Phosphorylation of Cofilin-1 Enhances Paclitaxel Resistance of Epithelial Ovarian Cancer Cells by Inhibiting Apoptosis / 生物医学与环境科学（英文）

Objective@#To investigate the molecular mechanism of high phosphorylation levels of cofilin-1 (p-CFL-1) associated with paclitaxel resistance in epithelial ovarian cancer (EOC) cells.@*Methods@#Cells displaying varying levels of p-CFL-1 and CFL-1 were created by plasmid transfection and shRNA interference. Cell inhibition rate indicating paclitaxel efficacy was assessed by Cell Counting Kit-8 (CCK-8) assay. Apoptosis was assessed by flow cytometry and protein levels were detected by western blotting. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the expression levels of phosphokinases and phosphatases of CFL-1. Survival analysis evaluated the correlation between the prognosis of EOC patients and the levels of p-CFL-1 and slingshot-1 (SSH-1).@*Results@#High levels of p-CFL-1 were observed in EOC cells that survived treatment with high doses of paclitaxel. SKOV3 cell mutants with upregulated p-CFL-1 showed impaired paclitaxel efficacy, as well as decreased apoptosis rates and pro-survival patterns of apoptosis-specific protein expression. Cytoplasmic accumulation of p-CFL-1 inhibited paclitaxel-induced mitochondrial apoptosis. SSH-1 silencing mediated CFL-1 phosphorylation in paclitaxel-resistant SKOV3 cells. Clinically, the high level of p-CFL-1 and the low level of SSH-1 in EOC tissues were closely related to chemotherapy resistance and poor prognosis in EOC patients.@*Conclusion@#The SSH-1/p-CFL-1 signaling pathway mediates paclitaxel resistance by apoptosis inhibition in EOC and is expected to be a potential prognostic predictor.

Modulated Gene Expression of Toxoplasma gondii Infected Retinal Pigment Epithelial Cell Line (ARPE-19) via PI3K/Akt or mTOR Signal Pathway

Due to the critical location and physiological activities of the retinal pigment epithelial (RPE) cell, it is constantly subjected to contact with various infectious agents and inflammatory mediators. However, little is known about the signaling events in RPE involved in Toxoplasma gondii infection and development. The aim of the study is to screen the host mRNA transcriptional change of 3 inflammation-related gene categories, PI3K/Akt pathway regulatory components, blood vessel development factors and ROS regulators, to prove that PI3K/Akt or mTOR signaling pathway play an essential role in regulating the selected inflammation-related genes. The selected genes include PH domain and leucine- rich-repeat protein phosphatases (PHLPP), casein kinase2 (CK2), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), glutamate-cysteine ligase (GCL), glutathione S-transferase (GST), and NAD(P)H: quinone oxidoreductase (NQO1). Using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), we found that T. gondii up-regulates PHLPP2, CK2β, VEGF, GCL, GST, and NQO1 gene expression levels, but down-regulates PHLPP1 and PEDF mRNA transcription levels. PI3K inhibition and mTOR inhibition by specific inhibitors showed that most of these host gene expression patterns were due to activation of PI3K/Akt or mTOR pathways with some exceptional cases. Taken together, our results reveal a new molecular mechanism of these gene expression change dependent on PI3K/Akt or mTOR pathways and highlight more systematical insight of how an intracellular T. gondii can manipulate host genes to avoid host defense.

