Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 49
Filtrar
1.
Diabetes Metab J ; 2024 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-38527457

RESUMO

Background: To investigate associations between variations in the co-expression-based brain insulin receptor polygenic score and cardiometabolic risk factors and diabetes mellitus. Methods: This cross-sectional study included 1,573 participants from the Helsinki Birth Cohort Study. Biologically informed expression-based polygenic risk scores for the insulin receptor gene network were calculated for the hippocampal (hePRS-IR) and the mesocorticolimbic (mePRS-IR) regions. Cardiometabolic markers included body composition, waist circumference, circulating lipids, insulin-like growth factor 1 (IGF-1), and insulin-like growth factor-binding protein 1 and 3 (IGFBP-1 and -3). Glucose and insulin levels were measured during a standardized 2-hour 75 g oral glucose tolerance test and impaired glucose regulation status was defined by the World Health Organization 2019 criteria. Analyzes were adjusted for population stratification, age, smoking, alcohol consumption, socioeconomic status, chronic diseases, birth weight, and leisure-time physical activity. Results: Multinomial logistic regression indicated that one standard deviation increase in hePRS-IR was associated with increased risk of diabetes mellitus in all participants (adjusted relative risk ratio, 1.17; 95% confidence interval, 1.01 to 1.35). In women, higher hePRS-IR was associated with greater waist circumference and higher body fat percentage, levels of glucose, insulin, total cholesterol, low-density lipoprotein cholesterol, triglycerides, apolipoprotein B, insulin, and IGFBP-1 (all P≤0.02). The mePRS-IR was associated with decreased IGF-1 level in women (P=0.02). No associations were detected in men and studied outcomes. Conclusion: hePRS-IR is associated with sex-specific differences in cardiometabolic risk factor profiles including impaired glucose regulation, abnormal metabolic markers, and unfavorable body composition in women.

2.
Anal Biochem ; 648: 114681, 2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35417678

RESUMO

Trypsin has been identified as a pancreatic protease comprising three isoenzymes, trypsin-1, -2, and -3. However, the gene for trypsinogen-3, PRSS3, also gives rise to additional variants, trypsinogen-4A and B, which differ from trypsinogen-3 only with respect to the leader-peptide part, and when activated are identical to trypsin-3. The unique overlapping leader peptides of trypsinogen-4A and B allowed us to develop a specific sandwich-type immunofluorometric assay that detects both these isoforms, but not trypsinogen-3 or activated trypsinogen-4. We measured the concentrations of trypsinogen-4 in various cell line lysates and bile of primary sclerosing cholangitis patients. Lysates of cell lines MDA-MB-231 and PC-3, and astrocytes contained trypsinogen-4, while the conditioned media from these cells did not, suggesting that trypsinogen-4, lacking a classical signal sequence, is not secreted from the cells. Interestingly, 5.7% of the 212 bile samples analyzed contained measurable (>2.4 µg/l) trypsinogen-4. In conclusion, we have established a specific assay for trypsinogen-4 and demonstrated that trypsinogen-4 can be found in biological samples. However, the clinical utility of the assay remains to be established.


Assuntos
Bile , Tripsinogênio , Humanos , Imunoensaio , Isoenzimas/metabolismo , Tripsina/metabolismo
3.
Scand J Clin Lab Invest ; 80(8): 640-643, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32967482

RESUMO

The concentrations of several diagnostic markers have been found to increase dramatically in critically ill patients with a severe disturbance of normal physiological homeostasis, without indication of the diseases they are normally associated with. To prevent false diagnoses and inappropriate treatments of critically ill patients, it is important that the markers aiding the selection of second-line treatments are evaluated in such patients and not only in the healthy population and patients with diseases the markers are associated with. The levels of trypsinogen isoenzymes, the trypsin inhibitor serine peptidase inhibitor Kazal type 1 (SPINK1), hCG and hCGß, which are used as pancreatitis and cancer markers, were analyzed by immunoassays from serum samples of 17 adult patients who have undergone surgery of the ascending aorta during hypothermic circulatory arrest (HCA) with optional selective cerebral perfusion. Highly elevated levels of trypsinogen-1, -2 and -3, SPINK1 and hCGß were observed in patients after HCA. This was accompanied by increased concentrations of S100ß and NSE. In conclusion, this study highlights the importance of critically evaluating the markers used for aiding selection of second line of treatments in critically ill patients.


