T cell receptor signaling pathway is overexpressed in CD4 + T cells from HAM/TSP individuals

ABSTRACT Human T-lymphotropic virus type 1 (HTLV-1) is a human retrovirus related to the chronic neuroinflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). CD4+ T cells activation appears to play a key role on HTLV-1 infection. Here we investigated the expression of genes associated to T cell activation CD3e molecule, epsilon (CD3?), lymphocyte-specific protein tyrosine kinase (LCK), vav 1 guanine nucleotide exchange factor (VAV1), and zeta-chain (TCR) associated protein kinase 70 kDa (ZAP70) on T lymphocytes of HTLV-1-infected individuals and compared to healthy uninfected individuals (CT). We observed that CD3?, LCK, ZAP70, and VAV1 gene expression were increased in CD4+ T cells from HAM/TSP group compared to HTLV-1 asymptomatic patients (HAC). Moreover, ZAP70 and VAV1 were also upregulated in HAM/TSP compared to CT group. We detected a positive correlation among all these genes. We also observed that CD3?, LCK, and VAV1 genes had a positive correlation with the proviral load (PVL) and Tax expression. These results suggest that PVL and Tax protein could drive CD3?, LCK, and VAV1 gene expression in CD4+ T cells, and these genes function on a synchronized way on the CD4+ T cell activation. The elucidation of the mechanisms underlying T cell receptor signaling pathway is of considerable interest and might lead to new insights into the mechanism of HAM/TSP.

Colonização nasal em profissionais de enfermagem de unidades especializadas em HIV/aids / Colonización nasal en unidades de enfermeras especializadas en el VIH/SIDA / Nasal colonization in nursing professionals from units specialized in HIV/AIDS

RESUMO Objetivo: investigar a presença de micro-organismos nas narinas dos profissionais de enfermagem de um hospital de ensino brasileiro. Método: estudo transversal, em duas unidades de internação especializadas em HIV/aids. Foram coletadas amostras de secreção nasal de profissionais de enfermagem no período de um mês. As amostras foram processadas no laboratório de microbiologia da instituição e a análise dos dados resultantes por meio do software Statistical Package for the Social Sciences (SPSS) versão 19.0. Os aspectos éticos foram contemplados. Resultados: dos 73 profissionais de enfermagem do serviço, foram coletadas amostras de secreção nasal de 61 (80,2%). Foram isolados seis tipos de micro-organismos em 22 (41,0%) culturas positivas. Destaca-se que o Staphylococcus aureus representou 22,9%, sendo quatro resistentes à oxacilina (MRSA). Conclusão: o Staphylococcus aureus foi o micro-organismo de maior prevalência nos indivíduos deste estudo. .

RESUMEN Objetivo: investigar la presencia de microorganismos en las fosas nasales del personal de enfermería de un hospital universitario brasileño. Método: estudio transversal en dos unidades de hospitalización especializados en VIH/SIDA. Muestras de secreción nasal de enfermeras fueron recolectados durante un mes. Las muestras fueron procesadas en el laboratorio de microbiología de la institución y se analizaron con el paquete estadístico para el software de Ciencias Sociales (SPSS) versión 19.0. Los aspectos éticos fueron cubiertos. Resultados: 73 de los profesionales de enfermería, se recogieron muestras de las secreciones nasales de 61 (80,2%). Se aislaron seis tipos de microorganismos en 22 (41,0%) cultivos positivos. Es de destacar que el Staphylococcus aureus representó el 22,9%, cuatro oxacilina-resistente (MRSA). Conclusión: Staphylococcus aureus fue la prevalencia más microorganismo en los individuos de este estudio. .

ABSTRACT Objective: to investigate the presence of microorganisms in the nostrils of the nursing professionals of a Brazilian teaching hospital. Method: cross-sectional study in two inpatient units specialized in HIV/AIDS. Nasal secretion samples of nursing professionals were collected in one month. The samples were processed at the microbiology laboratory of the institution and analyzed using the Statistical Package for the Social Sciences (SPSS) software, version 19.0. Ethical aspects were abided. Results: from the 73 members of the nursing staff, samples of nasal secretions were collected from 61 (80.2%). Six types of microorganisms were isolated in 22 (41.0%) positive cultures. It is noteworthy that Staphylococcus aureus accounted for 22.9%, four of them oxacillin-resistant (MRSA). Conclusion: Staphylococcus aureus microorganism accounted for the largest prevalence in individuals of this study. .