Expression of PH Domain Leucine-rich Repeat Protein Phosphatase, Forkhead Homeobox Type O 3a and RAD51, and their Relationships with Clinicopathologic Features and Prognosis in Ovarian Serous Adenocarcinoma / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Ovarian serous adenocarcinoma can be divided into low- and high-grade tumors, which exhibit substantial differences in pathogenesis, clinicopathology, and prognosis. This study aimed to investigate the differences in the PH domain leucine-rich repeat protein phosphatase (PHLPP), forkhead homeobox type O 3a (FoxO3a), and RAD51 protein expressions, and their associations with prognosis in patients with low- and high-grade ovarian serous adenocarcinomas.</p><p><b>METHODS</b>The PHLPP, FoxO3a, and RAD51 protein expressions were examined in 94 high- and 26 low-grade ovarian serous adenocarcinomas by immunohistochemistry. The differences in expression and their relationships with pathological features and prognosis were analyzed.</p><p><b>RESULTS</b>In high-grade serous adenocarcinomas, the positive rates of PHLPP and FoxO3a were 24.5% and 26.6%, while in low-grade tumors, they were 23.1% and 26.9%, respectively (P < 0.05 vs. the control specimens; low- vs. high-grade: P > 0.05). The positive rates of RAD51 were 70.2% and 65.4% in high- and low-grade serous adenocarcinomas, respectively (P < 0.05 vs. the control specimens; low- vs. high-grade: P > 0.05). Meanwhile, in high-grade tumors, Stage III/IV tumors and lymph node and omental metastases were significantly associated with lower PHLPP and FoxO3a and higher RAD51 expression. The 5-year survival rates of patients with PHLPP- and FoxO3a-positive high-grade tumors (43.5% and 36.0%) were significantly higher than in patients with PHLPP-negative tumors (5.6% and 7.2%, respectively; P< 0.05). Similarly, the 5-year survival rate of RAD51-positive patients (3.0%) was significantly lower than in negative patients (42.9%; P< 0.05). In low-grade tumors, the PHLPP, FoxO3a, and RAD51 expressions were not significantly correlated with lymph node metastasis, omental metastasis, Federation of Gynecology and Obstetrics stage, or prognosis.</p><p><b>CONCLUSIONS</b>Abnormal PHLPP, FoxO3a, and RAD51 protein expressions may be involved in the development of high- and low-grade ovarian serous adenocarcinomas, suggesting common molecular pathways. Decreased PHLPP and FoxO3a and increased RAD51 protein expression may be important molecular markers for poor prognosis, and RAD51 may be an independent prognosis factor, of high-grade, but not low-grade, ovarian serous adenocarcinomas.</p>

Reversible phosphorylation of the 26S proteasome

The 26S proteasome at the center of the ubiquitin-proteasome system (UPS) is essential for virtually all cellular processes of eukaryotes. A common misconception about the proteasome is that, once made, it remains as a static and uniform complex with spontaneous and constitutive activity for protein degradation. Recent discoveries have provided compelling evidence to support the exact opposite insomuch as the 26S proteasome undergoes dynamic and reversible phosphorylation under a variety of physiopathological conditions. In this review, we summarize the history and current understanding of proteasome phosphorylation, and advocate the idea of targeting proteasome kinases/phosphatases as a new strategy for clinical interventions of several human diseases.

Transcriptome analyses of chronic traumatic encephalopathy show alterations in protein phosphatase expression associated with tauopathy

Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative disorder that is associated with repetitive head injury and has distinctive neuropathological features that differentiate this disease from other neurodegenerative diseases. Intraneuronal tau aggregates, although they occur in different patterns, are diagnostic neuropathological features of CTE, but the precise mechanism of tauopathy is not known in CTE. We performed whole RNA sequencing analysis of post-mortem brain tissue from patients with CTE and compared the results to normal controls to determine the transcriptome signature changes associated with CTE. The results showed that the genes related to the MAP kinase and calcium-signaling pathways were significantly downregulated in CTE. The altered expression of protein phosphatases (PPs) in these networks further suggested that the tauopathy observed in CTE involves common pathological mechanisms similar to Alzheimer's disease (AD). Using cell lines and animal models, we also showed that reduced PPP3CA/PP2B phosphatase activity is directly associated with increases in phosphorylated (p)-tau proteins. These findings provide important insights into PP-dependent neurodegeneration and may lead to novel therapeutic approaches to reduce the tauopathy associated with CTE.