Assuntos
Aneurisma Aórtico/sangue , Dissecção Aórtica/sangue , Ponte Cardiopulmonar/efeitos adversos , Gonadotropina Coriônica Humana Subunidade beta/sangue , Parada Circulatória Induzida por Hipotermia Profunda/efeitos adversos , Inibidor da Tripsina Pancreática de Kazal/sangue , Adulto , Idoso , Dissecção Aórtica/patologia , Dissecção Aórtica/cirurgia , Aorta/patologia , Aorta/cirurgia , Aneurisma Aórtico/patologia , Aneurisma Aórtico/cirurgia , Biomarcadores/sangue , Ponte Cardiopulmonar/métodos , Circulação Cerebrovascular , Parada Circulatória Induzida por Hipotermia Profunda/métodos , Estado Terminal , Feminino , Humanos , Imunoensaio , Masculino , Pessoa de Meia-Idade , Perfusão/métodos , Estudos Prospectivos , Tripsina/sangue , Tripsinogênio/sangue
4.
Cell Signal ; 28(10): 1463-9, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27373413

RESUMO

Glycodelin is a glycoprotein mainly expressed in well-differentiated epithelial cells in reproductive tissues. In normal secretory endometrium, the expression of glycodelin is abundant and regulated by progesterone. In hormone-related cancers glycodelin expression is associated with well-differentiated tumors. We have previously found that glycodelin drives epithelial differentiation of HEC-1B endometrial adenocarcinoma cells, resulting in reduced tumor growth in a preclinical mouse model. Here we show that glycodelin-transfected HEC-1B cells have repressed protein kinase C delta (PKCδ) activation, likely due to downregulation of PDK1, and are resistant to phenotypic change and enhanced migration induced by phorbol 12-myristate 13-acetate (PMA). In control cells, which do not express glycodelin, the effects of PMA were abolished by using PKCδ and PDK1 inhibitors, and knockdown of PKCδ, MEK1 and 2, or ERK1 and 2 by siRNAs. Similarly, transforming growth factor ß (TGFß)-induced phenotypic change was only seen in control cells, not in glycodelin-producing cells, and it was mediated by PKCδ. Taken together, these results strongly suggest that PKCδ, via MAPK pathway, is involved in the glycodelin-driven cell differentiation rendering the cells resistant to stimulation by PMA and TGFß.


Assuntos
Glicodelina/metabolismo , Proteína Quinase C-delta/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Fenótipo , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo
5.
Clin Chem ; 57(11): 1506-13, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21920915

RESUMO

BACKGROUND: Trypsinogen 3 is a minor trypsinogen isoform in the pancreas. In contrast with trypsin 1 and 2, trypsin 3 degrades pancreatic secretory trypsin inhibitor, which may lead to an excess of active trypsin and acute pancreatitis (AP). We developed an immunoassay for trypsinogen 3 and studied whether an assay of serum trypsinogen 3 is of clinical utility in the diagnosis of AP. METHODS: Monoclonal antibodies were generated using recombinant human trypsinogen 3 as the antigen and used to establish a sandwich-type immunoassay. We analyzed serum trypsinogen 3 concentrations in 82 patients with AP and 63 patients with upper abdominal pain (controls). The reference interval was determined using serum samples from 172 apparently healthy individuals. RESULTS: The measuring range of the trypsinogen 3 assay was 1.0-250 µg/L. Intra- and interassay CVs were <11%, and cross-reactivity with other trypsinogen isoenzymes was <0.1%. The median trypsinogen 3 concentration in serum from healthy individuals was <1.0 µg/L, and the upper reference limit was 4.4 µg/L. We observed increased trypsinogen 3 concentrations in patients with mild (median 9.5 µg/L) and severe (15.0 µg/L) AP; in both groups, the concentrations were significantly higher than in controls (median <1.0 µg/L) (P < 0.0001). In ROC analysis, the area under the curve of trypsinogen 3 for separation between AP and controls was 0.90 (P < 0.0001). CONCLUSIONS: We established for the first time a specific immunoassay for trypsinogen 3 using monoclonal antibodies. Patients with AP were found to have increased serum concentrations of trypsinogen 3. The availability of this assay will be useful for studies of the clinical utility of trypsinogen 3.


