Expressão dos protooncogenes c-fos, c-myc e c-jun em miométrio normal e mioma humanos

OBJETIVO: Comparar a expressão gênica (mRNA) e protéica dos protooncogenes c-fos, c-myc e c-jun em miométrio normal e mioma humanos. MÉTODOS: Foi realizado um estudo do tipo caso-controle. O material foi coletado de 12 pacientes submetidas a histerectomia no Hospital de Clínicas de Porto Alegre. A expressão do mRNA específico para c-myc, c-fos, c-jun e beta-microglobulina foi avaliada pela técnica de RT-PCR, utilizando primers específicos para cada gene. A expressão protéica destes protooncogenes foi avaliada através de Western blot com anticorpos específicos. RESULTADOS: Não houve diferença significativa para expressão gênica desses protooncogenes entre miométrio normal e mioma (c-myc: 0,87 ± 0,08 vs 0,87 ± 0,08, p = 0,952; c-fos: 1,10 ± 0,17 vs 1,01 ± 0,11, p = 0,21; c-jun: 1,03 ± 0,12 vs 0,96 ± 0,09, p = 0,168, respectivamente). Não houve diferença significativa para expressão protéica desses protooncogenes entre miométrio normal e mioma (c-myc: 1,36 ± 0,48 vs 1,53 ± 0,29, p = 0,569; c-fos: 8,85 ± 5,5 vs 6,56 ± 4,22, p = 0,434; e c-jun: 6,47 ± 3,04 vs 5,42 ± 2,03, p = 0,266, respectivamente). CONCLUSÃO: A expressão gênica (transcrição) e a expressão protéica (tradução) dos protooncogenes c-myc, c-fos e c-jun em mioma e miométrio normal são semelhantes.

Immunoregulation in human malaria: the challenge of understanding asymptomatic infection

Asymptomatic Plasmodium infection carriers represent a major threat to malaria control worldwide as they are silent natural reservoirs and do not seek medical care. There are no standard criteria for asymptomaticPlasmodium infection; therefore, its diagnosis relies on the presence of the parasite during a specific period of symptomless infection. The antiparasitic immune response can result in reducedPlasmodium sp. load with control of disease manifestations, which leads to asymptomatic infection. Both the innate and adaptive immune responses seem to play major roles in asymptomatic Plasmodiuminfection; T regulatory cell activity (through the production of interleukin-10 and transforming growth factor-β) and B-cells (with a broad antibody response) both play prominent roles. Furthermore, molecules involved in the haem detoxification pathway (such as haptoglobin and haeme oxygenase-1) and iron metabolism (ferritin and activated c-Jun N-terminal kinase) have emerged in recent years as potential biomarkers and thus are helping to unravel the immune response underlying asymptomatic Plasmodium infection. The acquisition of large data sets and the use of robust statistical tools, including network analysis, associated with well-designed malaria studies will likely help elucidate the immune mechanisms responsible for asymptomatic infection.

Proliferative signaling initiated in ACTH receptors

This article reviews recent results of studies aiming to elucidate modes of integrating signals initiated in ACTH receptors and FGF2 receptors, within the network system of signal transduction found in Y1 adrenocortical cells. These modes of signal integration should be central to the mechanisms underlying the regulation of the G0->G1->S transition in the adrenal cell cycle. FGF2 elicits a strong mitogenic response in G0/G1-arrested Y1 adrenocortical cells, that includes a) rapid and transient activation of extracellular signal-regulated kinases-mitogen-activated protein kinases (ERK-MAPK) (2 to 10 min), b) transcription activation of c-fos, c-jun and c-myc genes (10 to 30 min), c) induction of c-Fos and c-Myc proteins by 1 h and cyclin D1 protein by 5 h, and d) onset of DNA synthesis stimulation within 8 h. ACTH, itself a weak mitogen, interacts with FGF2 in a complex manner, blocking the FGF2 mitogenic response during the early and middle G1 phase, keeping ERK-MAPK activation and c-Fos and cyclin D1 induction at maximal levels, but post-transcriptionally inhibiting c-Myc expression. c-Fos and c-Jun proteins are mediators in both the strong and the weak mitogenic responses respectively triggered by FGF2 and ACTH. Induction of c-Fos and stimulation of DNA synthesis by ACTH are independent of PKA and are inhibited by the PKC inhibitor GF109203X. In addition, ACTH is a poor activator of ERK-MAPK, but c-Fos induction and DNA synthesis stimulation by ACTH are strongly inhibited by the inhibitor of MEK1 PD98059.