A serine/threonine phosphatase 1 of Streptococcus suis type 2 is an important virulence factor

Streptococcus suis is regarded as one of the major pathogens of pigs, and Streptococcus suis type 2 (SS2) is considered a zoonotic bacterium based on its ability to cause meningitis and streptococcal toxic shock-like syndrome in humans. Many bacterial species contain genes encoding serine/threonine protein phosphatases (STPs) responsible for dephosphorylation of their substrates in a single reaction step. This study investigated the role of stp1 in the pathogenesis of SS2. An isogenic stp1 mutant (Δstp1) was constructed from SS2 strain ZJ081101. The Δstp1 mutant exhibited a significant increase in adhesion to HEp-2 and bEnd.3 cells as well as increased survival in RAW264.7 cells, as compared to the parent strain. Increased survival in macrophage cells might be related to resistance to reactive oxygen species since the Δstp1 mutant was more resistant than its parent strain to paraquat-induced oxidative stress. However, compared to parent strain virulence, deletion of stp1 significantly attenuated virulence of SS2 in mice, as shown by the nearly double lethal dose 50 value and the lower bacterial load in organs and blood in the murine model. We conclude that Stp1 has an essential role in SS2 virulence.

Can microcystins affect zooplankton structure community in tropical eutrophic reservoirs? / Microcistinas podem afetar a estrutura da comunidade zooplanctônica em reservatórios eutróficos tropicais?

Abstract The aim of our study was to assess whether cyanotoxins (microcystins) can affect the composition of the zooplankton community, leading to domination of microzooplankton forms (protozoans and rotifers). Temporal variations in concentrations of microcystins and zooplankton biomass were analyzed in three eutrophic reservoirs in the semi-arid northeast region of Brazil. The concentration of microcystins in water proved to be correlated with the cyanobacterial biovolume, indicating the contributions from colonial forms such as Microcystis in the production of cyanotoxins. At the community level, the total biomass of zooplankton was not correlated with the concentration of microcystin (r2 = 0.00; P > 0.001), but in a population-level analysis, the biomass of rotifers and cladocerans showed a weak positive correlation. Cyclopoid copepods, which are considered to be relatively inefficient in ingesting cyanobacteria, were negatively correlated (r2 = – 0.01; P > 0.01) with the concentration of cyanotoxins. Surprisingly, the biomass of calanoid copepods was positively correlated with the microcystin concentration (r2 = 0.44; P > 0.001). The results indicate that allelopathic control mechanisms (negative effects of microcystin on zooplankton biomass) do not seem to substantially affect the composition of mesozooplankton, which showed a constant and high biomass compared to the microzooplankton (rotifers). These results may be important to better understand the trophic interactions between zooplankton and cyanobacteria and the potential effects of allelopathic compounds on zooplankton.

Resumo Com o objetivo de avaliar se as cianotoxinas (microcistinas) podem afetar a composição da comunidade zooplanctônica, levando à dominância de formas microzooplanctônicas (protozoários e rotiferos), as variações nas concentrações de microcistina e a biomassa do zooplâncton foram analisadas em três reservatórios eutróficos na região semi-árida do nordeste brasileiro. A concentração de microcistinas na água esteve correlacionada com o biovolume de cianobactérias, indicando a contribuição de formas coloniais como Microcystis na produção de cianotoxinas. A nível de comunidade, a biomassa total do zooplâncton não apresentou correlacão com a concentração de microcistina (r2 = 0.00; P > 0.001), mas em uma análise a nível de populações, a biomassa de rotíferos e cladóceros apresentou uma fraca correlação positiva. Copépodos Cyclopoida, os quais são considerados relativamente ineficientes na ingestão de cianobactérias, estiveram negativamente correlacionados com a concentração de microcistinas (r2 = - 0.01; P > 0.01). Surpreendentemente, a biomassa de copépodos Calanoida foi positivamente correlacionada com a concentração de cianotoxinas (r2 = 0.44; P > 0.001). Os resultados indicam que mecanismos de controle alelopáticos (efeitos negativos da microcistina sobre o zooplâncton) parecem não afetar substancialmente a composição do mesozooplâncton, que apresentou uma alta e constante biomassa, quando comparada à biomassa do microzooplâncton (rotíferos). Esses resultados podem ser importantes para um melhor entendimento das interações tróficas entre o zooplâncton e cianobactérias, e do efeito potencial de compostos alelopáticos sobre o zooplâncton.