Epidermal growth factor receptor (EGFR) mutations in lung cancer: preclinical and clinical data

Lung cancer leads cancer-related mortality worldwide. Non-small-cell lung cancer (NSCLC), the most prevalent subtype of this recalcitrant cancer, is usually diagnosed at advanced stages, and available systemic therapies are mostly palliative. The probing of the NSCLC kinome has identified numerous nonoverlapping driver genomic events, including epidermal growth factor receptor (EGFR) gene mutations. This review provides a synopsis of preclinical and clinical data on EGFR mutated NSCLC and EGFR tyrosine kinase inhibitors (TKIs). Classic somatic EGFR kinase domain mutations (such as L858R and exon 19 deletions) make tumors addicted to their signaling cascades and generate a therapeutic window for the use of ATP-mimetic EGFR TKIs. The latter inhibit these kinases and their downstream effectors, and induce apoptosis in preclinical models. The aforementioned EGFR mutations are stout predictors of response and augmentation of progression-free survival when gefitinib, erlotinib, and afatinib are used for patients with advanced NSCLC. The benefits associated with these EGFR TKIs are limited by the mechanisms of tumor resistance, such as the gatekeeper EGFR-T790M mutation, and bypass activation of signaling cascades. Ongoing preclinical efforts for treating resistance have started to translate into patient care (including clinical trials of the covalent EGFR-T790M TKIs AZD9291 and CO-1686) and hold promise to further boost the median survival of patients with EGFR mutated NSCLC.

Overview of hospitalizations by ambulatory care sensitive conditions in the municipality of Cotia, Brazil / Panorama de las hospitalizaciones por condiciones sensibles de la atención primaria en la ciudad de cotia / Panorama das Internações por Condições Sensíveis à Atenção Primária no município de Cotia

Objective To describe the profile of Hospitalizations by Amulatory Care Sensitive Conditions (HACSC), in the Municipality of Cotia, from 2008 to 2012. Method ecological, exploratory, longitudinal study with a quantitative approach. Data on HACSC, by age group and sex, were obtained from the Department of the Unified Health System. For data analysis descriptive statistics were used. Results During the period, there were 46,676 admissions, excluding deliveries, 7,753 (16.61%) by HACSC. The main causes were cerebrovascular diseases, 16.96%, heart failure, 15.50%, hypertension, 10.80% and infection of the kidney and urinary tract, 10.51%. Regarding gender, HACSC occurred predominantly in males. There was a greater number of HACSC at extreme age ranges, especially in the elderly. Conclusion Chronic diseases predominate among the leading causes of HACSC and there was no significant difference between sex.



 .

Objetivo Describir el perfil de las Hospitalizaciones por Condiciones Sensibles de la Atención Primaria (HCSAP), en el municipio de Cotia, entre 2008 y 2012. Método Estudio ecológico, exploratorio, longitudinal con un enfoque cuantitativo. Los datos sobre HCSAP, por grupo de edad y sexo, se obtuvieron del Departamento del Sistema Único de Salud. Para el análisis de los datos se utilizaron estadísticas descriptivas. Resultados Durante el período, hubo 46.676 admisiones, excluyendo entregas, 7.753 (16,61%) por HCSAP. Las principales causas fueron las enfermedades cerebrovascular, 16,96%, insuficiencia cardíaca, 15,50%, hipertensión arterial 10,80% y infección del riñón y las vías urinarias, el 10,51%. Cuanto al género, HCSAP ocurrió mayormente en los hombres. Un mayor número de HCSAP en grupos de edades extremas, especialmente en los ancianos. Conclusión Las enfermedades crónicas predominan entre las principales causas de HCSAP y no hubo diferencia significativa entre los sexo.
 .