Control of the rat angiotensin I converting enzyme gene by CRE-like sequences

We characterized the role of potential cAMP-responsive elements (CRE) in basal and in induced angiotensin converting enzyme (ACE) gene promoter activity in order to shed light on the regulation of somatic ACE expression. We identified stimulators and repressors of basal expression between 122 and 288 bp and between 415 and 1303 bp upstream from the transcription start site, respectively, using a rabbit endothelial cell (REC) line. These regions also contained elements associated with the response to 8BrcAMP. When screening for CRE motifs we found pCRE, a proximal sequence between 209 and 222 bp. dCRE, a distal tandem of two CRE-like sequences conserved between rats, mice and humans, was detected between 834 and 846 bp. Gel retardation analysis of nuclear extracts of REC indicated that pCRE and dCRE bind to the same protein complexes as bound by a canonical CRE. Mutation of pCRE and dCRE in REC established the former as a positive element and the latter as a negative element. In 293 cells, a renal cell line, pCRE and dCRE are negative regulators. Co-transfection of ATF-2 or ATF-2 plus c-Jun repressed ACE promoter activity, suggesting that the ACE gene is controlled by cellular stress. Although mapping of cAMP responsiveness was consistent with roles for pCRE and dCRE, mutation analysis indicated that they were not required for cAMP responsiveness. We conclude that the basal activity of the somatic ACE promoter is controlled by proximal and distal CREs that can act as enhancers or repressors depending on the cell context.

Protein-mediated surface structuring in biomembranes

The lipids and proteins of biomembranes exhibit highly dissimilar conformations, geometrical shapes, amphipathicity, and thermodynamic properties which constrain their two-dimensional molecular packing, electrostatics, and interaction preferences. This causes inevitable development of large local tensions that frequently relax into phase or compositional immiscibility along lateral and transverse planes of the membrane. On the other hand, these effects constitute the very codes that mediate molecular and structural changes determining and controlling the possibilities for enzymatic activity, apposition and recombination in biomembranes. The presence of proteins constitutes a major perturbing factor for the membrane sculpturing both in terms of its surface topography and dynamics. We will focus on some results from our group within this context and summarize some recent evidence for the active involvement of extrinsic (myelin basic protein), integral (Folch-Lees proteolipid protein) and amphitropic (c-Fos and c-Jun) proteins, as well as a membrane-active amphitropic phosphohydrolytic enzyme (neutral sphingomyelinase), in the process of lateral segregation and dynamics of phase domains, sculpturing of the surface topography, and the bi-directional modulation of the membrane biochemical reactivity.

The role of stress-activated protein kinase signaling in renal pathophysiology

Two major stress-activated protein kinases are the p38 mitogen-activated protein kinase (MAPK) and the c-Jun amino terminal kinase (JNK). p38 and JNK are widely expressed in different cell types in various tissues and can be activated by a diverse range of stimuli. Signaling through p38 and JNK is critical for embryonic development. In adult kidney, p38 and JNK signaling is evident in a restricted pattern suggesting a normal physiological role. Marked activation of both p38 and JNK pathways occurs in human renal disease, including glomerulonephritis, diabetic nephropathy and acute renal failure. Administration of small molecule inhibitors of p38 and JNK has been shown to provide protection from renal injury in different types of experimental kidney disease through inhibition of renal inflammation, fibrosis, and apoptosis. In particular, a role for JNK signaling has been identified in macrophage activation resulting in up-regulation of pro-inflammatory mediators and the induction of renal injury. The ability to provide renal protection by blocking either p38 or JNK indicates a lack of redundancy for these two signaling pathways despite their activation by common stimuli. Therefore, the stress-activated protein kinases, p38 and JNK, are promising candidates for therapeutic intervention in human renal diseases.