Changes in expression of Slingshot protein in hypoxic human intestinal epithelial cell and its relation with barrier function of the cells / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the effect of hypoxia on Slingshot protein expression in human intestinal epithelial cell and its relation with changes in barrier function of the cells.</p><p><b>METHODS</b>The human intestinal epithelial cell line Caco-2 was used to reproduce monolayer-cells. One portion of the monolayer-cell specimens were divided into six parts according to the random number table, and they were respectively exposed to hypoxia for 0 (without hypoxia), 1, 2, 6, 12, and 24 h. Transepithelial electrical resistance (TER) was determined with an ohmmeter. Another portion of the monolayer-cell specimens were exposed to hypoxia as above. Western blotting was used to detect the protein expressions of zonula occludens 1 (ZO-1), occludin, claudin-1, Slingshot-1, Slingshot-2, and Slingshot-3. The remaining portion of the monolayer-cell specimens were also exposed to hypoxia as above. The content of fibrous actin (F-actin) and globular actin (G-actin) was determined by fluorescence method. The sample number of above-mentioned 3 experiments was respectively 10, 10, and 18 at each time point. Data were processed with one-way analysis of variance and Dunnett test.</p><p><b>RESULTS</b>(1) Compared with that of cells exposed to hypoxia for 0 h, TER of cells exposed to hypoxia for 1 to 24 h was significantly reduced (P values below 0.01). (2) Compared with those of cells exposed to hypoxia for 0 h (all were 1.00), the protein expressions of ZO-1, occludin, and claudin-1 of cells exposed to hypoxia for 1 to 24 h were generally lower, especially those of cells exposed to hypoxia for 12 h or 24 h (respectively 0.69 ± 0.20, 0.47 ± 0.15, and 0.47 ± 0.22, P<0.05 or P<0.01). Compared with those of cells exposed to hypoxia for 0 h, the protein expressions of Slingshot-1 and Slingshot-3 of cells exposed to hypoxia for 1 to 24 h were not obviously changed (P values above 0.05). The protein expression of Slingshot-2 of cells was decreased at first and then gradually increased from hypoxia hour 1 to 24. The protein expression of Slingshot-2 of cells exposed to hypoxia for 24 h (1.54 ± 0.57) was significantly higher than that of cells exposed to hypoxia for 0 h (1.00, P<0.05). (3) Compared with those of cells exposed to hypoxia for 0 h, the content of F-actin of cells exposed to hypoxia for 1, 6, 12, and 24 h was significantly decreased, whereas the content of G-actin of cells exposed to hypoxia for 6-24 h was significantly increased, P<0.05 or P<0.01; the content of F-actin and G-actin of cells exposed to hypoxia for the other time points was not obviously changed (P values above 0.05).</p><p><b>CONCLUSIONS</b>Hypoxia may cause cofilin activation after dephosphorylation and the depolymerization of F-actin by inducing Slingshot-2 protein expression, which in turn affects the tight junction of human intestinal epithelial cells, thus leading to deterioration of barrier function of these cells.</p>

Manual de monitoramento da qualidade dos registros de enfermagem na assistência domiciliar / Manual de monitoreo de la calidad de los registros de enfermería en la atención domiciliaria / Manual for monitoring the quality of nursing home care records

RESUMO Objetivo: construir e validar um instrumento para monitorar a qualidade dos registros de enfermagem no Programa de Assistência Domiciliar (PAD) em um hospital universitário. Método: estudo metodológico envolvendo a elaboração de um manual e submetido à validação de conteúdo por seis juízes sob consenso ≥ 80%. A coleta ocorreu em 2012 por meio de questionário contendo: evolução de enfermagem, diagnóstico e prescrição de enfermagem e normas para os registros da equipe de enfermagem preconizadas pelo Conselho Regional de Enfermagem-SP e pela instituição. Os itens do manual foram julgados de acordo com as variáveis - relevância, pertinência, clareza e simplicidade. Resultados: das 39 proposições 100% atingiram consenso ≥ 80% em relevância, pertinência e clareza; 92,3% em simplicidade. Os itens sono/repouso, mobilidade e checagem nas atividades prescritas não atingiram consenso mínimo favorável, sendo aprimorados pelas sugestões dos juízes. Conclusão: acreditamos que o instrumento possibilitará a melhoria dos processos de trabalho no PAD. .

