytic domains. The exon following the PAL domain encodes the trans-membrane domain of PAM; alternative splicing at this site produces integral membrane or soluble PAM proteins. The COOH-terminal domain of PAM is comprised of a short exon subject to alternative splicing and a long exon encoding the final 68 amino acids present in all bifunctional PAM proteins along with the entire 3'-untranslated region. Analysis of hybrid cell panels indicates that the human PAM gene is situated on the long arm of chromosome 5.
Assuntos
Éxons , Íntrons , Oxigenases de Função Mista/genética , Complexos Multienzimáticos , Animais , Sequência de Bases , Southern Blotting , Células CHO , Mapeamento Cromossômico , Cromossomos Humanos Par 5 , Cricetinae , DNA/genética , DNA/isolamento & purificação , Dopamina beta-Hidroxilase/genética , Humanos , Células Híbridas , Fígado/enzimologia , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Oligonucleotídeos Antissenso , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Ratos , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
Recently, studies using somatostatin (SRIF) analogs preferential for either the SRIF receptor 2 (SSTR2) or the SSTR5 subtype demonstrated a variable suppression of GH and PRL release from GH-secreting human adenomas. These data suggested the concept of SSTR subtype specificity in such tumors. In the present study the quantitative expression of messenger ribonucleic acid (mRNA) for the 5 SSTR subtypes and the inhibitory effects of SRIF14; SRIF28; octreotide; the SSTR2-preferential analog, BIM-23197; and the SSTR5-preferential analog, BIM-23268, on GH and PRL secretion were analyzed in cells cultured from 15 acromegalic tumors. RT-PCR analysis revealed a consistent pattern of SSTR2 and SSTR5 mRNA expression. SSTR5 mRNA was expressed at a higher level (1052 +/- 405 pg/pg glyceraldehyde-3-phosphate dehydrogenase) than SSTR2 mRNA (100 +/- 30 pg/pg glyceraldehyde-3-phosphate dehydrogenase). However, only SSTR2 mRNA expression correlated with the degree of GH inhibition induced by SRIF14, SRIF28, and BIM-23197. The SSTR5-preferential compound inhibited GH release in only 7 of 15 cases. In cells cultured from the 10 mixed adenomas that secreted both GH and PRL, RT-PCR analysis revealed a consistent coexpression of SSTR5, SSTR2, and SSTR1 mRNA. In all cases SRIF14, SRIF28, and the SSTR5-preferential analog, BIM-23268, significantly suppressed PRL secretion, with a mean maximal inhibition of 48 +/- 4%. In contrast, the SSTR2-preferential analogs, BIM-23197 and octreotide, were effective in suppressing PRL in only 6 of 10 cases. In cells cultured from adenomas taken from patients partially responsive to the SRIF analog, octreotide, partial additivity in suppressing both GH and PRL secretion was observed when the SSTR2- and SSTR5-preferring analogs, BIM-23197 and BIM-23268, were tested in combination. Our data show a highly variable ratio of the SSTR2 and SSTR5 transcripts, according to tumors. The SSTR2-preferring compound consistently inhibits GH release, whereas the SSTR5-preferring compound is the main inhibitor of PRL secretion. When both drugs are combined, the partial additivity observed in mixed GH- plus PRL-secreting adenomas may be of interest in the therapeutic approach of such tumors.