Roles of mitogen-activated protein kinases and angiotensin II in renal development

Experimental and clinical evidence suggests that angiotensin II (AII) participates in renal development. Renal AII content is several-fold higher in newborn rats and mice than in adult animals. AII receptors are also expressed in higher amounts in the kidneys of newborn rats. The kidneys of fetuses whose mother received a type 1 AII receptor (AT1) antagonist during gestation present several morphological alterations. Mutations in genes that encode components of the renin-angiotensin system are associated with autosomal recessive renal tubular dysgenesis. Morphological changes were detected in the kidneys of 3-week-old angiotensin-deficient mice. Mitogen-activated protein kinases (MAPKs) are important mediators that transduce extracellular stimuli to intracellular responses. The MAPK family comprises three major subgroups, namely extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinases (JNK), and p38 MAPK (p38). Important events in renal growth during nephrogenesis such as cellular proliferation and differentiation accompanied by apoptosis on a large scale can be mediated by MAPK pathways. A decrease in glomerulus number was observed in embryos cultured for 48 and 120 h with ERK or p38 inhibitors. Many effects of AII are mediated by MAPK pathways. Treatment with losartan during lactation provoked changes in renal function and structure associated with alterations in AT1 and type 2 AII (AT2) receptors and p-JNK and p-p38 expression in the kidney. Several studies have shown that AII and MAPKs play an important role in renal development. However, the relationship between the effects of AII and MAPK activation on renal development is still unclear.

Modulation of rhodopsin gene expression and signaling mechanisms evoked by endothelins in goldfish and murine pigment cell lines

Endothelins (ETs) and sarafotoxins (SRTXs) belong to a family of vasoconstrictor peptides, which regulate pigment migration and/or production in vertebrate pigment cells. The teleost Carassius auratus erythrophoroma cell line, GEM-81, and Mus musculus B16 melanocytes express rhodopsin, as well as the ET receptors, ETB and ETA, respectively. Both cell lines are photoresponsive, and respond to light with a decreased proliferation rate. For B16, the doubling time of cells kept in 14-h light (14L):10-h darkness (10D) was higher compared to 10L:14D, or to DD. The doubling time of cells kept in 10L:14D was also higher compared to DD. Using real-time PCR, we demonstrated that SRTX S6c (12-h treatment, 100 pM and 1 nM; 24-h treatment, 1 nM) and ET-1 (12-h treatment, 10 and 100 pM; 24- and 48-h treatments, 100 pM) increased rhodopsin mRNA levels in GEM-81 and B16 cells, respectively. This modulation involves protein kinase C (PKC) and the mitogen-activated protein kinase cascade in GEM-81 cells, and phospholipase C, Ca2+, calmodulin, a Ca2+/calmodulin-dependent kinase, and PKC in B16 cells. Cells were kept under constant darkness throughout the gene expression experiments. These results show that rhodopsin mRNA levels can be modulated by SRTXs/ETs in vertebrate pigment cells. It is possible that SRTX S6c binding to the ETB receptors in GEM-81 cells, and ET-1 binding to ETA receptors in B16 melanocytes, although activating diverse intracellular signaling mechanisms, mobilize transcription factors such as c-Fos, c-Jun, c-Myc, and neural retina leucine zipper protein. These activated transcription factors may be involved in the positive regulation of rhodopsin mRNA levels in these cell lines.