RESUMEN Objetivo: construir y validar un instrumento para monitorear la calidad del registros de enfermería en Programa de Atención Domiciliaria (PAD) de un Hospital Universitario. Metodo: estudio metodológico. Fue construido un manual y sometió a validación de contenido por seis jueces bajo el consenso ≥80%. La recogida currió en 2012, con un cuestionario que contiene: evolución de enfermería, diagnóstico y prescripción de enfermería y normas para los registros del personal de enfermaria estabelecidas por Consejo Regional de Enfermería-SP y por la institución. Los artículos del manual fueran juzgadso conforme las variables relevancia, pertinencia, claridad y sencillez. Resultados: de las 39 proposiciones 100% alcanzó consenso ≥ 80% en la relevancia, pertinencia y claridad; 92,3% en la simplicidad. Los itens sueño/resto, movilidad y verificar las actividades prescritas no alcanzó consenso favorable, siendo mejoradas por las sugerencias de los jueces. Conclusión: creemos que el instrumento permitirá la mejora de los procesos de trabajo en PAD. .

ABSTRACT Objective: to build and validate an instrument aimed at monitoring the quality of nursing records in the Home Care Program (HCP) of a university hospital. Method: methodological study involving the elaboration of a manual, whose content was later submitted to six experts for validation, reaching a ≥ 80% consensus. The data collection process was carried out in 2012 by means of a questionnaire comprised of the following issues: nursing evolution, nursing diagnosis, and nursing prescription, and standards for the nursing team recommended by the Regional Nursing Council of São Paulo and by the assessed institution. Manual items were judged according to the following variables: relevance, pertinence, clarity and simplicity. Results: of the 39 propositions, 100% achieved ≥ 80% agreement in the relevance, pertinence and clarity variables; 92.3% in the simplicity variable. Sleep/rest, Mobility and Check-out variables did not reach a favorable minimum consensus in the prescribed activities and were improved following suggestions from the experts. Conclusion: we believe that the instrument will enable the improvement of the HCP’s work process. .

Curcumin treatment recovery the decrease of protein phosphatase 2A subunit B induced by focal cerebral ischemia in Sprague-Dawley rats / 한국실험동물학회지

Curcumin provides various biological effects through its anti-inflammatory and antioxidant properties. Moreover, curcumin exerts a neuroprotective effect against ischemic condition-induced brain damage. Protein phosphatase 2A (PP2A) is a ubiquitous serine and threonine phosphatase with various cell functions and broad substrate specificity. Especially PP2A subunit B plays an important role in nervous system. This study investigated whether curcumin regulates PP2A subunit B expression in focal cerebral ischemia. Cerebral ischemia was induced surgically by middle cerebral artery occlusion (MCAO). Adult male rats were injected with either vehicle or curcumin (50 mg/kg) 1 h after MCAO and cerebral cortex tissues were isolated 24 h after MCAO. A proteomics study, reverse transverse-PCR and Western blot analyses were performed to examine PP2A subunit B expression levels. We identified a reduction in PP2A subunit B expression in MCAO-operated animals using a proteomic approach. However, curcumin treatment prevented injury-induced reductions in PP2A subunit B levels. Reverse transverse-PCR and Western blot analyses confirmed that curcumin treatment attenuated the injury-induced reduction in PP2A subunit B levels. These findings can suggest that the possibility that curcumin maintains levels of PP2A subunit B in response to cerebral ischemia, which likely contributes to the neuroprotective function of curcumin in cerebral ischemic injury.

Phosphorylation and Reorganization of Keratin Networks: Implications for Carcinogenesis and Epithelial Mesenchymal Transition

Metastasis is one of hallmarks of cancer and a major cause of cancer death. Combatting metastasis is highly challenging. To overcome these difficulties, researchers have focused on physical properties of metastatic cancer cells. Metastatic cancer cells from patients are softer than benign cancer or normal cells. Changes of viscoelasticity of cancer cells are related to the keratin network. Unexpectedly, keratin network is dynamic and regulation of keratin network is important to the metastasis of cancer. Keratin is composed of heteropolymer of type I and II. Keratin connects from the plasma membrane to nucleus. Several proteins including kinases, and protein phosphatases bind to keratin intermediate filaments. Several endogenous compounds or toxic compounds induce phosphorylation and reorganization of keratin network in cancer cells, leading to increased migration. Continuous phosphorylation of keratin results in loss of keratin, which is one of the features of epithelial mesenchymal transition (EMT). Therefore, several proteins involved in phosphorylation and reorganization of keratin also have a role in EMT. It is likely that compounds controlling phosphorylation and reorganization of keratin are potential candidates for combating EMT and metastasis.