Assuntos
Acromegalia/metabolismo , RNA Mensageiro/metabolismo , Receptores da Somatotropina/genética , Adenoma/metabolismo , Adulto , Células Cultivadas , Combinação de Medicamentos , Feminino , Hormônios/farmacologia , Hormônio do Crescimento Humano/antagonistas & inibidores , Hormônio do Crescimento Humano/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Octreotida/farmacologia , Oligopeptídeos/farmacologia , Fenótipo , Piperazinas/farmacologia , Prolactina/antagonistas & inibidores , Receptores de Somatostatina/agonistas , Receptores de Somatostatina/genética , Somatostatina/análogos & derivados , Somatostatina/farmacologiaRESUMO
We studied the expression level and cell-specific expression patterns of 5alpha-reductase (5alpha-R) types 1 and 2 iso-enzymes in human hyperplastic and malignant prostate tissue by semi-quantitative RT-PCR and in situ hybridisation analyses. In situ hybridisation established that 5alpha-R1 mRNA is preferentially expressed by epithelial cells and little expressed by stromal cells whereas 5alpha-R2 mRNA is expressed by both epithelium and stroma. Semi-quantitative RT-PCR has been performed on total RNA from different zones of normal prostate, BPH tissues and liver. We found that 5alpha-R1 and 5alpha-R2 mRNAs expression was near the same in all zones of normal prostate. In BPH tissue, 5alpha-R1 and 5alpha-R2 mRNAs expression was slightly but significantly increased, when it was compared to the levels recorded for normal prostate. In cancer samples, 5alpha-R1 mRNA expression was higher than in normal and hyperplastic prostate but the level of 5alpha-R2 mRNA was not statistically different from that observed in the different zones of normal prostate. In liver, 5alpha-R2 mRNA level was similar to that measured in BPH but 5alpha-R1 mRNA expression was ten times higher. The increase observed in 5alpha-R isoenzymes expression in BPH tissue could play an important role in the pathogenesis and/or maintenance of the disease.
Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , Isoenzimas/metabolismo , Próstata/enzimologia , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Epitélio/enzimologia , Humanos , Hibridização In Situ , Masculino , Hiperplasia Prostática/enzimologia , Hiperplasia Prostática/etiologia , Neoplasias da Próstata/enzimologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The TRH secretory responsiveness of the pancreatic islet cell clusters from newborn rat in organ culture was studied. Basal TRH secretion was stable over a 9-day period. The response to various secretagogues was tested on day 4. TRH secretion was stimulated by high potassium-induced depolarization and also through both cAMP and protein kinase-C dependent pathways. Like insulin, TRH release was stimulated by glucose and arginine and inhibited by somatostatin. These data suggest the existence of a common mechanism for TRH and insulin secretion by the pancreatic beta-cells.
Assuntos
Animais Recém-Nascidos/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Hormônio Liberador de Tireotropina/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Arginina/farmacologia , Meios de Cultura , Técnicas de Cultura , Feminino , Glucose/farmacologia , Secreção de Insulina , Potássio/fisiologia , Radioimunoensaio , Ratos , Ratos Endogâmicos , Somatostatina/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The effect of maternal diabetes (induced by i.p. injections of 40-50 mg/kg BW Streptozotocin on the day of mating) on TRH in the pancreas of newborn rats was studied. Determination of peptide alpha amidation activity and TRH precursor level on the day of birth revealed decreased biosynthesis of TRH resulting in profoundly (10 times) lower pancreatic TRH and TRH-OH concentrations in pups of diabetic rats. Pancreatic His-Pro-diketopiperazine (His-Pro-DKP) remained unaffected by maternal diabetes. The depression of pancreatic TRH was less profound 24 h later, and even elevated TRH was measured in the pancreas of pups of diabetic mothers on postnatal day 5. Short term postnatal starvation or nursing of intact pups by the diabetic foster mother did not affect pancreatic TRH. It could be postulated that postnatal TRH development in the rat pancreas is retarded by maternal diabetes, while His-Pro-DKP remains unaltered.
Assuntos
Animais Recém-Nascidos/metabolismo , Diabetes Mellitus Experimental/metabolismo , Pâncreas/metabolismo , Peptídeos Cíclicos/metabolismo , Piperazinas/metabolismo , Gravidez em Diabéticas , Hormônio Liberador de Tireotropina/metabolismo , Envelhecimento/metabolismo , Animais , Feminino , Insulina/metabolismo , Gravidez , Precursores de Proteínas/metabolismo , Ratos , Ratos EndogâmicosRESUMO
We report the first mutational study of thymidine kinase 1 (TK1) performed in human solid tumors. We sequenced cDNAs representing the complete coding region of TK1 in human breast (n=22) and colorectal (n=26) cancer. Codon 106 near the ATP binding site constantly differed (ATG --> GTG; Met --> Val) from the one deposited by Bradshaw and Deininger in the Genbank database (Accession number NM_003258). Silent polymorphisms at codon 11 (CCC --> CCT; Pro --> Pro) and codon 75 (GCG --> GCA; Ala --> Ala) were frequently detected in tumors as well as in normal tissues. In breast cancer the two polymorphisms were observed in 63.6% of the samples analyzed. No significant association could be found between polymorphisms and TK activity. In colorectal cancer the incidence of the two changes was 73.1% and 69.2%, respectively. Interestingly, one colon cancer with high cytosolic TK activity displayed two missense mutations located in and near the putative phosphorylation site by tyrosine kinase (s) (TAT --> CAT; Tyr --> His) and by cAMP-, cGMP-dependent protein kinase (TAC --> TGC; Tyr --> Cys), respectively; adjacent normal mucosa showed no mutation. This may open new avenues that imply TK1 activity in tumor cell proliferation.
