RESUMO
Free-surface-induced L10 chemical long-range ordering phenomena in a nanolayer, a nanowire and a cubic nanoparticle of FePt were studied by means of Monte Carlo simulations. The system was modeled with nearest-neighbor and next-nearest-neighbor interatomic pair interactions deduced from ab initio calculations. The generated samples, the dimensionality of which was determined by appropriate periodic boundary conditions imposed upon the generated supercells, were initially either perfectly ordered in the c-variant L10 superstructure ((001)-oriented monatomic planes), or completely disordered in the fcc crystalline structure. Vacancy-mediated creation of equilibrium atomic configurations was modelled by relaxing the systems at temperatures below the 'order-disorder' transition point using the Glauber algorithm implemented with the vacancy mechanism of atomic migration. The (100)-type-surface-induced heterogeneous nucleation of L10-order domains was observed and quantified by means of an original parameterization enabling selective determination of volume fractions of particular L10-variants. Due to the specific competition between the three kinds of (100)-type free surfaces, the initial c-L10 variant long-range order appeared to be the most stable in the cubic nanoparticle. The initially disordered samples were transformed by the creation of a specific L10 domain structure with a mosaic of particular L10-variant domains at the surfaces and almost homogeneous long-range order in the inner volume. The analysis of correlation effects revealed that chemical ordering was initiated at the free surfaces.
RESUMO
It has been suggested that interleukin 6 (IL-6) plays a potential role in the growth and progression of tumors, including esophageal cancer (EC). The aim of the study was to compare clinical significance of serum IL-6 with classic tumor markers - carcinoembryonic antigen (CEA) and squamous cell cancer antigen (SCC-Ag) - in EC patients in relation to its histological types - squamous cell carcinoma of esophagus (ESCC) and adenocarcinoma (AD) of esophagus. The study included 53 EC patients and 90 healthy subjects. Serum IL-6 and CEA levels were determined using immunoenzyme assays, while SCC-Ag - chemiluminescent assay. The diagnostic criteria and prognostic values for markers were defined. The levels of all proteins tested in EC, ESCC, and AD were higher than in healthy subjects. The percentage of elevated results was substantially higher for IL-6 (86%) than for CEA (30%) and SCC-Ag (24%) in EC, similarly as in ESCC (87%, 23%, and 33%) and AD (87%, 39%, and 13%, respectively) patients. Concentrations of IL-6 depended on distant metastases and patients' survival in EC and were significantly higher in ESCC patients with more advanced tumor stage and nodal metastases. The IL-6 area under receiver operating characteristic curve (0.92) was larger than for CEA (0.84) and SCC-Ag (0.62) in EC, likewise in ESCC (0.92, 0.87, 0.77) and AD (0.91, 0.79, 0.57, respectively). Our findings indicate better usefulness of IL-6 than classic tumor markers in the diagnosis of EC, especially in patients with ESCC.
Assuntos
Adenocarcinoma/sangue , Adenocarcinoma/patologia , Biomarcadores Tumorais/sangue , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/patologia , Neoplasias Esofágicas/sangue , Neoplasias Esofágicas/patologia , Interleucina-6/sangue , Adenocarcinoma/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/sangue , Área Sob a Curva , Antígeno Carcinoembrionário/sangue , Carcinoma de Células Escamosas/diagnóstico , Neoplasias Esofágicas/diagnóstico , Feminino , Humanos , Estimativa de Kaplan-Meier , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Invasividade Neoplásica , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Modelos de Riscos Proporcionais , Curva ROC , Serpinas/sangueRESUMO
Vascular endothelial growth factors C (VEGF-C) and D (VEGF-D) are important lymphangiogenic factors in human cancers. We studied the expression of VEGF-C and VEGF-D using immunohistochemistry in 73 resected esophageal cancer specimens, and correlated the results with patient clinicopathologic features and survival. High expression of VEGF-C was identified in 40 (54.7%) patients, and it correlated positively with histological grade (p=0.038), tumor stage (p=0.01), depth of tumor invasion (p=0.036) and lymph node metastasis (p=0.001). In 48 of 73 (65.7%) tumors, the VEGF-D protein was also expressed at high levels. VEGF-D immunoreactivity significantly correlated with tumor location (p=0.027), size of tumor (p=0.015), histological grade (p=0.02), depth of invasion (p=0.001) and lymph node metastasis (p=0.018). In logistic multivariate analysis, high expression of VEGF-C (OR 1.941, 95% CI 1.263-7.289, p=0.024) was associated with lymph node metastasis. Calculating the prognostic relevance revealed that both VEGF-C and VEGF-D correlated with decreased overall survival (p=0.01, p=0.003), disease free survival (p=0.02, p=0.006), and cancer-specific survival (p=0.03, p=0.005). In conclusion, our results suggest that high levels of both VEGF-C and VEGF-D proteins are associated with lymph node involvement, and that VEGF-C expression is an independent predictor of risk for lymph node metastasis in esophageal cancer. In locally advanced disease, overexpression of VEGF-C and VEGF-D may be useful in identifying patients who are more likely to have a poor prognosis even after curative resection.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Metástase Linfática/patologia , Fator C de Crescimento do Endotélio Vascular/biossíntese , Fator D de Crescimento do Endotélio Vascular/biossíntese , Adulto , Idoso , Intervalo Livre de Doença , Neoplasias Esofágicas/mortalidade , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