Targeting PPM1D by lentivirus-mediated RNA interference inhibits the tumorigenicity of bladder cancer cells

Protein phosphatase magnesium/manganese-dependent 1D (PPM1D) is a p53-induced phosphatase that functions as a negative regulator of stress response pathways and has oncogenic properties. However, the functional role of PPM1D in bladder cancer (BC) remains largely unknown. In the present study, lentivirus vectors carrying small hairpin RNA (shRNA) targeting PPM1D were used to explore the effects of PPM1D knockdown on BC cell proliferation and tumorigenesis. shRNA-mediated knockdown of PPM1D significantly inhibited cell growth and colony forming ability in the BC cell lines 5637 and T24. Flow cytometric analysis showed that PPM1D silencing increased the proportion of cells in the G0/G1 phase. Downregulation of PPM1D also inhibited 5637 cell tumorigenicity in nude mice. The results of the present study suggest that PPM1D plays a potentially important role in BC tumorigenicity, and lentivirus-mediated delivery of shRNA against PPM1D might be a promising therapeutic strategy for the treatment of BC.

Tabagismo como fator de risco para a doença renal crônica: revisão sistemática / Smoking as risk factor for chronic kidney disease: systematic review

Introdução: A doença renal crônica (DRC) e o tabagismo são problemas de saúde pública. Objetivo: Analisar o tabagismo como fator risco para a progressão da DRC. Métodos: Realizou-se uma revisão sistemática nas bases Medline, LILACS, SciELO, Google Acadêmico, Trials.gov e Embase com artigos publicados até fevereiro de 2013. Incluíram-se estudos: tipo coorte, ensaios clínicos e caso-controle. Realizados em seres humanos com idade ≥ 18 anos tendo tabagismo como fator de risco para progressão da DRC. Excluíram-se estudos que não referiam tabagismo e DRC no título ou tinham proposta de combate ao fumo. Resultados: Das 94 citações, 12 artigos foram selecionados. Destes, seis eram multicêntricos realizados em países desenvolvidos e quatro foram aleatorizados. Predominou o sexo masculino 51%-76%. Houve progressão associada ao tabagismo em 11 estudos. Identificou-se que o consumo ≥ 15 maços/ ano aumenta o risco de progressão da DRC. Conclusão: Tabagismo é fator de risco para progressão da DRC. .

Introduction: Chronic kidney disease (CKD) and smoking are public health problems. Objective: To assess smoking as a risk factor for progression of CKD. Methods: We conducted a systematic review in Medline, LILACS, SciELO, Google Scholar, Embase and Trials.gov with articles published until February/2013. Were included: cohort, clinical trials and case-control. Performed in humans, aged ≥ 18 years with smoking as a risk factor for progression of CKD. We excluded studies that reported no smoking and CKD in the title or had proposed to reduce smoking. Results: Among 94 citations, 12 articles were selected. Of these, six were multicenter conducted in developed countries, four were randomized. Males predominated 51-76%. There was associated with smoking progression in 11 studies. It was found that the consumption ≥ 15 packs/ year increases the risk of progression of CKD. Conclusion: Smoking is a risk factor for progression of CKD. .