Assuntos
Pancreatite/sangue , Tripsina/sangue , Doença Aguda , Adolescente , Adulto , Idoso , Anticorpos Monoclonais , Biomarcadores/sangue , Feminino , Humanos , Imunoensaio/métodos , Isoenzimas/sangue , Masculino , Pessoa de Meia-Idade , Pancreatite Necrosante Aguda/sangue , Valores de Referência , Sensibilidade e Especificidade , Adulto Jovem
6.
Diabetes ; 60(3): 909-17, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21300843

RESUMO

OBJECTIVE: Gestational diabetes mellitus (GDM) is a common metabolic disorder of pregnancy. Patients with GDM are at risk for high fetal mortality and gestational complications associated with reduced immune tolerance and abnormal carbohydrate metabolism. Glycodelin-A (GdA) is an abundant decidual glycoprotein with glycosylation-dependent immunomodulatory activities. We hypothesized that aberrant carbohydrate metabolism in GDM was associated with changes in glycosylation of GdA, leading to defective immunomodulatory activities. RESEARCH DESIGN AND METHODS: GdA in the amniotic fluid from women with normal (NGdA) and GDM (DGdA) pregnancies was purified by affinity chromatography. Structural analysis of protein glycosylation was preformed by lectin-binding assay and mass spectrometry. Cytotoxicity, cell death, cytokine secretion, and GdA binding of the GdA-treated lymphocytes and natural killer (NK) cells were determined. The sialidase activity in the placental tissue from normal and GDM patients was measured. RESULTS: GDM affected the glycosylation but not the protein core of GdA. Specifically, DGdA had a lower abundance of α2-6-sialylated and high-mannose glycans and a higher abundance of glycans with Sda (NeuAcα2-3[GalNAcß1-4]Gal) epitopes compared with NGdA. DGdA had reduced immuosuppressive activities in terms of cytotoxicity on lymphocytes, inhibitory activities on interleukin (IL)-2 secretion by lymphocytes, stimulatory activities on IL-6 secretion by NK cells, and binding to these cells. Desialylation abolished the immunomodulation and binding of NGdA. Placental sialidase activity was increased in GDM patients, which may account for the reduced sialic acid content of DGdA. CONCLUSIONS: Taken together, this study provides the first direct evidence for altered enzymatic glycosylation and impaired bioactivity of GdA in GDM patients.


Assuntos
Líquido Amniótico/metabolismo , Diabetes Gestacional/metabolismo , Glicoproteínas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Proteínas da Gravidez/metabolismo , Adulto , Análise de Variância , Morte Celular , Feminino , Citometria de Fluxo , Cromatografia Gasosa-Espectrometria de Massas , Glicodelina , Glicosilação , Humanos , Interleucina-6/metabolismo , Lectinas/metabolismo , Gravidez
7.
Hum Reprod ; 26(3): 517-26, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21227941

RESUMO

BACKGROUND: The maternal-fetal interface has a unique immunological response towards the implanting placenta. It is generally accepted that a T-helper type-2 (Th-2) cytokine prevailing environment is important in pregnancy. The proportion of Th-2 cells in the peripheral blood and decidua is significantly higher in pregnant women in the first trimester than in non-pregnant women. Glycodelin-A (GdA) is a major endocrine-regulated decidual glycoprotein thought to be related to fetomaternal defence. Yet the relationship between its immunoregulatory activities and the shift towards Th-2 cytokine profile during pregnancy is unclear. METHODS: GdA was immunoaffinity purified from human amniotic fluid. T-helper, T-helper type-1 (Th-1) and Th-2 cells were isolated from the peripheral blood. The viability of these cells was studied by XTT assay. Immunophenotyping of CD4/CD294, cell death and GdA-binding were determined by flow cytometry. The mRNA expression, surface expression and secretion of Fas/Fas ligand (FasL) were determined by quantitative polymerase chain reaction, flow cytometry and ELISA, respectively. The activities of caspase-3, -8 and -9 were measured. The phosphorylation of extracellular signal-regulated kinases (ERK), p38 and, c-Jun N-terminal kinase was determined by western blotting. RESULTS: Although GdA bound to both Th-1 and Th-2 cells, it had differential actions on the two cell-types. GdA induced cell death of the Th-1 cells but not the Th-2 cells. The cell death was mediated through activation of caspase -3, -8 and -9 activities. GdA up-regulated the expression of Fas and inhibited ERK activation in the Th-1 cells, which might enhance the vulnerability of the cells to cell death caused by a trophoblast-derived FasL. CONCLUSIONS: The data suggest that GdA could be an endometrial factor that contributes to the Th-2/Th-1 shift during pregnancy.