Objetivo Descrever o perfil das Internações por Condições Sensíveis à Atenção Primária (ICSAP), no Município de Cotia, entre 2008 e 2012. Método Estudo ecológico, exploratório, longitudinal, de abordagem quantitativa. Dados sobre as ICSAP, segundo a faixa etária e sexo, foram obtidos no Departamento de Informática do Sistema Único de Saúde. Para a análise dos dados foi utilizada a estatística descritiva. Resultados No período, houve 46.676 internações, excluindo os partos, sendo 7.753 (16,61%) por ICSAP. As principais causas foram: doenças cerebrovasculares, 16,96%; insuficiência cardíaca, 15,50%; hipertensão, 10,80%; e infecção do rim e trato urinário, 10,51%. Quanto ao sexo, as ICSAP ocorreram predominantemente nos homens. Houve maior número de ICSAP nos extremos das faixas etárias, especialmente nos idosos. Conclusão As doenças crônicas predominaram entre as principais causas de ICSAP e não houve diferença importante entre os sexos. .

Simultaneously expressed miR-424 and miR-381 synergistically suppress the proliferation and survival of renal cancer cells---Cdc2 activity is up-regulated by targeting WEE1

OBJECTIVES: MiRNAs are intrinsic RNAs that interfere with protein translation. Few studies on the synergistic effects of miRNAs have been reported. Both miR-424 and miR-381 have been individually reported to be involved in carcinogenesis. They share a common putative target, WEE1, which is described as an inhibitor of G2/M progression. Here, we studied the synergistic effects of miR-424 and miR-381 on renal cancer cells. METHODS: The viability of 786-O cells was analyzed after transfection with either a combination of miR-424 and miR-381 or each miRNA alone. We investigated cell cycle progression and apoptosis with flow cytometry. To confirm apoptosis and the abrogation of G2/M arrest, we determined the level of pHH3, which is an indicator of mitosis, and caspase-3/7 activity. The expression levels of WEE1, Cdc25, γH2AX, and Cdc2 were manipulated to investigate the roles of these proteins in the miRNA-induced anti-tumor effects. To verify that WEE1 was a direct target of both miR-424 and miR-381, we performed a dual luciferase reporter assay. RESULTS: We showed that the combination of these miRNAs synergistically inhibited proliferation, abrogated G2/M arrest, and induced apoptosis. This combination led to Cdc2 activation through WEE1 inhibition. This regulation was more effective when cells were treated with both miRNAs than with either miRNA alone, indicating synergy between these miRNAs. WEE1 was verified to be a direct target of each miRNA according to the luciferase reporter assay. CONCLUSIONS: These data clearly demonstrate that these two miRNAs might synergistically act as novel modulators of tumorigenesis by down-regulating WEE1 expression in renal cell cancer cells. .

TNNI3K is a novel mediator of myofilament function and phosphorylates cardiac troponin I

The phosphorylation of cardiac troponin I (cTnI) plays an important role in the contractile dysfunction associated with heart failure. Human cardiac troponin I-interacting kinase (TNNI3K) is a novel cardiac-specific functional kinase that can bind to cTnI in a yeast two-hybrid screen. The purpose of this study was to investigate whether TNNI3K can phosphorylate cTnI at specific sites and to examine whether the phosphorylation of cTnI caused by TNNI3K can regulate cardiac myofilament contractile function. Co-immunoprecipitation was performed to confirm that TNNI3K could interact with cTnI. Kinase assays further indicated that TNNI3K did not phosphorylate cTnI at Ser23/24 and Ser44, but directly phosphorylated Ser43 and Thr143 in vitro. The results obtained for adult rat cardiomyocytes also indicated that enhanced phosphorylation of cTnI at Ser43 and Thr143 correlated with rTNNI3K (rat TNNI3K) overexpression, and phosphorylation was reduced when rTNNI3K was knocked down. To determine the contractile function modulated by TNNI3K-mediated phosphorylation of cTnI, cardiomyocyte contraction was studied in adult rat ventricular myocytes. The contraction of cardiomyocytes increased with rTNNI3K overexpression and decreased with rTNNI3K knockdown. We conclude that TNNI3K may be a novel mediator of cTnI phosphorylation and contribute to the regulation of cardiac myofilament contraction function.

Vaccinia virus regulates expression of p21WAF1/Cip1 in A431 cells

In this paper, we provide evidence that both the mRNA and protein levels of the cyclin-dependent kinase (CDK) inhibitor p21WAF1/CDK-interacting protein 1 (Cip1) increase upon infection of A431 cells with Vaccinia virus (VACV). In addition, the VACV growth factor (VGF) seems to be required for the gene expression because infection carried out with the mutant virus VACV-VGF- revealed that this strain was unable to stimulate its transcription. Our findings are also consistent with the notion that the VGF-mediated change in p21WAF1/Cip1 expression is dependent on tyrosine kinase pathway(s) and is partially dependent on mitogen-activated protein kinase/extracellular-signal regulated kinase 1/2. We believe that these pathways are biologically significant because VACV replication and dissemination was drastically affected when the infection was carried out in the presence of the relevant pharmacological inhibitors.

