RESUMO
We investigated whether intranasal immunization with amoebic lysates plus cholera toxin modified the populations of T and B lymphocytes, macrophages and dendritic cells by flow cytometry from nose-associated lymphoid tissue (NALT), cervical lymph nodes (CN), nasal passages (NP) and spleen (SP). In all immunized groups, the percentage of CD4 was higher than CD8 cells. CD45 was increased in B cells from mice immunized. We observed IgA antibody-forming cell (IgA-AFC) response, mainly in NALT and NP. Macrophages from NP and CN expressed the highest levels of CD80 and CD86 in N. fowleri lysates with either CT or CT alone immunized mice, whereas dendritic cells expressed high levels of CD80 and CD86 in all compartment from immunized mice. These were lower than those expressed by macrophages. Only in SP from CT-immunized mice, these costimulatory molecules were increased. These results suggest that N. fowleri and CT antigens are taking by APCs, and therefore, protective immunity depends on interactions between APCs and T cells from NP and CN. Consequently, CD4 cells stimulate the differentiation from B lymphocytes to AFC IgA-positive; antibody that we previously found interacting with trophozoites in the nasal lumen avoiding the N. fowleri attachment to nasal epithelium.
Assuntos
Administração Intranasal , Antígenos de Protozoários/administração & dosagem , Naegleria fowleri/fisiologia , Mucosa Nasal/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos de Protozoários/imunologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Toxina da Cólera/administração & dosagem , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Naegleria fowleri/crescimento & desenvolvimento , Naegleria fowleri/imunologia , Mucosa Nasal/citologiaRESUMO
Diabetes and hypertension have been associated with cardiovascular diseases and stroke. Some reports have related the coexistence of hypertension and diabetes with increase in the risk of developing vascular complications. Recently some studies have shown results suggesting that in the early stages of diabetes and hypertension exist a reduced functional response to vasopressor agents like angiotensin II (Ang II), which plays an important role in blood pressure regulation mechanism through the activation of its AT1 and AT2 receptors. For that reason, the aim of this work was to study the gene and protein expression of AT1 and AT2 receptors in aorta of diabetic SHR and WKY rats. Diabetes was induced by the administration of streptozotocin (60 mg/kg i.p.). After 4 weeks of the onset of diabetes, the protein expression was obtained by western blot and the mRNA expression by RT-PCR. Our results showed that the hypertensive rats have a higher mRNA and protein expression of AT1 receptors than normotensive rats while the AT2 expression remained unchanged. On the other hand, the combination of diabetes and hypertension increased the mRNA and protein expression of AT1 and AT2 receptors significantly. In conclusion, our results suggest that diabetes with hypertension modifies the mRNA and protein expression of AT1 and AT2 receptors. However, the overexpression of AT2 could be associated with the reduction in the response to Ang II in the early stage of diabetes.
Assuntos
Angiotensina II/metabolismo , Aorta/metabolismo , Diabetes Mellitus Experimental/metabolismo , Hipertensão/metabolismo , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Animais , Aorta/fisiopatologia , Pressão Sanguínea/fisiologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/fisiopatologia , Perfilação da Expressão Gênica , Hipertensão/complicações , Hipertensão/fisiopatologia , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/metabolismoRESUMO
Moderate exercise enhances resistance to pathogen-associated infections. However, its influence on intestinal IgA levels and resistance to Salmonella typhimurium in mice has not been reported. The aim of this study was to assess the impact of moderate exercise on bacterial resistance and the intestinal-IgA response in a murine typhoid model. Sedentary and exercised (under a protocol of moderate swimming) BALB/c mice were orally infected with Salmonella typhimurium and sacrificed on days 7 or 14 post-infection (n=5 per group). Compared with infected sedentary mice, infected exercised animals had i) lower intestinal and systemic bacterial loads; ii) higher total and specific intestinal-IgA levels, iii) a higher percentage of IgA plasma cells in lamina propria; iv) a higher level on day 7 and lower level on day 14 of intestinal α- and J-chain mRNA and plasma corticosterone, v) unchanged mRNA expression of intestinal pIgR, and vi) a higher mRNA expression of liver pIgR, α-chain and J-chain on day 7. Hence, it is likely that an increase in corticosterone levels (stress response) induced by moderate exercise increased intestinal IgA levels by enabling greater liver expression of pIgR mRNA, leading to a rise in IgA transcytosis from the liver to intestine. The overall effect of these changes is an enhanced resistance to infection.