Assuntos
Glicoproteínas/metabolismo , Proteínas da Gravidez/metabolismo , Células Th1/imunologia , Células Th2/imunologia , Líquido Amniótico/química , Caspases/metabolismo , Sobrevivência Celular , Células Cultivadas , Células do Cúmulo/química , Proteína Ligante Fas/genética , Proteína Ligante Fas/metabolismo , Feminino , Regulação da Expressão Gênica , Glicodelina , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Glicosilação , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Gravidez , Proteínas da Gravidez/química , Proteínas da Gravidez/isolamento & purificação , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Sêmen/química , Células Th1/metabolismo , Células Th2/metabolismo , Receptor fas/genética , Receptor fas/metabolismo
8.
Breast Cancer Res Treat ; 128(1): 85-95, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20676758

RESUMO

Glycodelin (encoded by PAEP gene) is a secreted lipocalin protein mainly expressed in reproductive tissues, but also in several tumour types. In the breast, glycodelin is expressed both in normal epithelial and cancerous tissue. To investigate the association of glycodelin with clinicopathological features of breast cancer and outcome of patients we evaluated the protein expression of glycodelin in a large series of breast tumours. Immunohistochemical analysis of tissue microarrays was used to study glycodelin expression on 399 sporadic and 436 familial non-BRCA1/2 tumours with strong family history. Gene expression analysis was used to define genes co-expressed with PAEP in sporadic and familial non-BRCA1/2 breast tumours. In the sporadic series, the glycodelin expression associated with low proliferation rate (P < 0.001), with a tendency towards well-differentiated tumours (grades 1 and 2, P = 0.012) and high cyclin D1 (P = 0.034) expression. However, in familial non-BRCA1/2 cases with strong family history glycodelin expression associated with a less favourable phenotype, i.e. positive lymph node status (P = 0.003) and HER2-positive tumours (P = 0.009). Moreover, the patients with glycodelin-positive tumours had an increased risk for distant metastases (P = 0.001) and in multivariate analysis glycodelin expression was an independent predictor of metastasis (hazard ratio (HR) = 2.22, 95% confidence interval (95% CI) = 1.22-4.03, P = 0.009) in familial non-BRCA1/2 breast cancer. Gene expression analysis further revealed different gene expression profiles correlating with the PAEP expression in the sporadic and familial non-BRCA1/2 breast cancers. Our findings suggest differential progression pathways in the sporadic and familial non-BRCA1/2 breast tumours expressing glycodelin.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/metabolismo , Fenótipo , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Neoplasias da Mama/mortalidade , Intervalo Livre de Doença , Feminino , Perfilação da Expressão Gênica , Estudos de Associação Genética , Glicodelina , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Pessoa de Meia-Idade , Análise Multivariada , Mutação , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Modelos de Riscos Proporcionais , Análise Serial de Tecidos , Adulto Jovem
9.
Contraception ; 82(6): 526-33, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21074015

RESUMO

BACKGROUND: The study was conducted to assess the effects of levonorgestrel (LNG) on hormonal behavior and on the secretory pattern of intrauterine glycodelin at the midcycle of ovulatory women. STUDY DESIGN: Thirty healthy sterilized women with normal ovarian function were studied during one control untreated cycle and one LNG-treated cycle. In the treated cycle, each woman received two doses of 0.75 mg of LNG 12 h apart during the preovulatory phase approximately 2 days before the LH surge. Daily follicle development recordings were performed until follicle rupture was observed, and serum glycodelin, LH, estradiol, estrone and progesterone were measured as well. In addition, glycodelin concentrations were assayed in uterine flushing obtained on Days LH+1 and LH+12. RESULTS: LNG did not modify follicle rupture in 20 of 30 women. In spite of ovulatory progesterone and the occurrence of follicle rupture in these women, luteal phase length was significantly decreased, as well as the serum concentrations of LH, estradiol and estrone in the periovulatory phase. Glycodelin in serum and uterine flushings was significantly elevated in the periovulatory phase when compared to control cycles. CONCLUSIONS: LNG taken at the dose used in emergency contraception before the LH surge increased prematurely serum and intrauterine concentrations of glycodelin at the time of ovulation. Since there are well established glycodelin inhibitory effects upon fertilization, these results may represent an additional action of LNG in situations where the intervention did not interfere with ovulation.