Insulin sensitizing actions of fenugreek seed polyphenols, quercetin & metformin in a rat model.

Background & objectives: Plant polyphenols have been known to exert anti-diabetic action and promote insulin action. The present study was carried out to compare the effects of administration of fenugreek seed polyphenolic extract (FPEt), quercetin and metformin (a positive control) in an acquired model of insulin resistance (IR). Methods: Adult male Wistar rats divided into seven groups (n=12). IR was induced in groups (groups 2, 3, 4 and 5) by feeding a high fructose diet (FRU) (60 g/100 g diet) for 60 days. From day 16, FRU rats were administered either FPEt (200 mg/kg bw) (group 3), quercetin (50mg/kg bw) (group 4) or metformin (50 mg/kg bw) (group 5) for the next 45 days. Group 1 served as normal control while groups 6 and 7 served as FPEt and quercetin controls respectively. Oral glucose tolerance test (OGTT) was done on day 59 to assess glucose tolerance. At the end of 60 days, the levels of glucose, insulin, triglycerides (TG) and free fatty acids (FFA) were measured in the blood and the activities of insulin-inducible and suppressible enzymes in cytosolic and mitochondrial fractions of liver and skeletal muscle. The extent of tyrosine phosphorylation of proteins in response to insulin was determined by assaying protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) in liver. Results: Fructose caused increased levels of glucose, insulin, TG and FFA, alterations in insulin sensitivity indices, enzyme activities and reduced glycogen content. Higher PTP activity and lower PTK activity suggest reduced tyrosine phosphorylation status. Administration of FPEt or quercetin improved insulin sensitivity and tyrosine phosphorylation in fructose-fed animals and the effect was comparable with that of metformin. Interpretation & conclusions: Our findings indicated that FPEt and quercetin improved insulin signaling and sensitivity and thereby promoted the cellular actions of insulin in this model.

Inibidores de tirosino quinase na leucemia mieloide crônica / Tyrosine kinase inhibitors in chronic myeloid leukemia

A leucemia mieloide crônica (LMC) é uma neoplasia da medula óssea originada da translocação entre os cromossomos 9 e 22 t(9:22)(q34;11) e forma o gene híbrido BCR-ABL, que possui intensa atividade tirosino quinase, sendo responsável pela proliferação das células tumorais. Um grande avanço no tratamento da LMC foi conquistado com o surgimento dos inibidores da tirosino quinase, entre eles o imatinibe, que vem demonstrando ser efetivo na maior parte dos pacientes com LMC por apresentar respostas duradouras. Entretanto, há pacientes resistentes ou que desenvolvem resistência durante o tratamento com esta droga; sendo assim, inibidores de tirosino quinase de segunda geração, como o dasatinibe e o nilotinibe, foram desenvolvidos apresentando maior potência com a finalidade de diminuir a chance de desenvolvimento de resistência. O bosutinibe e o INNO-406 estão sendo estudados para atender pacientes resistentes às drogas anteriormente citadas e também com a finalidade de diminuir efeitos colaterais das mesmas; entretanto, eles ainda estão em fase clínica de estudo. Há ainda outras drogas inibidoras da tirosino quinase que estão em desenvolvimento na fase clínica ou pré-clínica. A partir do desenvolvimento destas novas drogas, múltiplas opções de tratamento para os pacientes com LMC poderão ser propostas, podendo, desta forma, individualizar o tratamento de acordo com o que cada paciente necessita. Este estudo visa descrever as drogas antineoplásicas que têm como mecanismo de ação a inibição da enzima tirosino quinase na LMC.