Assuntos
Resistência à Doença/fisiologia , Imunoglobulina A/metabolismo , Mucosa Intestinal/metabolismo , Condicionamento Físico Animal/fisiologia , Infecções por Salmonella/prevenção & controle , Salmonella typhimurium , Animais , Carga Bacteriana , Corticosterona/sangue , Modelos Animais de Doenças , Cadeias J de Imunoglobulina/metabolismo , Cadeias alfa de Imunoglobulina/metabolismo , Intestinos/microbiologia , Fígado/metabolismo , Masculino , Camundongos Endogâmicos BALB C , RNA Mensageiro/metabolismo , Receptores de Imunoglobulina Polimérica/metabolismo , Natação/fisiologiaRESUMO
OBJECTIVE: This study aimed to compare two routes of administration and different dosages of streptozotocin (STZ) for the pharmacological induction of gestational diabetes mellitus (GDM) in pregnant CD1 females. MATERIALS AND METHODS: 35 female CD1 mice were divided into 5 groups (n = 7). Diabetes mellitus (DM) was induced with STZ by two routes and two doses: 1) Control Group without administration of STZ (CL), 2) Intraperitoneal Group with 200 mg of STZ/Kg of weight (IP200), 3) Intraperitoneal Group with 230 mg of STZ/Kg of weight (IP230), 4) Subcutaneous Group with 200 mg of STZ/Kg of weight (SC200) and 5) Subcutaneous Group with 230 mg of STZ/Kg of weight (SC230). Body weight, food and water intake, glycemia, Homeostatic Model Assessment of Insulin Resistance Index (HOMA-IR), survival, and birth rate were identified. RESULTS: The SC230 group turned out to be the most effective dose and route for the induction of GDM in pregnant females. This scheme managed to reproduce sustained hyperglycemia with high HOMA-IR, the presence of polyphagia, polydipsia, and weight loss. In addition, the birth rate and survival were high compared to the other doses and routes of administration. CONCLUSIONS: The administration of a single dose of 230 mg/kg of weight by subcutaneous route supposes advantages compared to previously used models since it decreases the physiological stress due to manipulation and the costs since it does not require repeated doses or adjuvants such as high lipid diets to potentiate the diabetogenic effect of STZ. Graphical Abstract: https://www.europeanreview.org/wp/wp-content/uploads/Graphical-abstract-12.jpg.
Assuntos
Diabetes Mellitus Experimental , Diabetes Gestacional , Estreptozocina , Animais , Feminino , Gravidez , Camundongos , Diabetes Mellitus Experimental/induzido quimicamente , Estreptozocina/administração & dosagem , Injeções Subcutâneas , Glicemia/metabolismo , Glicemia/efeitos dos fármacos , Relação Dose-Resposta a Droga , Injeções Intraperitoneais , Resistência à Insulina , Peso Corporal/efeitos dos fármacosRESUMO
The immune-suppression caused by acute stress can be reduced by a regular practice of moderate exercise which is known to modulate the expression of secretory-IgA. This antibody is essential for protection against infections and maintenance of homeostasis at the mucosal level. In order to explore the effects of moderate exercise on secretory-IgA production in ileum of the small intestine, 2 groups of mice were submitted to this protocol for 6 months, an exercise group and a sedentary group. After sacrifice, levels of secretory-IgA in intestinal fluid and levels of adrenal hormones in serum were determined by enzyme immunoenzymatic assay. IgA-plasma cells in lamina propria were evaluated by flow cytometry. Transcriptional mRNA expression in mucosa of alpha-chain, J-chain, pIgR and cytokines (Interleukin-2, -4, -6, -10, transforming growth factor-beta, interferon-gamma and tumor necrosis factor) were determined by RT-PCR. In comparison with sedentary mice, moderate exercised mice displayed an up-regulating effect on the production of secretory-IgA and IgA-plasma cells, on the expression of all mRNA transcripts from secretory-IgA associated proteins, and on all cytokines tested. However, serum levels of adrenal hormones were not altered. Future studies on secretory-IgA production are necessary to support the substantive effect of moderate exercise on protection and homeostasis at the intestinal level.