Assuntos
Anticoncepção Pós-Coito , Anticoncepcionais Femininos/farmacologia , Glicoproteínas/análise , Hormônios Esteroides Gonadais/sangue , Levanogestrel/farmacologia , Ciclo Menstrual/efeitos dos fármacos , Proteínas da Gravidez/análise , Útero/efeitos dos fármacos , Adulto , Endométrio/efeitos dos fármacos , Estradiol/sangue , Estrona/sangue , Feminino , Glicodelina , Glicoproteínas/sangue , Humanos , Fase Luteal/efeitos dos fármacos , Hormônio Luteinizante/sangue , Hormônio Luteinizante/urina , Folículo Ovariano/diagnóstico por imagem , Folículo Ovariano/efeitos dos fármacos , Ovulação/efeitos dos fármacos , Proteínas da Gravidez/sangue , Progesterona/sangue , Ultrassonografia , Útero/química , Útero/metabolismo , Adulto Jovem
10.
Hum Reprod ; 25(11): 2721-33, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20843776

RESUMO

BACKGROUND: Glycodelin-A interacts with spermatozoa before fertilization, but its role in modulating sperm functions is not known. Zona pellucida-induced acrosome reaction is crucial to fertilization and its dysfunction is a cause of male infertility. We hypothesized that glycodelin-A, a glycoprotein found in the female reproductive tract, potentiates human spermatozoa for zona pellucida-induced acrosome reaction. METHODS: Glycodelin isoforms were immunoaffinity purified. The sperm intracellular cAMP concentration, protein kinase-A (PKA) and extracellular signal-regulated kinase (ERK) activities, and intracellular calcium were measured by ELISA, kinase activity assay kits and Fluo-4AM technique, respectively. The phosphorylation of inositol 1,4,5-trisphosphate type-1 receptor (IP3R1) mediated by ERK was determined by western blotting. Zona pellucida-induced acrosome reaction was detected by Pisum sativum staining. RESULTS: Pretreatment of spermatozoa with glycodelin-A significantly up-regulated adenylyl cyclase/PKA activity and down-regulated the activity of ERK and its phosphorylation of IP3R1, thereby enhancing zona pellucida-induced calcium influx and zona pellucida-induced acrosome reaction. Glycodelin-F or deglycosylated glycodelin-A did not have these actions. Treatment of spermatozoa with a protein kinase inhibitor abolished the priming activity of glycodelin-A, whilst ERK pathway inhibitors mimic the stimulatory effect of glycodelin-A on zona pellucida-induced acrosome reaction. CONCLUSIONS: Glycodelin-A in the female reproductive tract sensitizes spermatozoa for zona pellucida-induced acrosome reaction in a glycosylation-specific manner through activation of the adenylyl cyclase/PKA pathway, suppression of extracellular signal-regulated kinase activation and up-regulation of zona pellucida-induced calcium influx. The action of glycodelin-A may be important in vivo to ensure full responsiveness of human spermatozoa to the zona pellucida.


Assuntos
Reação Acrossômica/fisiologia , Adenilil Ciclases/metabolismo , Cálcio/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glicoproteínas/fisiologia , Proteínas da Gravidez/fisiologia , Espermatozoides/fisiologia , Zona Pelúcida/fisiologia , Reação Acrossômica/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação para Baixo , Feminino , Glicodelina , Glicosilação , Humanos , Receptores de Inositol 1,4,5-Trifosfato/efeitos dos fármacos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Espermatozoides/efeitos dos fármacos , Regulação para Cima
11.
Biol Chem ; 391(2-3): 283-293, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20128688

RESUMO

Human trypsin isoenzymes share extensive sequence similarity, but certain differences in their activity and susceptibility to inhibitors have been observed. Using phage display technology, we identified seven different peptides that bind to and inhibit the activity of trypsin-3, a minor trypsin isoform expressed in pancreas and brain. All of the peptides contain at least two of the amino acids tryptophan, alanine and arginine, whereas proline was found closer to the N-terminus in all but one peptide. All peptides contain two or more cysteines, suggesting a cyclic structure. However, we were able to make synthetic linear variants of these peptides without losing bioactivity. Alanine replacement experiments for one of the peptides suggest that the IPXXWFR motif is important for activity. By molecular modeling the same amino acids were found to interact with trypsin-3. The peptides also inhibit trypsin-1, but only weakly, if at all, trypsin-2 and -C. As trypsin is a highly active enzyme which can activate protease-activated receptors and enzymes that participate in proteolytic cascades involved in tumor invasion and metastasis, these peptides might be useful lead molecules for the development of drugs for diseases associated with increased trypsin activity.