Chronic myeloid leukemia (CML) is a neoplastic transformation of the hematopoietic system resulting from a t(9;22)(q34;q11) translocation forming a BCR-ABL hybrid gene which has intense enzyme tyrosine kinase activity responsible for the proliferation of tumor cells. A dramatic positive response was achieved in CML patients with imatinib. This drug is effective in most patients because it presents long-lasting responses. However, some patients are resistant or develop resistance during treatment, particularly in the late-stage disease, thus second generation tyrosine kinase inhibitors such as dasatinib and nilotinib were developed to reduce the risk of developing resistance. Bosutinib and INNO-406 are being developed to treat resistant patients and also to reduce the side effects of the aforementioned drugs. Additionally, novel tyrosine kinase inhibitors are in clinical or preclinical development stages. In the future, multiple treatment options will be available for patients with CML, with the possibility to individualize the treatment according to the needs of each patient. In the current study we describe antineoplastic drugs that act as tyrosine kinase enzyme inhibitors in CML.

Primary Systemic Anaplastic Large Cell Lymphoma in Korean Adults: 11 Years' Experience at Asan Medical Center

PURPOSE: Anaplastic large cell lymphoma (ALCL), a CD30+ T-cell non-Hodgkin's lymphoma, represents only 2-8% of lymphoma overall. Information on the clinical findings of primary systemic ALCL in Korea is limited. Our aims were to report the clinical features and outcomes of primary systemic ALCL. PATIENTS and METHODS: We retrospectively reviewed the medical records of 36 adult patients diagnosed with primary systemic ALCL at Asan Medical Center from February 1995 through June 2006. RESULTS: Of 36 patients, 29 were male. The median age was 39 years (range, 17-67 years), and 26 (72%) presented with Ann Arbor stages III and IV. The most commonly involved extranodal sites were bone (n = 7) and soft tissue (n = 6). Thirty-two of all patients (89%) were treated with an anthracycline-based regimen including cyclophosphamide/doxorubicin/vincristine/prednisone (CHOP) as induction chemotherapy; 16 (50%) achieved complete remission (CR), and 13 (41%) achieved partial remission (PR). Median overall survival (OS) and event-free survival (EFS) were 49 and 17 months, respectively. Univariate analysis showed that performance status (p = 0.035), international prognostic index (IPI) (p = 0.025), and age-adjusted IPI (p = 0.034) were significant prognostic factors for OS, whereas anaplastic lymphoma kinase (ALK) expression did not affect OS (p = 0.483). CONCLUSION: Our retrospective analysis of Korean primary systemic ALCL patients showed that median OS was 49 months and overall response to CHOP was 91%. Performance, IPI, and age-adjusted IPI were predictors of OS, whereas ALK expression did not have prognostic significance.

La inhibición de Rho kinasa disminuye la presión arterial y activa la vía vasodilatadora del sistema renina angiotensina en la pared arterial de ratas hipertensas / Inhibition of Rho-kynase decreases systolic blood pressure and activates the vasodilating pathway

Antecedentes: La activación de Rho kinasa disminuye la formación de óxido nítrico al inhibir eNOS. Por otro lado, el rol de la vía de señalización RhoA/Rho kinasa en la actividad y expresión génica de la enzima clave en la vía vasodilatadora del sistema renina angiotensina (SRA), denominada ECA2, no es conocido. Objetivo: Determinar la participación de la vía RhoA/Rho kinasa en la actividad enzimática y en la expresión de ECA2 y de eNOS en la pared arterial en ratas hipertensas (DOCA-sal). Métodos: Se usaron ratas Sprague Dawley de 150 grs. unifrectomizadas tratadas con desoxicorticosterona (DOCA, 100 mg/Kg/sem sbc) durante 6 semanas. Como controles se usaron ratas unifrectomizadas. Un tercer grupo recibió DOCA y además el inhibidor específico de Rho, fasudil (100 mg/kg/día) por gavage durante 21 días. Al finalizar los tratamientos se determinó la presión arterial sistólica (PAS), la masa relativa del ventrículo izquierdo (MRVI mg*100/g), las actividades de ECA y ECA2 por fluorimetría y la expresión de genes de ECA, ECA2 y eNOS por RT-PCR en la aorta. Conclusión: La mayor expresión de ECA2 inducida por fasudil indujo un aumento significativo de la expresióngénica de eNOS en la pared arterial, lo que pudiera explicar el efecto de fasudil sobre ECA2. La inhibición de Rhokinasa activa la vía vasodilatadora del SRA en la pared arterial de ratas hipertensas aumentando los niveles de ECA2 y de eNOS, y disminuye los niveles de ECA.