Assuntos
Íleo/imunologia , Imunoglobulina A Secretora/imunologia , Condicionamento Físico Animal/fisiologia , Esforço Físico/imunologia , Animais , Íleo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Esforço Físico/fisiologiaRESUMO
AIM: The aim of this paper was to evaluate the effect of a treatment with glycophosphopeptide on Olympic high platform divers during training and competition by measuring lymphocytes and cortisol in peripheral blood, and secretory immunoglobin A in saliva (sIgA). METHODS: Two groups of 8 divers were given a 14-day treatment of capsules (Gp or placebo) three times per day. Measurements of the peripheral blood lymphocytes (TCD3+, TCD4+ and T CD8+), plasma cortisol and IgA levels in saliva were made on day 0, 21 and 150. RESULTS: There was no significant difference found between the Gp- and placebo-treated groups regarding the increase in IgA between basal and first, or first and second measurements. The fact that there was a significant increase in S-IgA (9.89 ± 0.44 to 10.59±0.55, P=0.001) and B CD19+ (345.13±108.24 to 484.75±120.54, P=0.025) in the Gp- and not in the placebo-treated group between the basal and first measurement was due to the variation among the athletes of the latter group, and not the increase itself, indicating that Gp acted as an immunomodulator. It was apparently the exercise and not the Gp treatment that caused the increase in S-IgA and B CD19+ at the first and second measurements. CONCLUSION: The current study reports that with athletes who practiced moderately intense exercise, which stimulated the immune response, a Gp treatment of two weeks seems to have acted only as an immunomodulator that reduced the variation in the increased levels of IgA and B CD19+.
Assuntos
Adjuvantes Imunológicos/farmacologia , Mergulho/fisiologia , Exercício Físico/fisiologia , Imunidade Inata/efeitos dos fármacos , Imunoglobulina A Secretora/metabolismo , Saliva/metabolismo , Linfócitos T/efeitos dos fármacos , Adolescente , Método Duplo-Cego , Feminino , Seguimentos , Humanos , Contagem de Linfócitos , Masculino , Estudos Prospectivos , Infecções Respiratórias/imunologia , Infecções Respiratórias/metabolismo , Infecções Respiratórias/prevenção & controleRESUMO
The consumption of sweeteners has increased as a measure to reduce the consumption of calories and thus combat obesity and diabetes. Sweeteners are found in a large number of products, so chronic consumption has been little explored. The objective of the study was to evaluate the effect of chronic sweetener consumption on the microbiota and immunity of the small intestine in young mice. We used 72 CD1 mice of 21 days old, divided into 3 groups: (i) No treatment, (ii) Group A (6 weeks of treatment), and (iii) Group B (12 weeks of treatment). Groups A and B were divided into 4 subgroups: Control (CL), Sucrose (Suc), Splenda® (Spl), and Svetia® (Sv). The following were determined: anthropometric parameters, percentage of lymphocytes of Peyer's patches and lamina propria, IL-6, IL-17, leptin, resistin, C-peptide, and TNF-α. From feces, the microbiota of the small intestine was identified. The BMI was not modified; the mice preferred the consumption of Splenda® and Svetia®. The percentage of CD3+ lymphocytes in Peyer's patches was increased. In the lamina propria, Svetia® increased the percentage of CD3+ lymphocytes, but Splenda® decreases it. The Splenda® and Svetia® subgroups elevate leptin, C-peptide, IL-6, and IL-17, with reduction of resistin. The predominant genus in all groups was Bacillus. The chronic consumption of sweeteners increases the population of lymphocytes in the mucosa of the small intestine. Maybe, Bacillus have the ability to adapt to sweeteners regardless of the origin or nutritional contribution of the same.