Interactions between extracellular matrix (ECM) and epithelial cells are necessary for proper organisation and function of the epithelium. In the present study we show that bovine mammary epithelial cell line BME-UV1 cultured on ECM components, commercially available as Matrigel, constitutes a good model for studying mechanisms controlling functional differentiation of the bovine mammary gland. In contact with Matrigel BME-UV1 cells induce apicobasal polarity, and within 16 days form three dimensional (3D) acinar structures with a centrally localized hollow lumen, which structurally resemble mammary alveoli present in the functionally active mammary gland. We have shown that the 3D culture system enables a high expression and proper localisation of integrin receptors and tight junction proteins in BME-UV1 cells to be induced. This effect was not obtained in cells grown in the classical 2D culture system on plastic. Moreover, ECM highly stimulated the synthesis of one of the major milk proteins, beta-casein, even in the absence of prolactin. Our results show that contact with ECM plays an important role in the lactogenic activity of bovine MECs, however, prolactin is necessary for the efficient secretion of milk proteins.
Assuntos
Diferenciação Celular , Meios de Cultura/farmacologia , Células Epiteliais/citologia , Matriz Extracelular/metabolismo , Glândulas Mamárias Animais/citologia , Prolactina/farmacologia , Animais , Membrana Basal/fisiologia , Caseínas/metabolismo , Bovinos , Técnicas de Cultura de Células , Linhagem Celular , Meios de Cultura/química , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Prolactina/químicaRESUMO
Mutations in the gene encoding the phosphotyrosine phosphatase PTP1C, a cytoplasmic protein containing a COOH-terminal catalytic and two NH2-terminal Src homology 2 (SH2) domains, have been identified in motheaten (me) and viable motheaten (mev) mice and are associated with severe hemopoietic dysregulation. The me mutation is predicted to result in termination of the PTP1C polypeptide within the first SH2 domain, whereas the mev mutation creates an insertion or deletion in the phosphatase domain. No PTP1C RNA or protein could be detected in the hemopoietic tissues of me mice, nor could PTP1C phosphotyrosine phosphatase activity be isolated from cells homozygous for the me mutation. In contrast, mice homozygous for the less severe mev mutation expressed levels of full-length PTP1C protein comparable to those detected in wild type mice and the SH2 domains of mev PTP1C bound normally to phosphotyrosine-containing ligands in vitro. Nevertheless, the mev mutation induced a marked reduction in PTP1C activity. These observations provide strong evidence that the motheaten phenotypic results from loss-of-function mutations in the PTP1C gene and imply a critical role for PTP1C in the regulation of hemopoietic differentiation and immune function.
Assuntos
Hematopoese , Camundongos Mutantes/genética , Proteínas Tirosina Fosfatases/metabolismo , Animais , Sequência de Bases , Primers do DNA/química , Receptores ErbB/metabolismo , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Linhagem , Fosfoproteínas/metabolismo , Mutação Puntual , Proteínas Tirosina Fosfatases/genética , RNA Mensageiro/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de SinaisRESUMO
Recent data implicating loss of PTP1C tyrosine phosphatase activity in the genesis of the multiple hemopoietic cell defects found in systemic autoimmune/immunodeficient motheaten (me) and viable motheaten (mev) mice suggest that PTP1C plays an important role in modulating intracellular signaling events regulating cell activation and differentiation. To begin elucidating the role for this cytosolic phosphatase in lymphoid cell signal transduction, we have examined early signaling events and mitogenic responses induced by B cell antigen receptor (BCR) ligation in me and mev splenic B cells and in CD5+ CH12 lymphoma cells, which represent the lymphoid population amplified in motheaten mice. Despite their lack of functional PTP1C, me and mev B cells proliferated normally in response to LPS. However, compared with wild-type B cells, cells from the mutant mice were hyperresponsive to normally submitogenic concentrations of F(ab')2 anti-Ig antibody, and they exhibited reduced susceptibility to the inhibitory effects of Fc gamma IIRB cross-linking on BCR-induced proliferation. Additional studies of unstimulated CH12 and wild-type splenic B cells revealed the constitutive association of PTP1C with the resting BCR complex, as evidenced by coprecipitation of PTP1C protein and phosphatase activity with BCR components and the depletion of BCR-associated tyrosine phosphatase activity by anti-PTP1C antibodies. These results suggest a role for PTP1C in regulating the tyrosine phosphorylation state of the resting BCR complex components, a hypothesis supported by the observation that PTP1C specifically induces dephosphorylation of a 35-kD BCR-associated protein likely representing Ig-alpha. In contrast, whereas membrane Ig cross-linking was associated with an increase in the tyrosine phosphorylation of PTP1C and an approximately 140-kD coprecipitated protein, PTP1C was no longer detected in the BCR complex after receptor engagement, suggesting that PTP1C dissociates from the activated receptor complex. Together these results suggest a critical role for PTP1C in modulating BCR signaling capacity, and they indicate that the PTP1C influence on B cell signaling is likely to be realized in both resting and activated cells.