Background: Through nitric oxide synthase (eNOS) inhibition, Rho-kynase decreases the formation of NO. The role of the RhoA/Rho kynase signaling pathway upon the activity and gene expression of the enzyme responsible for the vasodilating effects of the renin-angiotensin system (RAS), named ACE2 , is unknown. Aim: To determine the role of the RhoA/Rho kynase pathway on the activity and expression of ACE2 and eNOS in the arterial wall of rats with DOCA-salt induced hypertension. Methods: Sprague Dawley uninephrectomized DOCA hypertensive rats ( DOCA, 100mg/Kg/week sbc during 6 weeks) were used as controls. A third group received the specific Rho inhibitor fasudil (100 mg/Kg/day) in addition to DOCA for 21 days. At the end of the treatment period, blood pressure, relative left ventricular mass (RLVIM,mg*100/g), ACE and ACE2 activities (fluorometry) were determined. The expression of ACE and ACE2 genes, along with eNOS in the aortic wall were determined by RT-PCR. Results: Are expressed as mean +/- SEM.

Protein tyrosine kinases in Schistosoma mansoni

The identification and description of signal transduction molecules and mechanisms are essential to elucidate Schistosoma mansoni host-parasite interactions and parasite biology. This mini review focuses on recent advancements in the study of signalling molecules and transduction mechanisms in S. mansoni, drawing special attention to the recently identified and characterised protein tyrosine kinases of S. mansoni.

Sitios de acción inhibitoria de la prolactina sobre la síntesis de estradiol inducida por la hormona folículo estimulante en las células de la granulosa / Sites of prolactin inhibition on gonadotropin-induced estrogen production in cultured rat granulosa cells

En este estudio se investigaron los sitios probables de la acción inhibitoria de prolactina (Prl) sobre la esteroidogénesis ovárica inducida por la hormona folículo estimulante (FSH). Para esta finalidad se estudió la capacidad de cultivos primarios de células de la granulosa de la rata de sintetizar estradiol y AMPc bajo la estimulación con FSH o de activadores de la vía dependiente de AMPc en presencia de Prl humana. La participación de otros sistemas de transducción de señal como los dependientes de PKC y proteínas Gi en los mecanismos de acción inhibitoria de la Prl fue también investigada utilizando inhibidores de estos sistemas como la calfostina C y la toxina pertusis. Los resultados demostraron la habilidad de la Prl de alterar la esteroidogénesis previa y posterior a la generación de AMPc, muy probablemente por mecanismos que involucran la activación de la subunidad catalítica de la adenilato ciclasa, así como a través de interactuar con sistemas de transducción de señal dependientes de PKC y proteínas sensibles a la toxina pertusis. Nuestros resultados sugieren un mecanismo de interacción entre receptores acoplados a proteínas G con aquéllos acoplados a cinasas de tirosinas mediado muy probablemente por vías de señalización dependientes de proteínas Gi.

We studied the sites of prolactin inhibition upon FSH induced ovarian steroidogenesis and the ability of prolactin (Prl) to inhibit the synthesis of estradiol and cAMP accumulation under the stimulation of FSH or cAMP dependent activators. The participation of other signal pathways such as PKC and Gi proteins on the inhibitory actions of Prl was also investigated using calfostine C andpertusis toxin as inhibitors. Results showed a dose dependent prolactin decrease in FSH-induced estradiol and cAMP production prior and after the generation of the cyclic nucleotide by a mechanism involving the catalytic subunit of adenyl cyclase and/or through activation of PKC or by the interaction with pertusin toxin sensitive G proteins. Our results suggest a mechanism by which G protein coupled receptors are linked with those coupled with tyrosine kinase through the involvement of a Gi protein mediated mechanism.