Assuntos
Neoplasias da Mama/genética , Neoplasias Colorretais/genética , Análise Mutacional de DNA , Timidina Quinase/genética , Trifosfato de Adenosina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Sítios de Ligação , Linhagem Celular Tumoral , Códon , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Citosol/metabolismo , DNA Complementar/metabolismo , Bases de Dados Genéticas , Humanos , Pessoa de Meia-Idade , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Mutação de Sentido Incorreto , Polimorfismo GenéticoRESUMO
The sheep is a valuable model to study growth hormone (GH) neuroregulation since its GH secretion pattern is close to that in humans and an integrated physiological approach is possible in this species. Somatostatin receptor subtype 5 (sst5) appears to be important in GH regulation but the ovine sst5 gene (osst5) has not yet been cloned. We report here the cloning of sst5 in that species. We screened a cDNA sheep library and isolated a 1.24 kb cDNA, which includes the whole coding region of osst5. The predicted protein consists of 367 amino acids exhibiting a putative seven transmembrane domain topology typical of G protein-coupled receptors. Nucleotide sequence comparisons with that of other species sst5 showed that osst5 displays 83.8, 81 and 79.7% homology with human, rat, and mice sst5, respectively. Southern blot analysis of ovine cortex DNA demonstrated that osst5 is encoded by a single gene. Osst5 transiently expressed in Chinese Hamster ovary (CHO) cells exhibit a high affinity for somatostatin-14. Reverse transcriptase-polymerase chain reaction (RT-PCR) studies demonstrated that osst5 mRNAs are present in pituitary, cortex, hypothalamus, hippocampus, colon and adrenal gland. The cloning of osst5 should provide a useful tool to study the mechanisms through which somatostatin inhibits hormone secretion in the sheep.
Assuntos
Clonagem Molecular , Receptores de Somatostatina/genética , Ovinos/genética , Glândulas Suprarrenais/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Córtex Cerebral/química , Colo/química , Cricetinae , DNA Complementar/isolamento & purificação , Expressão Gênica , Biblioteca Gênica , Hipocampo/química , Humanos , Hipotálamo/química , Dados de Sequência Molecular , Hipófise/química , RNA Mensageiro/análise , Receptores de Somatostatina/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Distribuição Tecidual , TransfecçãoRESUMO
It is possible to assay hypothalamic factors in hypophysial portal blood from several species. However, hypophysial portal blood collection must be performed after anesthesia and surgical stress which can both modify the regulation of hypothalamo-pituitary secretion. However, this method has allowed some progress in our knowledge of the hypothalamic control of pituitary secretion. The methodology, advantages and limits of hypophysial portal blood collection are briefly described in this review.
Assuntos
Coleta de Amostras Sanguíneas/métodos , Hipotálamo/irrigação sanguínea , Hipófise/irrigação sanguínea , Sistema Porta , Animais , Hormônios Hipotalâmicos/sangue , Hormônios Hipofisários/sangue , RatosRESUMO
BACKGROUND: Currently, the benefit of chemotherapy (CT) in node-negative breast carcinoma (NNBC) is discussed. The evaluation of classical clinical and histological factors is limited to assess individual outcome. A statistical model was developed to improve the prognostic accuracy of NNBC. METHODS: A total of 305 node-negative breast carcinomas who underwent surgery (+/- radiotherapy) but no adjuvant treatment were selected. Putative prognosis factors including age, tumour size, oestrogen receptor (ER), progesterone receptor (PgR), Scarff-Bloom-Richardon (SBR) grading, urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1) and thymidine kinase (TK) were evaluated. The developed model was internally validated using Harrell's concordance index. A prognosis index (PI) was proposed and compared with Adjuvant! Online program. RESULTS: Age (p < 0.001), pathological tumour size (pT) (p < 0.001), PgR (p = 0.02), and PAI-1 (p ≤ 0.001) were included in the Cox regression model predicting Breast cancer specific survival (BCSS) at 5-years. Internal validation revealed a concordance index of 0.71. A PI score was derived from our nomogram. The PI score was significantly associated with BCSS (hazard ratio (HR): 4.1 for intermediate, p=0.02, HR: 8.8, p < 0.001 for high group) as compared to Adjuvant! Online score (HR: 1.4, p=0.14). CONCLUSION: A nomogram can be used to predict probability survival curves for individual breast cancer patients.