Assuntos
Linfócitos B/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais/imunologia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linhagem Celular , Células Cultivadas , Cruzamentos Genéticos , Feminino , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Linfoma , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Fosfotirosina , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/análise , Proteínas Tirosina Fosfatases/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Baço/imunologia , Tirosina/análogos & derivados , Tirosina/análiseRESUMO
We report on the development of structural and magnetic order in epitaxially grown L10 FePt thin films. Upon annealing, the easy axis of magnetization changes from the out-of-plain into the in-plain direction. We found that the overall fraction of reoriented domains first increases but after certain time decreases before achieving a saturated state. The results are based on conversion electron Mössbauer spectroscopy studies and confirm Monte Carlo simulations in nano-layered FePt. We present a modified version of the Johnson-Mehl-Avrami (JMA) model adequately describing the experimental findings. Two dynamical processes, the first being a 2D-growth, dominate the initial state of sample annealing and the second being a 3D-growth, dominate the late stage close to saturation. From an Arrhenius plots of JMA coefficients for both processes we extracted the activation energies of the underlying dynamics which are 1.5(1) eV for disordering and 0.8(2) eV for ordering.
RESUMO
Composite films of nano-size nickel grains embedded in a carbonaceous matrix are synthesized by a PVD process of C(60) fullerenes and Ni acetate. The morphology of the nano-composite films is characterized by TEM, selected area electron diffraction, chemical analysis and AFM. Correlations with deposition parameters and typical structure changes are found. The mechanical properties are analyzed by nanoindentation. The load-displacement charts show typical pop-ins correlated with the heterogeneous nano-structure. The depth dependent hardness and indentation modulus vary according to the nano-composite structure and reflect the changes of the mechanical properties in the film.
RESUMO
BACKGROUND: The p70 S6 kinase, an enzyme critical for cell-cycle progression through the G1 phase, is activated in vivo by insulin and mitogens through coordinate phosphorylation at multiple sites, regulated by signaling pathways, some of which depend on and some of which are independent of phosphoinositide 3-kinase (Pl 3-kinase). It is not known which protein kinases phosphorylate and activate p70. RESULTS: Co-expression of p70 with 3-phosphoinositide-dependent protein kinase 1 (PDK1), a protein kinase that has previously been shown to phosphorylate and activate protein kinase B (PKB, also known as c-Akt), resulted in strong activation of the S6 kinase in vivo. In vitro, PDK1 directly phosphorylated Thr252 in the activation loop of the p70 catalytic domain, the phosphorylation of which is stimulated by PI 3-kinase in vivo and is indispensable for p70 activity. Whereas PDK1-catalyzed phosphorylation and activation of PKB in vitro was highly dependent on the presence of phosphatidylinositol 3,4,5-trisphosphate (Ptdlns (3,4,5)P3), PDK1 catalyzed rapid phosphorylation and activation of p70 in vitro, independent of the presence of Ptdlns(3,4,5)P3. The ability of PDK1 to phosphorylate p70 Thr252 was strongly dependent on the phosphorylation of the p70 noncatalytic carboxy-terminal tail (amino acids 422-525) and of amino acid Thr412. Moreover, once Thr252 was phosphorylated, its ability to cause activation of the p70 S6 kinase was also controlled by the p70 carboxy-terminal tail and by phosphorylation of p70 Ser394, and most importantly, Thr412. The overriding determinant of the absolute p70 activity was the strong positive cooperativity between Thr252 and Thr412 phosphorylation; both sites must be phosphorylated to achieve substantial p70 activation. CONCLUSIONS: PDK1 is one of the components of the signaling pathway recruited by Pl 3-kinase for the activation of p70 S6 kinase as well as of PKB, and serves as a multifunctional effector downstream of the Pl 3-kinase.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Sequência de Aminoácidos , Animais , Células CHO , Linhagem Celular Transformada , Cricetinae , Ativação Enzimática , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Dados de Sequência Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Quinases S6 Ribossômicas/genética , Serina/metabolismo , Treonina/metabolismo , TransfecçãoRESUMO