Hypoxia activates the cyclin D1 promoter via the Jak2/STAT5b pathway in breast cancer cells

Hypoxia, a common consequence of solid tumor growth in breast cancer or other cancers, serves to propagate a cascade of molecular pathways which include angiogenesis, glycolysis, and various cellcycle control proteins. As we have shown previously, hypoxia activates STAT5 (signal transducer and activator of transcription 5) and increases its binding activity to the GAS element in mammary epithelial cells. In this study we attempted to elucidate the mechanism by which cyclin D1 is regulated by the STAT5 protein under hypoxic conditions. Our data demonstrate that hypoxia (2% O2) or desferrioxamine (DFO) induces tyrosine and serine phosphorylation of STAT5 in human breast cancer cells (MCF-7) and mammary epithelial cells (HC11). Imunoprecipitation and subsequent Western analysis showed that Jak2 leads to the tyrosine phosphorylation and activation of STAT5a or STAT5b under hypoxic conditions. Using a transfected COS-7 cell model system, we demonstrate that the activity of a cyclin D1 promoter-luciferase construct increased under hypoxic conditions or DFO treatment. The activity of the STAT5b/cyclin D1 promoter increased significantly by 12 h of hypoxia, whereas the activity of the STAT5a/cyclin D1 promoter was unaffected under hypoxic conditions. These increases in promoter activity are predominantly mediated by the Jak2/ STAT5b signaling pathway. We have shown by EMSA that hypoxia induces STAT5 to bind to the cyclin D1 promoter (GAS-1) in MCF-7 and HC11 cells. These data suggest that STAT5b may mediate the transcriptional activation of cyclin D1 after hypoxic stimulation.

A Case of Primary Gastric CD30-Positive Anaplastic Large-Cell Lymphoma

Gastric CD30-positive anaplastic large-cell lymphoma is a very rare disease. It is sometimes difficult to distinguish it from undifferentiated carcinoma, sarcoma and so on. We report here on a case of primary gastric anaplastic large-cell lymphoma. A 50-yr-old woman complained of epigastric pain and severe chest pain for 1 week. The gastroendoscopic examination revealed geographic mucosal irregularities with shallow ulceration at the antrum. She underwent a total gastrectomy. The gross finding of the resected stomach was an 8 x 4.5 cm sized ulceroinfiltrative lesion at the pyloric antrum along the lesser curvature. The microscopic examination revealed diffuse and solid proliferations of large atypical cells with pleomorphic nuclei. Immunohistochemically, the tumor cells were positive for CD30, vimentin and CD3, and this was a finding compatible with anaplastic large-cell lymphoma. To the best of our knowledge, this is the first such reported case in Korea.

Increased Expression of Focal Adhesion Kinase in Thyroid Cancer: Immunohistochemical Study

Focal adhesion kinase (FAK) is a tyrosine kinase that is found in cellular structures called focal adhesions. FAK appears to be a key element in signal transduction pathways involved in cell adhesion and locomotion. FAK is overexpressed in various tumors, including tumors derived from regions of the head and neck, colon, breast, prostate, and liver. In this study, we investigated immunohistochemically whether FAK expression was increased in thyroid cancers. FAK staining was not seen in any of the 20 normal thyroid tissues or the 6 nodular hyperplasia specimens. In contrast, FAK staining was observed in all of 17 papillary carcinomas, 9 follicular carcinomas, 8 medullary carcinomas, and 2 anaplastic carcinomas. Nine of 17 follicular adenomas showed FAK immunoreactivity. FAK was not expressed in normal tissue and nodular hyperplasia, but was expressed in some of the follicular adenoma, and all of the follicular, papillary, medullary and anaplastic thyroid carcinoma. This result indicates that the up-regulation of FAK may play a role in the development of thyroid carcinogenesis.

Cellular Proliferative Effect of Dexamethasone in Immortalized Trabecular Meshwork Cell (TM5) Line

Dexamethasone (DEX), one of the corticosteroid hormones, is one of the most common therapeutic strategies in ophthalmological treatment. Despite its widespread use and clinical efficiency, little is known regarding the specific effects of DEX on cell growth, differentiation and cell death in human trabecular meshwork cells. The presence of the glucocorticoid receptor (GR, dexamethasone receptor) in TM-5 cell line, which was derived from the primary human trabecular meshwork cells, was verified by RT-PCR and western blot analysis. The effects of DEX on the cellular proliferation of TM5 cells were measured by a BrdU incorporation assay. Western blot analysis were used to examine the effects of DEX on the Ras/MEK/ERK signaling pathway. The total Ras, MEK1/2 and ERK1/2 protein levels as well as the levels of activated (phosphorylated) form were both significantly increased by the DEX treatment for 5 days. Both MEK1/2 and ERK1/2 were significantly activated by phosphorylation after 10 minutes. The dependence of this increased cell proliferation on GR activation by DEX and the sustained activation of ERK was examined using RU486 (a GR inhibitor) and U0126 (a MEK inhibitor). Both RU486 and U0126 prevented the induction of cell proliferation by the DEX treatment in the TM5 cells. In conclusion this study demonstrated that GR is expressed in TM5 cells. Secondly, DEX treatment for 5 days stimulates cell proliferation in TM5 cells, and that this increased proliferation effect is mediated by the Ras/MEK/ERK pathway.