Assuntos
Neoplasias da Mama/cirurgia , Carcinoma/cirurgia , Técnicas de Apoio para a Decisão , Linfonodos/patologia , Mastectomia Radical Modificada , Mastectomia Segmentar , Nomogramas , Fatores Etários , Biomarcadores Tumorais/análise , Neoplasias da Mama/química , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Carcinoma/química , Carcinoma/mortalidade , Carcinoma/patologia , Quimioterapia Adjuvante , Intervalo Livre de Doença , Feminino , França , Humanos , Estimativa de Kaplan-Meier , Excisão de Linfonodo , Mastectomia Radical Modificada/efeitos adversos , Mastectomia Radical Modificada/mortalidade , Mastectomia Segmentar/efeitos adversos , Mastectomia Segmentar/mortalidade , Pessoa de Meia-Idade , Análise Multivariada , Seleção de Pacientes , Inibidor 1 de Ativador de Plasminogênio/análise , Modelos de Riscos Proporcionais , Radioterapia Adjuvante , Receptores de Progesterona/análise , Medição de Risco , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Carga TumoralRESUMO
Thyrotropin-Releasing hormone (TRH)-degrading pyroglutamyl peptidase I (PGP I) and prolylendopeptidase (PE) activities have been demonstrated in rat insulinoma RINm 5F cell line. These two enzymes catalyze the conversion of TRH to Histydyl-Proline-Diketopiperazine and to acid TRH respectively. After cell fractionation, we found all the PGP I and PE activities in the cytosolic fraction. The membrane-bound PGP II activity is not detectable in the RINm 5F cells. Further investigations on these two cytosolic enzymes show that pyroglutamyl- and proline-containing peptides are inhibitors of each TRH-degrading enzyme. Gel filtration chromatography on Sephadex G100 shows that PGP I and PE activity have an apparent molecular mass of about 18 kDa and 57 kDa, respectively. Kinetic analysis with TRH as substrate, gives a Km of 44 microM and 235 microM, and a Vmax of 1.49 and 8.80 pmol/min/micrograms protein for PGP I and PE, respectively. Immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH levels in the cell line extracts are 2.2 +/- 0.9, 22.5 +/- 11.1 and 28.7 +/- 14.6 pg/1O6 cells, respectively. When cells have been incubated for 2 to 72 hours with a P.E. inhibitor (Z-Gly-Pro-CHN2) at 5 x 10(-7) M, both cell PGP I and PE activities are inhibited. No change in the cellular content of immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH have been observed in treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Adenoma de Células das Ilhotas Pancreáticas/enzimologia , Endopeptidases/metabolismo , Piroglutamil-Peptidase I/metabolismo , Serina Endopeptidases , Hormônio Liberador de Tireotropina/metabolismo , Adenoma de Células das Ilhotas Pancreáticas/tratamento farmacológico , Sequência de Aminoácidos , Animais , Linhagem Celular , Diazometano/análogos & derivados , Diazometano/farmacologia , Cinética , Dados de Sequência Molecular , Peso Molecular , Prolil Oligopeptidases , Inibidores de Proteases/farmacologia , Radioimunoensaio , Ratos , Frações Subcelulares/enzimologia , Especificidade por SubstratoRESUMO
We demonstrate that two enzymes, soluble unspecific pyroglutamyl peptidase I and prolyl endopeptidase, able to degrade thyrotropin-releasing hormone (TRH) in vitro were present in pancreas at the early stage of rat development. Specific particulate pyroglutamyl peptidase II remained undetectable during ontogenesis. Pyroglutamyl peptidase I specific activity increased until day 3 and decreased after day 5. Furthermore, prolyl endopeptidase specific activity rose slightly to a peak on postnatal day 20. A good correlation between immunoreactive TRH and deaminated TRH (TRH-OH) was found in the 1st wk after birth. However, His-Pro diketopiperazine (DKP) levels were stable and low during development. We show that hot acidic extraction conditions could artefactually generate His-Pro DKP. In vivo, active site-directed inhibitors of pyroglutamyl peptidase I and prolyl endopeptidase enzymes do not show any TRH-deamidating and/or pyroglutamyl peptidase I pathways in neonatal rat pancreas. The data suggest that these two enzymes are not involved in intra- or extracellular control of TRH levels in neonatal rat pancreas and that pancreatic TRH content appears to be principally regulated by biosynthetic steps. Nevertheless, low levels of endogenous His-Pro DKP and TRH-OH identified in neonatal rat pancreas suggest that TRH or TRH-like peptides may be metabolized in this tissue in intact rats, albeit at low rates.