Previous studies have shown that the noncatalytic carboxy-terminal tail of the p70 S6 kinase (amino acids 422 to 525) contains an autoinhibitory pseudosubstrate domain that is phosphorylated in situ during activation and in vitro by mitogen-activated protein kinases. The present study shows that a recombinant p70 deleted of the carboxy-terminal tail (p70 delta CT104) nevertheless exhibits a basal and serum-stimulated 40S kinase activity and susceptibility to inhibition by wortmannin very similar to those of the parent, full-length p70 kinase. Carboxy-terminal deletion reduces the extent of maximal inhibition produced by rapamycin, from > 95% in the full-length p70 to 60 to 80% in p70 delta CT104, without altering the sensitivity to rapamycin inhibition (50% inhibitory concentration of 2 nM). Serum activation of p70 delta CT104, as with the parent, full-length p70, is accompanied by an increase in 32P content (about twofold) in situ and a slowing in electrophoretic mobility; both modifications are inhibited by pretreatment with wortmannin or rapamycin. 32P-peptide maps of p70 delta CT104 show multisite phosphorylation, and wortmannin and rapamycin appear to cause preferential dephosphorylation of the same subset of sites. Thus, it is likely that activation of the kinase requires phosphorylation of p70 at sites in addition to those previously identified in the carboxy-terminal tail. Evidence that the carboxy-terminal tail actually functions as a potent intramolecular inhibitor of kinase activity in situ is uncovered by deletion of a short acidic segment (amino acids 29 to 46) from the p70 amino-terminal noncatalytic region. Deletion of amino acids 29 to 46 causes a >95% inhibition of p70 activity despite continue phosphorylation of the carboxy-terminal tail in situ; additional deletion of the carboxy-terminal tail (yielding p70 delta 29-46/ delta CT104) increases activity 10-fold, to a level approaching that of p70 delta CT104. Deletion of residues 29 to 46 also abolishes completely the sensitivity of p70 to inhibition by rapamycin but does not alter the susceptibility to activation by serum of inhibition by wortmannin. Although the mechanisms underlying the effects of the delta 29-46 deletion are not known, they are not attributable to loss of the major in situ p70 phosphorylation site at Ser-40. Thus, activation of the p70 S6 kinase involves multiple, independent inputs directed at different domains of the p70 polypeptide. Disinhibition from the carboxy-terminal tail requires, in addition to its multisite phosphorylation, an activating input dependent on the presence of amino acids 29 to 46; this p70-activating input may be the same as that inhibited by rapamycin but is distinct from that arising from the wortmannin-inhibitable phosphatidylinositol 3-kinase. In addition, as exemplified by the rapamycin-resistant but mitogen- and wortmannin-sensitive p70 delta 29-46/ delta CT104 mutant, a further activating input, which probably involves site-specific phosphorylation in the segment between amino acids 46 to 421, is necessary.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Células 3T3 , Sequência de Aminoácidos , Androstadienos/farmacologia , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Primers do DNA/genética , DNA Complementar/genética , Ativação Enzimática , Camundongos , Mitógenos/farmacologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Polienos/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Quinases S6 Ribossômicas , Deleção de Sequência , Sirolimo , Relação Estrutura-Atividade , WortmaninaRESUMO
The SH2 domain-containing SHP-1 tyrosine phosphatase has been shown to negatively regulate a broad spectrum of growth factor- and cytokine-driven mitogenic signaling pathways. Included among these is the cascade of intracellular events evoked by stem cell factor binding to c-Kit, a tyrosine kinase receptor which associates with and is dephosphorylated by SHP-1. Using a series of glutathione S-transferase (GST) fusion proteins containing either tyrosine-phosphorylated segments of the c-Kit cytosolic region or the SH2 domains of SHP-1, we have shown that SHP-1 interacts with c-Kit by binding selectively to the phosphorylated c-Kit juxtamembrane region and that the association of c-Kit with the larger of the two SHP-1 isoforms may be mediated through either the N-terminal or C-terminal SHP-1 SH2 domain. The results of binding assays with mutagenized GST-Kit juxtamembrane fusion proteins and competitive inhibition assays with phosphopeptides encompassing each c-Kit juxtamembrane region identified the tyrosine residue at position 569 as the major site for binding of SHP-1 to c-Kit and suggested that tyrosine 567 contributes to, but is not required for, this interaction. By analysis of Ba/F3 cells retrovirally transduced to express c-Kit receptors, phenylalanine substitution of c-Kit tyrosine residue 569 was shown to be associated with disruption of c-Kit-SHP-1 binding and induction of hyperproliferative responses to stem cell factor. Although phenylalanine substitution of c-Kit tyrosine residue 567 in the Ba/F3-c-Kit cells did not alter SHP-1 binding to c-Kit, the capacity of a second c-Kit-binding tyrosine phosphatase, SHP-2, to associate with c-Kit was markedly reduced, and the cells again showed hyperproliferative responses to stem cell factor. These data therefore identify SHP-1 binding to tyrosine 569 on c-Kit as an interaction pivotal to SHP-1 inhibitory effects on c-Kit signaling, but they indicate as well that cytosolic protein tyrosine phosphatases other than SHP-1 may also negatively regulate the coupling of c-Kit engagement to proliferation.