Regulation of lovastatin on a key inflammation-related microRNA in myocardial cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Advances in the understanding of cardiovascular pathogenesis have highlighted that inflammation plays a central role in atherosclerotic coronary heart disease. Therefore, exploring pharmacologically based anti-inflammatory treatments to be used in cardiovascular therapeutics is worthwhile to promote the discovery of novel ways of treating cardiovascular disorders.</p><p><b>METHODS</b>The myocardial cell line H9c2(2-1) was exposed to lipopolysaccharide (LPS) in culture and resulted in a cellular pro-inflammation status. miR-21 microRNA levels were detected using quantitative real-time polymerase chain reaction (Q-RT-PCR). The influence of lovastatin on miR-21 under normal and pro-inflammatory conditions was tested after being added to the cell culture mixture for 24 hours. Conditional gene function of two predicted cardiovascular system relevant downstream targets of miR-21, protein phosphatase 1 regulatory subunit 3A (PPP1R3A) and signal transducer and activator of transcription 3 (STAT3), were analyzed with immunoblotting.</p><p><b>RESULTS</b>Forty-eight hours of LPS treatment significantly increased the miR-21 to 170.71%± 34.32% of control levels (P = 0.002). Co-treatment with lovastatin for 24 hours before harvesting attenuated the up-regulation of miR-21 (P = 0.013). Twenty-four hours of lovastatin exposure up-regulated PPP1R3A to 143.85%± 21.89% of control levels in cardiomyocytes (P = 0.023). Lovastatin up-regulated the phosphorylation level of STAT3 compared to the background LPS pretreatment (P = 0.0077), this effect was significantly (P = 0.018) blunted when miR-21 was functionally inhibited.</p><p><b>CONCLUSIONS</b>miR-21 plays a major role in the regulation of the cellular anti-inflammation effects of lovastatin.</p>

In Vivo Effects of Adipose-Derived Stem Cells in Inducing Neuronal Regeneration in Sprague-Dawley Rats Undergoing Nerve Defect Bridged with Polycaprolactone Nanotubes

There have been many attempts for regeneration of peripheral nerve injury. In this study, we examined the in vivo effects of non-differentiated and neuronal differentiated adipose-derived stem cells (ADSCs) in inducing the neuronal regeneration in the Sprague-Dawley (SD) rats undergoing nerve defect bridged with the PCL nanotubes. Then, we performed immunohistochemical and histopathologic examinations, as well as the electromyography, in three groups: the control group (14 sciatic nerves transplanted with the PCL nanotube scaffold), the experimental group I (14 sciatic nerves with the non-differentiated ADSCs at a density of 7x105 cells/0.1 mL) and the experimental group II (14 sciatic nerves with the neuronal differentiated ADSCs at 7x105 cells/0.1 mL). Six weeks postoperatively, the degree of the neuronal induction and that of immunoreactivity to nestin, MAP-2 and GFAP was significantly higher in the experimental group I and II as compared with the control group. In addition, the nerve conduction velocity (NCV) was significantly higher in the experimental group I and II as compared with the control group (P=0.021 and P=0.020, respectively). On the other hand, there was no significant difference in the NCV between the two experimental groups (P>0.05). Thus, our results will contribute to treating patients with peripheral nerve defects using PCL nanotubes with ADSCs.

Protein Phosphatase 2C of Toxoplasma Gondii Interacts with Human SSRP1 and Negatively Regulates Cell Apoptosis / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>The protozoan Toxoplasma gondii expresses large amounts of a 37 kDa Type 2C serine-threonine phosphatase, the so-called TgPP2C which has been suggested to contribute to parasite growth regulation. Ectopic expression in mammalian cells also indicated that the enzyme could regulate growth and survival. In this study, we aimed to investigate the interaction of TgPP2C with human SSRP1 (structure-specific recognition protein 1) and the effects of TgPP2C on cell viability.</p><p><b>METHODS</b>The yeast two hybrid system, His-tag pull-down and co-immunoprecipitation assays were used to confirm the interaction of TgPP2C with SSRP1 and determine the binding domain on SSRP1. The evaluation of cell apoptosis was performed using cleaved caspase-3 antibody and Annexin-V/PI kit combined with flow cytometry.</p><p><b>RESULTS</b>We identified human SSRP1 as an interacting partner of TgPP2C. The C-terminal region of SSRP1 including the amino acids 471 to 538 was specifically mapped as the region responsible for interaction with TgPP2C. The overexpression of TgPP2C down-regulated cell apoptosis and negatively regulated apoptosis induced by DRB, casein kinase II (CKII) inhibitor, through enhanced interaction with SSRP1.</p><p><b>CONCLUSION</b>TgPP2C may be a parasitic factor capable of promoting cell survival through interaction with the host protein SSRP1, thereby creating a favorable environment for parasite growth.</p>