RESUMO
BACKGROUND: Diabetes mellitus is considered a chronic noncommunicable disease in which inflammation plays a main role in the progression of the disease and it is known that n-3 fatty acids have anti-inflammatory properties. One of the most recent approaches is the study of the fatty acids of microalgae as a substitute for fish oil and a source rich in fatty acids EPA and DHA. OBJECTIVE: To analyze the effect of supplementation with n-3 fatty acids extracted from microalgae on the inflammatory markers from two different strains of mice. METHODS: Mice of two strains, db/db and CD1, were supplemented with n-3 fatty acids extracted from microalgae in lyophilized form and added to food; the experiment was carried out from week 8 to 16 of life. Flow cytometry was performed to determine the percentage of TCD4+ cells producing Th1 and Th2 cytokines. RESULTS: Supplementation with microalgae fatty acids decreased the percentage of TCD4+ cells producing IFN-γ and TNF-α and increased the ones producing IL-17A and IL-12 in both strains; on the other hand, supplementation decreased percentage of TCD4+ cells producing IL-4 and increased the ones producing TGF-ß. CONCLUSIONS: Microalgae n-3 fatty acids could be a useful tool in the treatment of diabetes as well as in the prevention of the appearance of health complications caused by inflammatory states.
RESUMO
BACKGROUND: Cutaneous leishmaniasis lacks effective and well-tolerated treatments. The current therapies mainly rely on antimonial drugs that are inadequate because of their poor efficacy. Traditional medicine offers a complementary alternative for the treatment of various diseases. Additionally, several plants have shown success as anti-leishmanial agents. Therefore, we sought to evaluate the in vitro and in vivo activity of MEBA against Leishmania mexicana. MATERIALS AND METHODS: Methanolic extract of B. aptera was obtained by macetration, after we determined in vitro anti-leishmanial activity of MEBA by MTT assay and the induced apoptosis in promastigotes by flow cytometry. To analyze the in vivo anti-leishmanial activity, we used infected mice that were treated and not treated with MEBA and we determined the levels of cytokines using ELISA. The phytochemical properties were determined by CG-MS and DPPH assay. RESULTS: We determined of LC50 of 0.408 mg/mL of MEBA for in vitro anti-leishmanial activity. MEBA induced apoptosis in promastigotes (15.3% ± 0.86). Treated mice exhibited smaller lesions and contained significantly fewer parasites than did untreated mice; in addition, we found that IFN-γ and TNF-α increased in the sera of MEBA-treated mice. GC-MS analysis showed that podophyllotoxin was the most abundant compound. Evaluation of the activity by DPPH assay demonstrated an SC50 of 11.72 µg/mL. CONCLUSION: Based on the above data, it was concluded that MEBA is a good candidate in the search for new anti-leishmanial agents.
Assuntos
Bursera/química , Leishmania mexicana , Leishmaniose Cutânea/tratamento farmacológico , Fitoterapia , Extratos Vegetais/uso terapêutico , Animais , Feminino , Interferon gama/sangue , Leishmaniose Cutânea/sangue , Leishmaniose Cutânea/parasitologia , Medicina Tradicional , Camundongos Endogâmicos BALB C , Casca de Planta , Extratos Vegetais/farmacologia , Podofilotoxina/análise , Podofilotoxina/farmacologia , Podofilotoxina/uso terapêutico , Fator de Necrose Tumoral alfa/sangueRESUMO
Genotoxic stress activation of the tumor suppressor transcription factor p53 involves post-translational C-terminal modifications that increase both protein stability and DNA binding activity. We compared the requirement for p53 protein activation of p53 target sequences in two major p53-regulated genes, p21/WAF1 (encoding a cell cycle inhibitory protein) and Mdm2 (encoding a ubiquitin ligase that targets p53 for proteolytic degradation). The p53 binding site in the proximal p21/WAF1 promoter contains a single p53 binding consensus sequence, while the p53 binding site in the Mdm2 promoter contains two consensus sequences linked by a 17 bp spacer. Binding of recombinant p53 protein to the p21/WAF1 binding site required monoclonal antibody PAb421, which can mimic activating phosphorylation and/or acetylation events at the C-terminus. In contrast, recombinant p53 bound strongly to the Mdm2 binding site in the absence of PAb421 antibody. Separate binding to each consensus sequence of the Mdm2 binding site still required PAb421, indicating that p53 binding was not simply due to greater affinity to the Mdm2 consensus sequences. Linking two p21/WAF1 binding sites with the 17 bp spacer region from the Mdm2 gene eliminated the PAb421 requirement for p53 binding to the p21/WAF1 site. These results suggest a mechanism for regulation of Mdm2 gene transcription that differs from that other p53-induced genes by its lack of a requirement for C-terminal activation of p53 protein. A steady induction of Mdm2 protein would maintain p53 protein at low levels until post-translational modifications following DNA damage increased p53 activity towards other genes, mediating p53 growth inhibitory and apoptotic activities.