Assuntos
Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Tirosina/metabolismo , Membrana Celular/metabolismo , Regulação para Baixo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Fenilalanina/metabolismo , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Transdução de Sinais , Fator de Células-Tronco/metabolismo , Células Tumorais Cultivadas , Domínios de Homologia de srcRESUMO
The aim of our study was to examine whether non beta(1)-/beta(2)-adrenoceptors participate in the relaxation of the human pulmonary artery. For this purpose the vasodilatory effect of the non-conventional partial beta-adrenoceptor agonist cyanopindolol was examined. Cyanopindolol (1-300 microM), studied in the presence of the beta(1)-/beta(2)-adrenoceptor antagonist propranolol, relaxed the human pulmonary artery preconstricted with serotonin 1 microM in a concentration-dependent manner (maximally by about 80%). This effect was diminished by bupranolol 10 microM (an antagonist of beta(1)-beta(3)-adrenoceptors and the low affinity state of the beta(1)-adrenoceptor) and CGP 20712 10 microM (known to antagonize the low-affinity state of the beta(1)-adrenoceptor at high concentrations). In further experiments, the effect of beta-adrenoceptor ligands on the serotonin-induced vasoconstriction was examined. The concentration-response curve for serotonin was not affected by cyanopindolol 30 microM, bupranolol 10 microM and CGP 20712 10 microM but shifted to the right by cyanopindolol 100 and 300 microM; the serotonin 5-HT(2A) receptor antagonist ketanserin 0.3 microM abolished the maximum contraction elicited by serotonin. In conclusion, the present study reveals that the vasodilatory effect of cyanopindolol in the human pulmonary artery consists of two components, i.e. activation of a propranolol-insensitive atypical beta-adrenoceptor and antagonism against 5-HT(2A) receptors.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Pindolol/análogos & derivados , Artéria Pulmonar/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Análise de Variância , Bupranolol/farmacologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Imidazóis/farmacologia , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Pindolol/farmacologia , Artéria Pulmonar/fisiologia , Receptor 5-HT2A de Serotonina/efeitos dos fármacos , Receptor 5-HT2A de Serotonina/fisiologia , Receptores Adrenérgicos beta 1/efeitos dos fármacos , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Antagonistas do Receptor 5-HT2 de Serotonina , Vasoconstrição/efeitos dos fármacos , Vasodilatação/fisiologiaRESUMO
In type 2 diabetes, impaired insulin signaling leads to hyperglycemia and other metabolic abnormalities. To study a new class of antidiabetic agents, we compared two small, nonpeptide molecules that activate insulin receptor (IR) beta-subunit tyrosine kinase activity: Merck L7, a direct IR agonist, and Telik's TLK16998, an IR sensitizer. In rat hepatoma cells (HTCs) that overexpress the IR (HTC-IR), IR autophosphorylation was directly activated by L7 in the absence of insulin. TLK16998 did not directly activate IR autophosphorylation, but it enhanced IR autophosphorylation in the presence of insulin. Tyrosine phosphorylation of an endogenous 185-kDa IR substrate was also significantly enhanced by both Merck L7 alone and TLK16998 plus insulin. Adding TLK16998 to L7 produced synergistic effects, further indicating that these two compounds act on the IR through separate mechanisms. We next studied HTC-IR(Delta485-599) cells, which overexpress a mutant IR with a deletion in the alpha-subunit connecting domain that does not undergo autophosphorylation in response to insulin binding. L7 was able to directly activate autophosphorylation of the deletion mutant IR in these cells, whereas TLK16998 had no effect. Compounds were then tested in three other cell models of impaired IR function. Both TLK16998 and Merck L7 improved IR autophosphorylation in cells with diminished IR signaling due to either treatment with tumor necrosis factor-alpha or overexpression of membrane glycoprotein PC-1. However, in TPA (tetradecanoylphorbol acetate)-treated cells, TLK16998 but not Merck L7 was able to significantly reverse the impaired insulin-stimulated IR autophosphorylation. In summary, these two classes of IR activators selectively increased IR function in a variety of insulin-resistant cell lines.