Induction of nitric oxid esynthase(NOS) by soluble glucocorticoid induced tumor necrosis factor receptor(sGITR) is modulated by IFN-g in murine macrophage

Earlier study showed that glucocorticoid induced tumor necrosis factor receptor (GITR), a new TNFR family, activated murine macrophages to express inducible nitric oxide synthase (iNOS) and to generate nitric oxide (NO). A possible involvement of pro-inflammatory cytokines on NO production by GITR was investigated in vitro systems and signaling molecules contributing to sGITR-induced iNOS production are determined in Raw 264.7 cells, a murine macrophage cell line. The result showed that the synergy was afforded by the combination of GITR with IFN-gamma in a dose-dependent manner but IFN-gamma alone was not able to induce NOS. No effects were observed with TNF-alpha, IL-1beta, or IL-6 co-treated with GITR. To determine signaling molecules contributing to sGITR-induced iNOS production, a specific inhibitor for signal pathway proteins tested showed that PDTC (NF- kB) and genistein (tyrosine kinase) inhibited NOS induction significantly, while sodium orthovanadate (tyrosine phosphatase) potentiated NOS expression. These results suggest that activations of NF-kB were involved in induction of iNOS by GITR and IFN-gamma priming caused earlier and stronger NF-kB activation.

Insulin receptor tyrosine kinase activity in monocytes of type 2 diabetes mellitus patients receiving oral L-lysine.

The action of lysine as an antidiabetic agent was examined in human volunteers. Eight patients with type 2 DM were orally supplemented with L-lysine hydrochloride 1 g/day in two doses along with antidiabetic tablets (glyciphage or chlorformine), for a period of two months. Periodically their plasma fasting sugar and insulin receptor tyrosine kinase activity was measured in their monocytes. Eight normal healthy volunteers served as controls for comparison of receptor tyrosine kinase activity. Insulin receptor tyrosine kinase was isolated from monocytes by immunoprecipitation and the activity was determined using exogenous substrate poly glu-tyr (4:1) and radioactive ATP. Phosphorylated peptide was separated by electrophoresis and quantified using a liquid scintillation system. The enzyme activity was significantly low (22074 +/- 1728 dpm/ml immunoprecipitate) in subjects with diabetes when compared to non-diabetic control group (50,775 +/- 3597). Lysine treatment enhanced the enzyme activity by 31% in patients with diabetes and decreased their blood sugar by 27%.

ATP-induced focal adhesion kinase activity is negatively modulated by phospholipase D2 in PC12 cells

Extracellular ATP has been known to modulate various cellular responses including mitogenesis, secretion and morphogenic activity in neuronal cells. In the ATP-induced morphogenic activity, focal adhesion kinase(s) such as Fak have been suggested to play a critical role. Binding of ATP to its specific cell surface receptor in PC12 cells induces phospholipase D (PLD) activity. However, the role of PLD on ATP-induced Fak activation in PC12 cells remains unclear. In this study, we investigated the role of PLD on the ATP-induced Fak activation and paxillin phosphorylation using two established cell lines: wild type PLD2- and lipase-inactive mutant PLD2-inducible PC12 cells. Stimulation of cells with ATP caused PLD2 activation via classical protein kinase C activation. ATP also induced Fak activation, and paxillin phosphorylation, and were dramatically reduced by wild type PLD2 overexpression but not by lipase-inactive mutant PLD2 overexpression. When the PC12 cells were pretreated with propranolol, a specific inhibitor for phosphatidic acid phosphohydrolase resulting in the accumulation of PA, ATP-induced Fak activation and paxillin phosphorylation were also reduced. We found that inhibition of tyrosine phosphatases by pervanadate completely blocked PLD2-dependent Fak and paxillin dephosphorylation. Taken together, we suggest that PLD2 activity might play a negative role in ATP-induced Fak and paxillin phosphorylation possibly through tyrosine phosphatases.