Assuntos
Proteínas Nucleares , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteína Supressora de Tumor p53/metabolismo , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Sequência Consenso , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-mdm2 , Deleção de Sequência , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genéticaRESUMO
The expression of the tumor suppressor gene p53 was studied in Syrian hamster embryo cells neoplastically initiated with a single dose of 3-methylcholanthrene. Ten randomly selected individual 3-methylcholanthrene-transformed colonies were established in culture independently. Eight of these cell lines contained levels of p53 mRNA similar to those in primary embryo cells (p53+ cell lines), as measured by Northern blot analysis of total RNA, whereas two of them (81C43 and 81C47) showed no detectable levels of p53 mRNA (p53- cell lines). However, Southern blot and karyotype analyses did not reveal any significant changes in copy number or gross rearrangements of the p53 gene in any of the p53- cell lines. A 3-kilobase genomic fragment cloned from p53- cells (81C47) containing both upstream and downstream promoters of the p53 gene was able to drive the expression of a CAT reporter gene when transfected into either p53+ or p53- cells. Furthermore, run-on assays performed on nuclei of p53- cells showed that the p53 gene was transcriptionally active, demonstrating that the genetic defect leading to the lack of p53 expression was not due to alterations in the promoter region. Detection of mRNA species corresponding to p53 mRNA precursors in Northern blot analysis of polyadenylated RNA from both p53+ and p53- cells indicated that the lack of p53 expression was not caused by mutations in the 3' regulatory region of the p53 gene affecting transcription termination and/or polyadenylation of p53 precursor mRNA. PCR amplification and nucleotide sequence analysis of extensive internal regions of the gene revealed that both p53- cell lines were homozygous for the same unique point mutation on the splice acceptor site of the fifth intron, a G to C transversion in the last nucleotide of the intron. The presence of this mutation in both p53- cell lines strongly suggests that it was induced specifically by 3-methyl-cholanthrene treatment and indicates that the resulting splicing malfunction may account for the lack of p53 gene expression.