Assuntos
Resistência à Insulina/fisiologia , Insulina/fisiologia , Receptor de Insulina/fisiologia , Animais , Western Blotting , Deleção de Genes , Humanos , Insulina/farmacologia , Fosforilação/efeitos dos fármacos , Ratos , Receptor de Insulina/agonistas , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Células Tumorais Cultivadas , Tirosina/metabolismoRESUMO
Insulin resistance, an important feature of type 2 diabetes, is manifested as attenuated insulin receptor (IR) signaling in response to insulin binding. A drug that promotes the initiation of IR signaling by enhancing IR autophosphorylation should, therefore, be useful for treating type 2 diabetes. This report describes the effect of a small molecule IR sensitizer, TLK16998, on IR signaling. This compound activated the tyrosine kinase domain of the IR beta-subunit at concentrations of 1 micromol/l or less but had no effect on insulin binding to the IR alpha-subunit even at much higher concentrations. TLK16998 alone had no effect on IR signaling in mouse 3T3-L1 adipocytes but, at concentrations as low as 3.2 micromol/l, enhanced the effects of insulin on the phosphorylation of the IR beta-subunit and IR substrate 1, and on the amount of phosphatidylinositol 3-kinase that coimmunoprecipitated with IRS-1. Phosphopeptide mapping revealed that the effect of TLK16998 on the IR was associated with increased tyrosine phosphorylation of the activation loop of the beta-subunit tyrosine kinase domain. TLK16998 also increased the potency of insulin in stimulating 2-deoxy-D-glucose uptake in 3T3-L1 adipocytes, with a detectable effect at 8 micromol/l and a 10-fold increase at 40 micromol/l. In contrast, only small effects were observed on IGF-1-stimulated 2-deoxy-D-glucose uptake. In diabetic mice, TLK16998, at a dose of 10 mg/kg, lowered blood glucose levels for up to 6 h. These results suggest, therefore, that small nonpeptide molecules that directly sensitize the IR may be useful for treating type 2 diabetes.
Assuntos
Compostos Azo/farmacologia , Proteínas Musculares , Naftalenos/farmacologia , Receptor de Insulina/efeitos dos fármacos , Células 3T3 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Glicemia/análise , Diabetes Mellitus/sangue , Diabetes Mellitus/genética , Diabetes Mellitus Experimental/sangue , Transportador de Glucose Tipo 4 , Fator de Crescimento Insulin-Like I/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Transporte de Monossacarídeos/metabolismo , Fosforilação/efeitos dos fármacos , Receptor de Insulina/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Expression of the aboral ectoderm-specific LpS1 gene in Lytechinus was used to study lineage-specific transcriptional regulation during sea urchin development. Band shift assays using anti-USF antibody showed that a USF-like protein bound the USF core sequence 5'-CACGTG-3' in the promoter of the LpS1 gene. DNA constructs consisting of a wild-type LpS1 promoter and the same LpS1 promoter with a mutated USF binding site fused to the bacterial chloramphenicol acetyltransferase reporter gene were tested. The mutation in the USF binding site caused an increase in chloramphenicol acetyltransferse activity. We selected a clone that encodes USF, LvUSF, from a gastrula-stage cDNA library representing Lytechinus variegatus. Transactivation experiments, in which LvUSF RNA or a DNA construct consisting of the LvUSF cDNA clone fused to the Lytechinus pictus metallothionein promoter coinjected with the wild-type or mutated LpS1 promoter-chloramphenicol acetyltransferase gene construct, showed that chloramphenicol acetyltransferase activity from the wild-type construct was repressed, while the construct mutated at the USF binding site was active. The same wild-type and mutated LpS1 promoter DNA fragments ligated to the green fluorescent protein reporter gene were used to examine spatial expression. The reporter gene constructs containing the mutated USF binding site were expressed inappropriately in all cell types including the gut and oral ectoderm in gastrula and larva stage embryos, while the wild-type constructs were expressed primarily in the aboral ectoderm. USF was expressed in all cells of the early embryo and in all tissues except the aboral ectoderm in later embryos. The data are consistent with a model depicting Lytechinus USF, as a temporal and spatial regulator by repressing LpS1 gene transcription in non-aboral ectoderm cells.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação a DNA , Proteínas Repressoras/genética , Ouriços-do-Mar/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Clonagem Molecular , DNA Complementar/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA/genética , RNA/metabolismo , Proteínas Repressoras/metabolismo , Ouriços-do-Mar/crescimento & desenvolvimento , Ouriços-do-Mar/metabolismo , Fatores de Transcrição/metabolismo , Fatores Estimuladores UpstreamRESUMO
During sea urchin development, esophageal muscle arises from secondary mesenchyme cells, descendants of the vegetal plate that delaminate from the coelomic epithelium at the end of gastrulation. In lithium-induced exogastrulae, where vegetal plate descendants evert rather than invaginate, myogenesis occurs normally, indicating that myocyte progenitors do not have to be near the future stomodeum for differentiation to occur. Vegetal plate descendants isolated along with the extracellular matrix at different times during gastrulation produce differentiated myocytes in culture as monitored by staining with a myosin heavy chain antibody. Vegetal isolates prepared at mid-gastrulation or later consistently produce differentiated myocytes whose form and position resembled their counterparts in the intact embryo, whereas vegetal isolates prepared a few hours earlier while capable of gut differentiation, as evidenced by the de novo synthesis of the endodermal surface marker Endo 1, did not produce differentiated myocytes. These results suggest that sometime after early gastrulation, a subset of secondary mesenchyme cells are competent to differentiate into muscle cells. RNase protection assays showed that the accumulation of sea urchin myogenic factor (SUM-1) mRNA is likely to be coincident with the earliest demonstrable commitment of myogenic precursors. Premature expression of SUM-1 coding sequences in mesenchyme blastulae resulted in the activation of muscle-specific enhancer elements, demonstrating that SUM-1 can function precociously in the early embryo. However, SUM-1 expressed in this manner did not activate the endogenous MHC gene, nor induce premature or ectopic production of muscle cells.