Silibinin improves palmitate-induced insulin resistance in C2C12 myotubes by attenuating IRS-1/PI3K/Akt pathway inhibition

The present study investigated the effect of silibinin, the principal potential anti-inflammatory flavonoid contained in silymarin, a mixture of flavonolignans extracted from Silybum marianum seeds, on palmitate-induced insulin resistance in C2C12 myotubes and its potential molecular mechanisms. Silibinin prevented the decrease of insulin-stimulated 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose) uptake and the downregulation of glutamate transporter type 4 (GLUT4) translocation in C2C12 myotubes induced by palmitate. Meanwhile, silibinin suppressed the palmitate-induced decrease of insulin-stimulated Akt Ser473 phosphorylation, which was reversed by wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase (PI3K). We also found that palmitate downregulated insulin-stimulated Tyr632 phosphorylation of insulin receptor substrate 1 (IRS-1) and up-regulated IRS-1 Ser307 phosphorylation. These effects were rebalanced by silibinin. Considering several serine/threonine kinases reported to phosphorylate IRS-1 at Ser307, treatment with silibinin downregulated the phosphorylation of both c-Jun N-terminal kinase (JNK) and nuclear factor-κB kinase β (IKKβ), which was increased by palmitate in C2C12 myotubes mediating inflammatory status, whereas the phosphorylation of PKC-θ was not significantly modulated by silibinin. Collectively, the results indicated that silibinin prevented inhibition of the IRS-1/PI3K/Akt pathway, thus ameliorating palmitate-induced insulin resistance in C2C12 myotubes.

Effect of microRNA-21 on the proliferation of human degenerated nucleus pulposus by targeting programmed cell death 4

This study aims to explore the effect of microRNA-21 (miR-21) on the proliferation of human degenerated nucleus pulposus (NP) by targeting programmed cell death 4 (PDCD4) tumor suppressor. NP tissues were collected from 20 intervertebral disc degeneration (IDD) patients, and from 5 patients with traumatic spine fracture. MiR-21 expressions were tested. NP cells from IDD patients were collected and divided into blank control group, negative control group (transfected with miR-21 negative sequences), miR-21 inhibitor group (transfected with miR-21 inhibitors), miR-21 mimics group (transfected with miR-21 mimics) and PDCD4 siRNA group (transfected with PDCD4 siRNAs). Cell growth was estimated by Cell Counting Kit-8; PDCD4, MMP-2,MMP-9 mRNA expressions were evaluated by qRT-PCR; PDCD4, c-Jun and p-c-Jun expressions were tested using western blot. In IDD patients, the expressions of miR-21 and PDCD4 mRNA were respectively elevated and decreased (both P<0.05). The miR-21 expressions were positively correlated with Pfirrmann grades, but negatively correlated with PDCD4 mRNA (both P<0.001). In miR-21 inhibitor group, cell growth, MMP-2 and MMP-9 mRNA expressions, and p-c-Jun protein expressions were significantly lower, while PDCD4 mRNA and protein expressions were higher than the other groups (all P<0.05). These expressions in the PDCD4 siRNA and miR-21 mimics groups was inverted compared to that in the miR-21 inhibitor group (all P<0.05). MiR-21 could promote the proliferation of human degenerated NP cells by targeting PDCD4, increasing phosphorylation of c-Jun protein, and activating AP-1-dependent transcription of MMPs, indicating that miR-21 may be a crucial biomarker in the pathogenesis of IDD.