Assuntos
Genes p53/efeitos dos fármacos , Íntrons/genética , Mutação Puntual/genética , Regiões Promotoras Genéticas/genética , Animais , Sequência de Bases , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Cricetinae , Embrião de Mamíferos , Fibroblastos/metabolismo , Genes Reporter , Genes p53/genética , Íntrons/efeitos dos fármacos , Mesocricetus , Metilcolantreno , Dados de Sequência Molecular , Regiões Promotoras Genéticas/fisiologia , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Transcrição Gênica/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
p53-interacting proteins from mouse epidermal cells and human myelogenous leukemia cells were isolated by affinity chromatography using glutathione S-transferase (GST)-p53 fusion proteins. One of these proteins was topoisomerase I, whose interaction with p53 was recently reported. A carboxyl-terminal fragment containing the last 92 amino acids of p53 (GST-299-390) was sufficient for binding to topoisomerase I. Nanomolar concentrations of either GST-p53 or GST-299-390 enhanced the catalytic activity of purified human topoisomerase I. Purified wild-type human p53 and point mutants Ser-239, Ser-245, and His-273 were equivalent in their enhancement of human topoisomerase I activity. Because topoisomerase I is thought to promote genetic recombination, competence to enhance topoisomerase I catalytic activity coupled with a deficiency in transcriptional activity may be a mechanism for gain of function in mutant p53 proteins.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Animais , Sítios de Ligação , Células Cultivadas , Ativação Enzimática , Humanos , Camundongos , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
To investigate the mechanisms of radiation-induced neoplastic conversion, DNA from X-ray transformed human epidermal keratinocytes (RHEK-1) was used in sequential cycles of NIH3T3 transfection followed by nude mice tumorigenicity assays. NIH3T3-derived transformants retained discrete DNA fragments hybridizing to human alu probes. Four clones were isolated from a cosmid library prepared from one of these transformants (49-7G) using human DNA as the probe. Analyses of DNAs from 49-7G cells and the four cosmid clones with probes for a number of human oncogenes demonstrated that the cloned sequences were related to the trk oncogene. Transfection of NIH3T3 cells with the cosmid DNAs did not result in the appearance of transformed foci when the murine fibroblasts were cultured on plastic. However, foci developed when transfected cells were cultured on plates coated with various extracellular matrix (ECM) components. Neomycin-resistant cosmid-transfected NIH3T3 cells did induce tumors in nude mice, and their tumorigenicity correlated with their level of trk expression. Nucleotide sequence analyses of cDNA clones isolated from a 49-7G library with a human trk probe revealed that the cloned sequences resulted from the fusion between 5' sequences from the human beta-1,4-galactosyltransferase gene, which encodes a membrane protein involved in cell-cell and cell-matrix interactions, and 3' sequences from the human trk proto-oncogene. The 76 kDa protein product of the chimeric gene, designated bgt-trk, has been identified in NIH3T3 cells transfected with cosmid 19/2 or with bgt-trk cDNA expression constructs, and its phosphorylation in tyrosine has been found to increase when the transfected cells were seeded on plates coated with ECM components which also elicited foci formation in NIH3T3 transformation assays. The fusion of the trk tyrosine kinase domain to a cell adhesion molecule may explain the ECM dependence for the expression of the full transforming potential of the resulting oncogene product.
Assuntos
Moléculas de Adesão Celular/metabolismo , Expressão Gênica , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Receptores Proteína Tirosina Quinases/genética , Receptores de Fator de Crescimento Neural/genética , Células 3T3 , Animais , Clonagem Molecular , Humanos , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Camundongos , Camundongos Nus , Ligação Proteica , Proteínas Tirosina Quinases/química , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptor trkA , Receptores de Fator de Crescimento Neural/metabolismoRESUMO
Ewing's sarcoma (EWS) cells accumulate elevated steady-state levels of poly (ADP-ribose) polymerase (PARP) mRNA and protein. To understand the molecular mechanisms underlying PARP upregulation, we cloned and analysed the 5'-flanking region of the PARP gene from EWS cells. Nucleotide sequence analysis demonstrated no variations in the PARP promoter region in EWS cells. The PARP promoter encompasses multiple binding motifs for the ETS transcription factor. We have also observed that there is a coordinated up-regulation of the expression of both PARP and ETS1, relative to cells of other human tumor types expressing lower levels of PARP. Transient co-expression of ETS1 in EWS cells resulted in a strong enhancement of PARP-promoter activity. The participation of ETS in the regulation of PARP gene expression was further demonstrated in EWS cells stably transfected with Ets1 antisense cDNA constructs. Antisense-mediated down-regulation of endogenous ETS1 resulted in the inhibition of PARP expression in EWS cells, and sensitized these cells to ionizing radiation. These data provide support for ETS regulation of PARP expression levels, and implicate ETS transcription factors in the radiation response of EWS cells.