Assuntos
Proteínas Recombinantes de Fusão/farmacologia , Inibidores da Tripsina/farmacologia , Tripsina/metabolismo , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Relação Estrutura-Atividade , Inibidores da Tripsina/química , Inibidores da Tripsina/genética , Tripsinogênio/antagonistas & inibidores , Tripsinogênio/metabolismo
12.
Biol Chem ; 391(4): 475-9, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20180640

RESUMO

Kallikrein-related peptidase 2 (KLK2) degrades insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) in vitro. IGFBP-3 forms complexes with IGFs, preventing them from binding to their receptors and stimulating cell proliferation and survival. IGF-independent actions have also been described for IGFBP-3. The degradation of IGFBP-3 by KLK2 or other proteases in the prostate may promote the growth of prostate cancer. We studied IGFBP-3 degradation by immunoblotting and two specific immunoassays, one recognizing only native non-fragmented IGFBP-3 and the other one recognizing both intact and proteolytically cleaved IGFBP-3. Peptides were used to inhibit the enzyme activity of KLK2 and cleavage sites in IGFBP-3 were identified by mass spectrometry. KLK2 proteolyzed IGFBP-3 into several small fragments, mostly after Arg residues, in keeping with the trypsin-like activity of KLK2. The fragmentation could be inhibited by KLK2-inhibiting peptides in a dose-dependent fashion. As degradation of IGFBP-3 could lead to a more aggressive cancer phenotype, inhibition of KLK2 activity might be useful for treatment of prostate cancer and other diseases associated with increased KLK2 activity.


Assuntos
Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/química , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptídeos/farmacologia , Inibidores de Serina Proteinase/farmacologia , Calicreínas Teciduais/antagonistas & inibidores , Calicreínas Teciduais/metabolismo , Sequência de Aminoácidos , Animais , Fluorimunoensaio , Humanos , Immunoblotting , Espectrometria de Massas , Dados de Sequência Molecular
13.
Fertil Steril ; 94(2): 769-71, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19945098
14.
J Reprod Immunol ; 83(1-2): 26-30, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19857900

RESUMO

Glycodelin is a glycoprotein with 4 known glycoforms, namely glycodelin-S, glycodelin-A, glycodelin-F and glycodelin-C. The glycoforms are present in the female reproductive tract which the spermatozoa must pass through before fertilizing the oocyte. Thus the spermatozoa interact with each of these glycoforms in succession. The glycoforms have different effects on sperm function. Glycodelin-S in the seminal plasma suppresses albumin-induced cholesterol efflux from the spermatozoa and thereby regulates the initiation of capacitation. Glycodelin-A in the oviductal fluid suppresses extracellular signal-regulated kinase making the spermatozoa more sensitive to zona pellucida-induced acrosome reaction. Follicular fluid glycodelin-F suppresses progesterone-induced acrosome reaction and prevents premature acrosome reaction. Glycodelin-A and glycodelin-F also inhibit spermatozoa-zona pellucida binding by interacting with sperm surface fucosyltransferase-5, which also binds to zona pellucida glycoproteins. The physiological implication of this phenomenon remains to be determined. The inhibitory activities of glycodelin-A and glycodelin-F on spermatozoa-zona pellucida binding is removed by glycodelin-C in the cumulus matrix. Glycodelin-C not only displaces sperm-bound glycodelin-A and glycodelin-F, it also enhances spermatozoa-zona pellucida binding. These different biological activities of the glycodelin isoforms are determined by glycosylation of the proteins. Deglycosylation abolishes the binding and therefore the action of the glycodelins on spermatozoa. Knowledge of the mechanism of actions of glycodelins may enable development of novel strategies for fertility regulation.


Assuntos
Reação Acrossômica , Endométrio/metabolismo , Tubas Uterinas/metabolismo , Glicoproteínas/fisiologia , Proteínas da Gravidez/fisiologia , Capacitação Espermática/fisiologia , Animais , Feminino , Glicodelina , Glicosilação , Humanos , Masculino , Sêmen , Interações Espermatozoide-Óvulo/fisiologia
15.
Hum Reprod ; 24(11): 2856-67, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19625311