Assuntos
Endopeptidases/metabolismo , Pâncreas/crescimento & desenvolvimento , Piroglutamil-Peptidase I/metabolismo , Serina Endopeptidases , Hormônio Liberador de Tireotropina/metabolismo , Envelhecimento , Animais , Membrana Celular/enzimologia , Feminino , Cinética , Masculino , Pâncreas/enzimologia , Pâncreas/metabolismo , Prolil Oligopeptidases , Inibidores de Proteases/farmacologia , Ratos , Ratos EndogâmicosRESUMO
A case of watery diarrhoea hypokalaemia achlorhydria (WDHA) syndrome due to a pancreatic tumour and identified by VIP plasma level, VIP immunocytochemistry, and ultrastructural analysis of tumour sections, is reported. Since VIP is the mediator of the syndrome and is biologically active under its amidated form, the enzymatic alpha-amidating activity was investigated and characterized in tumour extract; using the synthetic substrate D-Tyr-Val-Gly, the enzyme displayed an optimal activity at pH 7.0, under aerobic conditions and with 35 microM CuSO4 and 3 mM ascorbate as co-factors. The Kmax and Vmax values of the enzymatic activity were 133.7 microM and 26.9 pmol/h/micrograms protein respectively. Its molecular weight, determined by molecular sieving, was close to 36 kDa. Other tumours of the human endocrine pancreas were also investigated for the enzymatic activity. The clinical interest of studying the regulation of the alpha-amidating activity in such tumours is discussed.
Assuntos
Oxigenases de Função Mista/análise , Complexos Multienzimáticos , Neoplasias Pancreáticas/enzimologia , Vipoma/enzimologia , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Peso Molecular , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/patologia , Peptídeo Intestinal Vasoativo/sangue , Vipoma/sangue , Vipoma/patologiaRESUMO
Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides. Southern blot analysis of human placental DNA demonstrated that PAM is encoded by a single gene. The chromosomal localization of the PAM gene was established using in situ hybridization. A 2.2-kb human PAM cDNA hybridized to human metaphase chromosomes revealed a significant clustering of silver grains over chromosome 5 bands q14-q21. The gene encoding another enzyme important in the post-translational processing of neuroendocrine precursors, prohormone convertase 1 (PC1), is localized in the same region (5q15-q21).
Assuntos
Cromossomos Humanos Par 5 , Oxigenases de Função Mista/genética , Complexos Multienzimáticos , Southern Blotting , Células Cultivadas , Bandeamento Cromossômico , Mapeamento Cromossômico , HumanosRESUMO
Qualitative and quantitative expression of m.RNA coding for Peptidyl-Glycine alpha-Amidating Monooxygenase (PAM) in the developing rat pancreas was investigated by Northern and dot blot hybridization, with a bovine PAM c.DNA probe (0.7 kb fragment). A specific hybridization signal was evidenced for a 3.7 kb m.RNA species. Measurement of PAM m. RNA rate during the rat pancreas ontogenesis revealed a biphasic profile which appeared corelated with that of gastrin and TRH m.RNA respectively. On the other hand, streptozotocin-treatment resulted in a 50% decrease of PAM m.RNA levels.