Efeito da mitomicina C em cultura de estroma de pólipos nasais eosinofílicos: indução de apoptose em eosinófilos

A polipose nasossinusal eosinofílica é uma afecção comum a várias doenças, determina acometimento extenso dos seios paranasais e possui grande tendência à recidiva após tratamento. Os eosinófilos exercem papel essencial na patogênese, relacionada a baixo índice de apoptose e a longa permanência destas células ativas nos tecidos. OBJETIVO: Este estudo teve por objetivo avaliar o efeito da mitomicina C na indução de apoptose em eosinófilos presentes no estroma de pólipos nasais eosinofílicos. FORMA DE ESTUDO: Caso controle. MATERIAL E MÉTODO: O estudo foi auto-pareado, com 9 amostras cultivadas em meio RPMI 1640 e avaliadas em zero, 12 e 24 horas. O grupo estudo recebeu mitomicina C numa concentração de 400µg/ml durante 5 minutos. Em cada tempo as duas culturas, controle e estudo, foram submetidas a estudo histopatológico para determinação do índice apoptótico. Utilizou-se a coloração hematoxilina-eosina com aumento microscópico de 1000x. RESULTADO: Pela análise de 674 campos digitalizados observou-se que as culturas tratadas com mitomicina C apresentaram índice apoptótico em 12 horas significativamente maior em relação ao grupo controle (p< 0,001). CONCLUSÃO: Concluiu-se que a mitomicina C é eficaz na indução de apoptose em eosinófilos presentes em estroma de pólipos nasais eosinofílicos.

Reabilitação vestibular personalizada: levantamento de prontuários dos pacientes atendidos no ambulatório de otoneurologia da I.S.C.M.S.P.

O OBJETIVO: o objetivo deste trabalho foi verificar a eficácia da reabilitação vestibular personalizada (RVP) em diferentes quadros otoneurológicos. FORMA DE ESTUDO: clínico retrospectivo. MATERIAL E MÉTODO: Foi realizado um estudo retrospectivo que constou de uma descrição do programa de reabilitação vestibular de 37 pacientes, com idade entre 21 a 87 anos, vinte e seis do sexo feminino, onze do sexo masculino, com quadros otoneurológicos variados, atendidos no Ambulatório de Otoneurologia do Departamento de Otorrinolaringologia da Irmandade da Santa Casa de Misericórdia de São Paulo, no período de 2002 a 2003. Estes foram submetidos à avaliação otoneurológica e depois de realizado o diagnóstico pelo otorrinolaringologista, encaminhados para a reabilitação vestibular, realizada semanalmente no ambulatório, e em casa diariamente. Cada paciente seguiu programas específicos de exercícios, considerando-se os achados ao exame vestibular, o quadro clínico e, principalmente, os sintomas apresentados. Os exercícios foram baseados em diferentes protocolos descritos na literatura. Foi realizada uma análise individual da evolução clínica e em conjunto, a fim de verificar a eficácia da RVP em diferentes quadros clínicos. CONCLUSÃO: Pudemos concluir que o programa de RVP mostrou-se um recurso terapêutico efetivo na diminuição e extinção dos sintomas e conseqüente melhora na qualidade de vida de pacientes portadores de diferentes quadros clínicos.

Efeito da mitomicina C na secreção de fator estimulador de granulócitos macrófagos e interleucina-5 em cultura de estroma de pólipos nasais eosinofílicos

O estudo de fatores teciduais, como a concentração de fator estimulador de colônias de macrófagos (GM-CSF) e interleucina 5 (IL-5), aponta para os mecanismos envolvidos na manutenção da eosinofilia em pólipos nasossinusais eosinofílicos. A mitomicina C (MMC) tem sido utilizada com bons resultados em otorrinolaringologia. OBJETIVO: Este estudo teve como objetivo avaliar a ação da Mitomicina C sobre a secreção de GM-CSF e IL-5 em pólipos eosinofílicos. FORMA DE ESTUDO: caso-controle. MATERIAL E MÉTODO: O estudo foi comparativo experimental autopareado, com amostras de pólipos biopsiados de pacientes portadores de polipose nasossinusal eosinofílica. Os fragmentos semeados como grupo experimental receberam mitomicina C por 5 minutos na dosagem de 400microg/ml e então lavadas em meio RPMI. Nos tempos zero, 12 e 24 horas, o sobrenadante foi retirado para determinação dos níveis de GM-CSF em 22 pacientes e IL-5, em 19 pacientes, utilizando o método de ELISA. RESULTO: Diminuição de secreção de GM-CSF nos grupos tratados com mitomicina C no tempo 24h (p<= 0,05); no grupo tratado houve expressão significativa de GM-CSF entre zero e 12 horas (p=0,013) demonstrando a viabilidade da cultura igualmente ao grupo não tratado; tendência à queda dos níveis de IL-5 no grupo tratado em 24h. CONCLUSÃO: O estudo demonstrou que a mitomicina C foi capaz de inibir a síntese de GM-CSF em culturas de pólipos nasais eosinofílicos e com provável ação sobre a secreção de IL-5, necessitando de estudos complementares.