Assuntos
Proteínas Musculares/farmacologia , Músculos/embriologia , Ouriços-do-Mar/embriologia , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Diferenciação Celular/efeitos dos fármacos , Cloretos/farmacologia , Sequência Consenso , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Lítio/farmacologia , Cloreto de Lítio , Dados de Sequência Molecular , Miosinas/biossíntese , Células-Tronco/efeitos dos fármacosRESUMO
Previous studies showed that the core promoter of the mouse cAMP-dependent protein kinase regulatory subunit type II beta (RII beta) gene was composed of two functional elements. One element was GC rich and bound the Sp1 transcription factor. The second element contained a helix-loop-helix (HLH)-motif. Each element conferred transcriptional activity when inserted upstream of a reporter gene, chloramphenicol acetyltransferase and transfected into mouse NB2a neuroblastoma cells and Chinese hamster ovary (CHO) cells. The core promoter was further characterized by mutational analysis using electrophoretic mobility shift assays and by transfection into CHO and NB2a cells. Electrophoretic mobility shift assays showed that the HLH-consensus motif, CACGTG, present in the RII beta gene bound nuclear factors present in NB2a and CHO cells. Mutations in the HLH-core motif decreased the binding of these factors and reduced the transcriptional activity of constructs containing the chloramphenicol acetyltransferase reporter when transfected into these cells. The results showed that the central nucleotides as well as the adjacent bases were important for the interaction with the nuclear binding factors. UV cross-linking, Southwestern blot analysis, and interference of the mobility shift patterns by specific antisera directed against USF and c-Myc indicated that both of these transcription factors were forming complexes with the HLH-consensus motif. The results suggest that RII beta transcription may be regulated, in part, by USF and c-Myc in NB2a and CHO cells.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas de Ligação a DNA , Sequências Hélice-Alça-Hélice , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Células CHO , Sequência Consenso , Cricetinae , Cricetulus , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico , Proteína Quinase Tipo II Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/química , Genes , Camundongos , Dados de Sequência Molecular , Neuroblastoma/patologia , Ratos , Células Tumorais Cultivadas , Fatores Estimuladores UpstreamRESUMO
Mexican Americans have a threefold greater prevalence of non-insulin-dependent diabetes mellitus (NIDDM) than non-Hispanic Whites as found in the San Antonio Heart Study, a population-based study of diabetes and cardiovascular disease. In addition, Mexican-American diabetic subjects have higher levels of glycemia than non-Hispanic White diabetic subjects. We therefore hypothesized that the prevalence of clinical proteinuria would be greater among Mexican-American diabetic subjects (n = 317) than among non-Hispanic White diabetic subjects (n = 67). Clinical proteinuria, defined as greater than or equal to 1+ on the Ames Albustix test, was 2.82 times more prevalent in Mexican-American diabetic subjects compared with non-Hispanic White diabetic subjects adjusting for age and duration (95% confidence interval [CI] = 1.05, 7.55; P = .039). After controlling for other possible confounding variables (i.e., glycemia, systolic blood pressure, smoking, and insulin use), the excess of proteinuria in Mexican-American diabetic subjects was only slightly attenuated, although the statistical significance became borderline (odds ratio [OR] = 2.59, 95% CI = 0.91, 7.32; P = .072). The prevalence of microalbuminuria (greater than 30 mg/L) was also significantly higher in Mexican-American diabetic subjects than in non-Hispanic White diabetic subjects (OR = 3.54, 95% CI = 1.28, 9.81; P = .015). We also compared previously diagnosed Mexican-American diabetic subjects (n = 243) from San Antonio with previously diagnosed non-Hispanic White diabetic subjects in Wisconsin (n = 476).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Diabetes Mellitus Tipo 2/urina , Proteinúria/epidemiologia , Adulto , Albuminúria/epidemiologia , Diabetes Mellitus Tipo 2/diagnóstico , Feminino , Hispânico ou Latino , Humanos , Masculino , México/etnologia , Pessoa de Meia-Idade , População BrancaRESUMO