Assuntos
Poli(ADP-Ribose) Polimerases/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/fisiologia , Fatores de Transcrição/fisiologia , Sequência de Bases , Divisão Celular/genética , Divisão Celular/efeitos da radiação , Radioisótopos de Césio , Regulação da Expressão Gênica , Inibidores do Crescimento/fisiologia , Humanos , Dados de Sequência Molecular , Poli(ADP-Ribose) Polimerases/biossíntese , Regiões Promotoras Genéticas/fisiologia , Proteína Proto-Oncogênica c-ets-1 , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-ets , Sarcoma de Ewing/enzimologia , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/biossíntese , Ativação Transcricional , Transfecção , Células Tumorais CultivadasRESUMO
Treatment of Syrian hamster embryo fibroblasts with a single dose of 3-methylcholanthrene caused the activation of the transforming potential of cellular sequences (Notario et al, Oncogene 5: 1425-1430, 1990), which were subsequently isolated by cosmid rescue techniques, and further identified as a novel oncogene, termed cph because of its involvement in the carcinogenic progression of hamster embryo cells (Velasco et al, Oncogene 9: 2065-2069, 1994). We have analysed the expression of the cph proto-oncogene in adult Syrian hamster tissues by northern hybridization using cph-specific genomic probes. The three cph transcripts expressed in normal and neoplastic Syrian hamster embryo cells in culture (5.0, 3.5 and 2.0 kb) were also present in most adult tissues, although different mRNA species, most likely resulting from alternative splicing events, were expressed in testes. The highest steady-state level of cph mRNA was found in kidney, whereas cph expression was nearly undetectable in skin and skeletal muscle. Southern blot analyses of DNAs from other eucaryotic organisms were performed under moderate stringency conditions with a Syrian hamster-specific cph probe. Discrete cph-hybridizing sequences were present in genomes from yeast to mammalian species, including humans, thus demonstrating that cph is a highly conserved gene in eucaryotic evolution. Using fluorescence in situ hybridization (FISH), we have determined also the chromosomal localization of the cph proto-oncogene in the hamster genome. FISH experiments demonstrated that cph is a single copy gene, localized on the euchromatic short arm of the X chromosome, at region Xpa7. Because chromosome X is frequently involved in structural alterations in neoplastic Syrian hamster cells transformed by chemical carcinogens and oncogenic viruses, the localization of the cph locus on this chromosome supports the notion that the cph oncogene plays a role in the malignant conversion of chemically transformed hamster fibroblasts. The wide range of tissue-specific expression and species-specific distribution of cph strongly suggest that the normal function of the cph protein product(s) may be essential for metabolic processes involved in the regulation of cell proliferation and survival.
Assuntos
Evolução Molecular , Mesocricetus/genética , Proto-Oncogenes/genética , Cromossomo X , Animais , Mapeamento Cromossômico , Sequência Conservada , Cricetinae , Embrião de Mamíferos/citologia , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Mesocricetus/embriologia , Mesocricetus/crescimento & desenvolvimento , Proto-Oncogene Mas , Proto-Oncogenes/fisiologia , Homologia de Sequência do Ácido Nucleico , Testículo/química , Testículo/metabolismo , Distribuição Tecidual , Transcrição GênicaRESUMO
Exogenous L-arginine and L-ornithine rapidly accumulate in rat pancreatic islets. L-Arginine is converted to L-ornithine and urea. Endogenous or exogenous L-ornithine generates di- and polyamines, the putrescine turnover being faster than that of spermidine and spermine. However, the major pathway for L-ornithine metabolism consists of its transamination to L-glutamaldehyde and further conversion to L-glutamate. The amines and L-glutamate derived from exogenous L-ornithine are incorporated into islet proteins at the intervention of transglutaminase and cycloheximide-sensitive biosynthetic processes, respectively. These findings suggest the hypothesis that the insulinotropic action of L-arginine and L-ornithine could somehow be related to the metabolism of these cationic amino acids in islet cells.