Efeito da mitomicina C em polipose nasossinusal eosinofílica, in vivo: dosagem de IL5 e GM-CSF, RT-PCR

A polipose nasossinusal eosinofílica (PNS) é manifestação de uma doença inflamatória crônica na mucosa do nariz e nos seios paranasais caracterizada por infiltração de granulócitos eosinófilos. O fator responsável pela eosinofilia e manutenção dessas células com a perpetuação do processo inflamatório e formação polipóide é objeto constante de estudos. As citocinas como IL5 (interleucina 5) e GM-CSF (fator estimulador de colônia granulócito macrófago) aumentam a sobrevida dos eosinófilos e prolongam a sua presença no tecido polipóide, diminuindo o índice de apoptose eosinofílica. OBJETIVO: Avaliar o efeito da mitomicina C - MMC - por meio de aplicação tópica em pacientes portadores de PNS eosinofílica quanto à presença de IL5 e GM-CSF. CASUÍSTICA E MÉTODOS: Quinze pacientes portadores de PNS eosinofílica foram submetidos à aplicação tópica de MMC na concentração de 0,5mg/ml, 1ml, durante cinco minutos, na cavidade nasal direita, e submetidos à biópsia para RT-PCR 24hs após. O grupo-controle foi a cavidade nasal esquerda. O perfil de citocinas foi analisado para IL5 e GM-CSF. RESULTADOS: A comparação dos resultados de GM-CSF pré e pós-uso de MMC quando usamos o teste t pareado apresenta p=0,041. A comparação para IL5 resulta em p < 0,001. CONCLUSÃO: O uso de MMC em pacientes com PNS mostra redução com significância estatística par GM-CSF e importante significância para IL5.

Efeito da mitomicina C tópica na cicatrização de prega vocal em modelo suíno

Várias terapias adjuvantes à cirurgia vêm sendo usadas para modular o processo cicatricial nas pregas vocais, entre elas a Mitomicina tópica (MTC). OBJETIVOS: Avaliar os efeitos da MTC no processo de cicatrização de pregas vocais de suínos 30 dias após exérese de fragmento de mucosa com laser de CO2 mediante a mensuração da deposição de colágeno. FORMA DE ESTUDO: Experimental em suínos. MATERIAIS E MÉTODOS: Dois grupos de 6 suínos cada foram avaliados após exérese de fragmento de mucosa de prega vocal a laser de CO2 (grupo controle sem uso e grupo experimento com uso de MTC tópica). Após 30 dias os animais foram submetidos à eutanásia, sendo coletadas amostras das pregas vocais para análise histológica, a fim de quantificar a deposição de colágeno mediante coloração de Picrosirius Red. RESULTADOS: A média da área do colágeno total das pregas vocais do grupo controle foi de 2648,03 µm2, enquanto a média do colágeno total das pregas vocais do grupo experimento foi de 2200,30 µm2 (p = 0,0043). CONCLUSÃO: A MTC usada topicamente após a exérese de fragmento de mucosa da prega vocal a laser de CO2 em suínos, diminui, significativamente, a deposição do colágeno total.