RESUMO

BACKGROUND: Glycodelin-C is a glycodelin isoform isolated from the cumulus matrix. It stimulates spermatozoa-zona pellucida binding. Here, we report the isolation and characterization of a novel glycodelin interacting protein (GIP) from human cumulus matrix. METHODS: GIP was purified by liquid chromatograph and identified by mass spectrometry. The interaction of GIP with glycodelin, matrix molecule and spermatozoa were investigated. RESULTS: Mass spectrometry analysis suggested that GIP contained the N-terminal region of alpha2-macroglobulin, confirmed by western blot with anti-alpha2-macroglobulin antibody. GIP bound to native but not deglycosylated glycodelin-C in native gel electrophoresis, suggesting that the binding was glycosylation-dependent. GIP did not bind to capacitated and uncapacitated human spermatozoa. The cumulus cells could convert exogenous labeled alpha2-macroglobulin into GIP in vitro. GIP interacted with hyaluronic acid, a major component of the cumulus matrix. Glycodelin-C bound to hyaluronic acid-coated agarose beads in the presence of GIP. Human spermatozoa acquired the hyaluronic acid-GIP-bound glycodelin-C during incubation in vitro. CONCLUSION: The hyaluronic acid-GIP complex formed in the cumulus matrix retains and concentrates glycodelin-C in the cumulus matrix for displacing sperm-bound glycodelin-A and -F and stimulating the zona binding activity of the spermatozoa traversing through the cumulus mass.


Assuntos
Células do Cúmulo/metabolismo , Glicoproteínas/metabolismo , Proteínas da Gravidez/metabolismo , alfa-Macroglobulinas/metabolismo , Cromatografia Líquida , Proteínas da Matriz Extracelular/metabolismo , Feminino , Glicodelina , Glicosilação , Humanos , Ácido Hialurônico/metabolismo , Masculino , Espectrometria de Massas , Isoformas de Proteínas/metabolismo , Espermatozoides/metabolismo
16.
Hum Reprod ; 24(9): 2093-103, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19520712

RESUMO

BACKGROUND: Trophoblast invasion is crucial to placentation. The relationship between decidual glycodelin-A and trophoblast invasion is not known. METHODS: Invasiveness of First trimester extravillous cytotrophoblast-1 (TEV-1) cell line, TEV-1, cells was determined by trans-well invasion assay. The gene expression, protein secretion and activities of the matrix metalloproteinase (MMP)-2 and -9, urokinase plasminogen activator (uPA), tissue inhibitor of metalloproteinase (TIMP)-1 and -2 and plasminogen activator inhibitor (PAI-1) of glycodelin-A-treated cells were measured by quantitative PCR, ELISA and gel zymography, respectively. RESULTS: Glycodelin-A bound to TEV-1 cells. At a concentration of 1 microg/ml, glycodelin-A, but not other glycodelin isoforms, suppressed the invasion of TEV-1 cells. The effect was glycosylation-dependent and was associated with reduction (P < 0.05) of MMP2, MMP9 and uPA activities in the conditioned medium from the treated culture. Glycodelin-A treatment suppressed the amount of MMP2 protein in the conditioned medium (P < 0.05) and MMP2 mRNA in the cells (P < 0.05), but did not affect that of MMP9. Glycodelin-A also significantly reduced the expression, secretion and activity of uPA (P < 0.05). The treatment did not affect the expression of TIMP-1, TIMP-2 or PAI-1, cell proliferation or survival of the cells. CONCLUSIONS: Glycodelin-A inhibits the invasion of extravillous cytotrophoblasts mainly by suppressing the activity of MMP2 and MMP9 in a glycosylation-dependent fashion.


Assuntos
Glicoproteínas/fisiologia , Placentação/fisiologia , Proteínas da Gravidez/fisiologia , Trofoblastos/metabolismo , Linhagem Celular , Feminino , Glicodelina , Humanos , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Placentação/efeitos dos fármacos , Gravidez , Primeiro Trimestre da Gravidez , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
17.
Scand J Clin Lab Invest ; 69(4): 452-9, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19551557

RESUMO

Glycodelin is a lipocalin family glycoprotein expressed mainly in reproductive tissues. It is involved in cell recognition, and its relationship with epithelial differentiation is well established. Glycodelin actually appears to drive epithelial differentiation. The evidence comes from studies employing endometrial and breast cancer cell lines. First, transfection of glycodelin cDNA into glycodelin-negative carcinoma cells results in reduced expression of oncogenes, increased expression of tumor suppressor genes, increased cell differentiation, and reduced carcinoma cell growth. Second, histone deacetylase inhibitors (HDACIs) induce glycodelin synthesis in endometrial cancer cells concomitantly with cell differentiation. This effect is blocked by specific down-regulation of glycodelin by RNA interference, suggesting that the effects of HDACIs are mediated by glycodelin. We recently found that glycodelin not only reduces carcinoma cell growth in vitro, but glycodelin cDNA transfection to MCF-7 breast carcinoma cells also reduces growth of these cells in vivo, demonstrated by xenograft tumor growth in mouse mammary fat pads. These results strongly suggest that glycodelin acts as a tumor suppressor in breast cancer. The findings are compatible with the observations that certain types of glycodelin-expressing ovarian and breast cancers have a more favorable prognosis compared to glycodelin non-expressing tumors. This research has therefore introduced a novel mechanism to control cancer cell growth. In this communication we review the differentiation-related effects of glycodelin.