Tumor-specific genes delivered to dendritic cells (DCs) have been used for the generation of cytotoxic T cells (CTLs), but their application has been limited on the one hand by low viral titers resulting in low transduction efficiency and poor protein production, and on the other hand by immunogenicity of the selectable marker and poor viability of the DCs. We addressed these limitations by creating a multipurpose master vector (pMV) and cloning the tumor gene NY-ESO-1, which is highly expressed in more than 50% of advanced myeloma patients. pMV was constructed from a Moloney murine leukemia virus (Mo-MuLV)-based retroviral backbone with the following features: (1) an extended packaging signal to achieve high viral titers, (2) a splice acceptor region to facilitate protein production, (3) a nonimmunogenic selectable marker, dihydrofolate reductase-L22Y (DHFR(L22Y)), to exclude the generation of CTLs against the selectable marker, (4) an internal ribosomal entry site between the tumor-specific gene (NY-ESO-1) and the selectable marker DHFR(L22Y) for coexpression of two heterologous gene products from a single bicistronic mRNA, minimizing the possibility of differential expression of these two genes, and (5) human granulocyte-macrophage colony-stimulating factor (hGM-CSF) cDNA driven by the human T-lymphotropic virus promoter to enhance DC function and viability. Recombinant virus of pMV-NY-ESO-1 was generated with vesicular stomatitis virus G envelope protein (VSV-G) in the GP2-293 cell line for efficient transduction. We present evidence that the DC phenotype is unaltered after transduction and that more than 85% of DCs express NY-ESO-1, which secrete approximately 40 ng of GM-CSF per 10(6) DCs.
Assuntos
Antígenos de Neoplasias/metabolismo , Células Dendríticas/metabolismo , Vetores Genéticos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Testículo/metabolismo , Antígenos CD34/metabolismo , Antígenos de Neoplasias/análise , Antígenos de Neoplasias/genética , Técnicas de Transferência de Genes , Marcadores Genéticos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Células HeLa , Humanos , Imuno-Histoquímica , Masculino , Vírus da Leucemia Murina de Moloney/genética , Mieloma Múltiplo/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Retroviridae/genética , Transdução GenéticaRESUMO
Tumor necrosis factor-alpha (TNF-alpha) has a key role in skeletal disease in which it promotes reduced bone formation by mature osteoblasts and increased osteoclastic resorption. Here we show that TNF inhibits differentiation of osteoblasts from precursor cells. TNF-alpha treatment of fetal calvaria precursor cells, which spontaneously differentiate to the osteoblast phenotype over 21 days, inhibited differentiation as shown by reduced formation of multilayered, mineralizing nodules and decreased secretion of the skeletal-specific matrix protein osteocalcin. The effect of TNF was dose dependent with an IC50 of 0.6 ng/ml, indicating a high sensitivity of these precursor cells. Addition of TNF-alpha from days 2-21, 2-14, 7-14, and 7-10 inhibited nodule formation but addition of TNF after day 14 had no effect. Partial inhibition of differentiation was observed with addition of TNF on only days 7-8, suggesting that TNF could act during a critical period of phenotype selection. Growth of cells on collagen-coated plates did not prevent TNF inhibition of differentiation, suggesting that inhibition of collagen deposition into matrix by proliferating cells could not, alone, explain the effect of TNF. Northern analysis revealed that TNF inhibited the expression of insulin-like growth factor I (IGF-I). TNF had no effect on expression of the osteogenic bone morphogenic proteins (BMPs-2, -4, and -6), or skeletal LIM protein (LMP-1), as determined by semiquantitative RT-PCR. Addition of IGF-I or BMP-6 to fetal calvaria precursor cell cultures enhanced differentiation but could not overcome TNF inhibition, suggesting that TNF acted downstream of these proteins in the differentiation pathway. The clonal osteoblastic cell line, MC3T3-E1-14, which acquires the osteoblast phenotype spontaneously in postconfluent culture, was also studied. TNF inhibited differentiation of MC3T3-E1-14 cells as shown by failure of mineralized matrix formation in the presence of calcium and phosphate. TNF was not cytotoxic to either cell type as shown by continued attachment and metabolism in culture, trypan blue exclusion, and Alamar Blue cytotoxicity assay. These results demonstrate that TNF-alpha is a potent inhibitor of osteoblast differentiation and suggest that TNF acts distal to IGF-I, BMPs, and LMP-1 in the progression toward the osteoblast phenotype.