Assuntos
Arginina/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Ornitina/metabolismo , Animais , Arginase/metabolismo , Arginina/farmacologia , Glucose/farmacologia , Glutamatos/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/enzimologia , Modelos Biológicos , Ornitina/análogos & derivados , Ornitina/farmacologia , Oxirredução , Poliaminas/biossíntese , Ratos , Ureia/biossínteseRESUMO
The metabolism of L-arginine and L-ornithine was examined in tumoral islet cells of the RINm5F line and compared to the situation previously characterized in normal rat islets. The maximal velocity of arginase in cell homogenates, as well as either the production of 14C-urea or the steady-state content of 14C-labelled ornithine in intact cells exposed to L-[U-14C]arginine were about one order of magnitude lower in tumoral than normal islet cells. The activity of ornithine-glutamate transaminase was similar in both cell types, and this coincided with a comparable rate of 14C-labelled L-glutamate generation by intact cells exposed to L-[1-14C]ornithine. Despite a comparable cell content in 14C-labelled ornithine of normal and tumoral cells exposed to exogenous ornithine, the rate of di- and polyamine generation was about one order of magnitude higher in tumoral than normal islet cells, this coinciding with a much higher activity of ornithine decarboxylase in RINm5F cell than islet homogenates.
Assuntos
Adenoma de Células das Ilhotas Pancreáticas/metabolismo , Arginina/metabolismo , Insulina/metabolismo , Ornitina/metabolismo , Neoplasias Pancreáticas/metabolismo , Adenoma de Células das Ilhotas Pancreáticas/patologia , Animais , Arginase/metabolismo , Arginina/farmacologia , Aminas Biogênicas/biossíntese , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Proteínas de Neoplasias/metabolismo , Ornitina/farmacologia , Ornitina Descarboxilase/metabolismo , Ornitina-Oxo-Ácido Transaminase/metabolismo , Oxirredução , Neoplasias Pancreáticas/patologia , Ratos , Estimulação Química , Células Tumorais Cultivadas/metabolismo , Ureia/metabolismoRESUMO
We have determined the major immunoglobulin isotypes (IgG, IgA, IgM) of antiamebic antibodies induced in the serum and in the large and small intestine after local (oral and rectal) or systemic (intraperitoneal and intramuscular) immunization of mice with glutaraldehyde-fixed Entamoeba histolytica trophozoites (GFT). IgA predominated in the small intestine after immunization through all routes, whereas in the large intestine similar antibody levels of the major isotypes were induced by rectal, intraperitoneal and intramuscular immunization. The intramuscular route elicited intestinal responses lower than those induced by the rectal and intraperitoneal routes, but higher than the slight IgA antibody increase observed after oral immunization. The differences in antiamebic antibody response patterns at the large and small intestine suggest that there are different mucosal effector compartments. They also indicate that isotype analysis of mucosal antibodies from the sites where an infectious agent resides is needed to evaluate whether a vaccine candidate induces responses of higher protective value in the appropriate site, and that the study of antibody responses must not be limited to sampling the serum or mucosal sites distant to the relevant one.
Assuntos
Anticorpos Antiprotozoários/biossíntese , Entamoeba histolytica/imunologia , Isotipos de Imunoglobulinas/biossíntese , Intestinos/imunologia , Animais , Glutaral , Imunidade nas Mucosas , Imunização , Imunoglobulina G/classificação , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Infants in developing countries are at high risk of developing severe clinical measles if they become infected during the "window of vulnerability" (age 4-9 months), when declining maternal antibodies do not protect against wild virus, yet impede successful immunization by attenuated measles vaccine. We developed two Sindbis replicon-based DNA vaccines expressing measles virus hemagglutinin and fusion protein with the goal of priming young infants to respond safely and effectively to subsequent boosting with attenuated measles vaccine. Intradermal prime with DNA vaccines by needle-free injection followed by aerosol or parenteral boost with licensed measles vaccine was well tolerated by juvenile and young infant rhesus macaques, and protected against clinical measles and viremia on wild-type virus challenge. A proteosome-measles vaccine administered alone (three doses) or as a boost following DNA vaccine priming was also safe and protective. These promising results pave the way for clinical trials to assess this prime-boost strategy.