Assuntos
Adenocarcinoma/patologia , Neoplasias da Mama/patologia , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Neoplasias do Endométrio/patologia , Glicoproteínas/fisiologia , Proteínas da Gravidez/fisiologia , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Glicodelina , Inibidores de Histona Desacetilases , Humanos , Camundongos , Interferência de RNA
18.
J Biol Chem ; 284(22): 15084-96, 2009 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-19240032

RESUMO

Glycodelin is a human glycoprotein with four reported glycoforms, namely glycodelin-A (GdA), glycodelin-F (GdF), glycodelin-C (GdC), and glycodelin-S (GdS). These glycoforms have the same protein core and appear to differ in their N-glycosylation. The glycosylation of GdA is completely different from that of GdS. GdA inhibits proliferation and induces cell death of T cells. However, the glycosylation and immunomodulating activities of GdF and GdC are not known. This study aimed to use ultra-high sensitivity mass spectrometry to compare the glycomes of GdA, GdC, and GdF and to study the relationship between the immunological activity and glycosylation pattern among glycodelin glycoforms. Using MALDI-TOF strategies, the glycoforms were shown to contain an enormous diversity of bi-, tri-, and tetra-antennary complex-type glycans carrying Galbeta1-4GlcNAc (lacNAc) and/or GalNAcbeta1-4GlcNAc (lacdiNAc) antennae backbones with varying levels of fucose and sialic acid substitution. Interestingly, they all carried a family of Sda (NeuAcalpha2-3(GalNAcbeta1-4)Gal)-containing glycans, which were not identified in the earlier study because of less sensitive methodologies used. Among the three glycodelins, GdA is the most heavily sialylated. Virtually all the sialic acid on GdC is located on the Sda antennae. With the exception of the Sda epitope, the GdC N-glycome appears to be the asialylated counterpart of the GdA/GdF glycomes. Sialidase activity, which may be responsible for transforming GdA/GdF to GdC, was detected in cumulus cells. Both GdA and GdF inhibited the proliferation, induced cell death, and suppressed interleukin-2 secretion of Jurkat cells and peripheral blood mononuclear cells. In contrast, no immunosuppressive effect was observed for GdS and GdC.


Assuntos
Glicoproteínas/metabolismo , Linfócitos/citologia , Linfócitos/metabolismo , Proteínas da Gravidez/metabolismo , Apoptose , Configuração de Carboidratos , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Células do Cúmulo/enzimologia , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Glicodelina , Glicoproteínas/isolamento & purificação , Glicosilação , Humanos , Interleucina-2/metabolismo , Lectinas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Necrose/patologia , Neuraminidase/metabolismo , Polissacarídeos/química , Proteínas da Gravidez/isolamento & purificação , Ligação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
19.
Int J Cancer ; 123(10): 2279-84, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-18720404

RESUMO

Malignant growth is characterized by loss of cell differentiation, uncontrolled proliferation and resistance to apoptosis. Many tumor suppressor genes that protect cells against malignant transformation regulate cell differentiation. Here, we show for the first time that glycodelin, a differentiation-related protein, reduces breast cancer tumor growth in vivo. We found that glycodelin cDNA-transfected MCF-7 breast cancer cells showed a differentiated phenotype and produced smaller tumors in mouse mammary fat pads compared with control-transfected cells. Glycodelin-induced differentiation was associated with reduced expression of oncogenes and increased expression of tumor suppressor genes. Our results suggest that glycodelin acts as a tumor suppressor in breast cancer. This may explain its reported association with a more favorable prognosis in some cancers.


Assuntos
Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Glicoproteínas/farmacologia , Proteínas da Gravidez/farmacologia , Animais , Neoplasias da Mama/genética , Diferenciação Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Glicodelina , Humanos , Camundongos , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...