BCL2 antagonist of cell death kinases, phosphatases, and ovarian cancer sensitivity to cisplatin / 부인종양

OBJECTIVE: The BCL2 family proteins are critical mediators of cellular apoptosis and, as such, have been implicated as determinants of cancer cell chemo-sensitivity. Recently, it has been demonstrated that the phosphorylation status of the BCL2 antagonist of cell death (BAD) protein may influence ovarian cancer (OVCA) cell sensitivity to cisplatin. Here, we sought to evaluate how kinase and phosphatase components of the BAD apoptosis pathway influence OVCA chemo-sensitivity. METHODS: Protein levels of cyclin-dependent kinase 1 (CDK1) and protein phosphatase 2C (PP2C) were measured by immunofluorescence in a series of 64 primary advanced-stage serous OVCA patient samples. In parallel, levels of cAMP-dependent protein kinase (PKA), AKT, and PP2C were quantified by Western blot analysis in paired mother/daughter platinum-sensitive/resistant OVCA cell lines (A2008/C13, A2780S/A2780CP, Chi/ChiR). BAD pathway kinase CDK1 was depleted using siRNA transfection, and the influence on BAD phosphorylation and cisplatin-induced apoptosis was evaluated. RESULTS: OVCA patient samples that demonstrated complete responses to primary platinum-based therapy demonstrated 4-fold higher CDK1 (p<0.0001) and 2-fold lower PP2C (p=0.14) protein levels than samples that demonstrated incomplete responses. Protein levels of PP2C were lower in the platinum-resistant versus that shown in the platinum-sensitive OVCA cell line sub-clones. Levels of PKA were higher in all platinum-resistant than in platinum-sensitive OVCA cell line sub-clones. Selective siRNA depletion of CDK1 increased sensitivity to cisplatin-induced apoptosis (p<0.002). CONCLUSION: BAD pathway kinases and phosphatases, including CDK1 and PP2C, are associated with OVCA sensitivity to platinum and may represent therapeutic opportunities to enhance cytotoxic efficacy.

New components of the necroptotic pathway

Programmed necrosis, also known as necroptosis, has recently drawn great attention. As an important cellular regulation mechanism, knowledge of its signaling components is expanding. Necroptosisis demonstrated to be regulated by the RIP1 and RIP3 kinases, and its pathophysiological importance has been confirmed in a number of disease models. Here we review the new members of this necroptosis pathway, MLKL, PGAM5, Drp1 and DAI, and discuss some of their possible applications according to recent findings.

Differential gene expression pattern in brains of acrylamide-administered mice / 대한수의학회지

The present study was performed to evaluate the relationship between the neurotoxicity of acrylamide and the differential gene expression pattern in mice. Both locomotor test and rota-rod test showed that the group treated with higher than 30 mg/kg/day of acrylamide caused impaired motor activity in mice. Based on cDNA microarray analysis of mouse brain, myelin basic protein gene, kinesin family member 5B gene, and fibroblast growth factor (FGF) 1 and its receptor genes were down-regulated by acrylamide. The genes are known to be essential for neurofilament synthesis, axonal transport, and neuro-protection, respectively. Interestingly, both FGF 1 and its receptor genes were down-regulated. Genes involved in nucleic acid binding such as AU RNA binding protein/enoyl-coA hydratase, translation initiation factor (TIF) 2 alpha kinase 4, activating transcription factor 2, and U2AF 1 related sequence 1 genes were down-regulated. More interesting finding was that genes of both catalytic and regulatory subunit of protein phosphatases which are important for signal transduction pathways were down-regulated. Here, we propose that acrylamide induces neurotoxicity by regulation of genes associated with neurofilament synthesis, axonal transport, neuro-protection, and signal transduction pathways.

AG490 inhibits the proliferation of K562 and down-regulates protein phosphatase PHLPP expression / 中国实验血液学杂志

This study was aimed to investigate the effect of AG490, a JAK2 inhibitor, on expression of PHLPP and p-Akt in K562. K562 cells were treated with different concentrations of AG490. The proliferation of K562 cells was examined by WST-1 assay and apoptosis of K562 cells was detected by flow cytometry with Annexin V-FITC/PI double staining. The expressions of PHLPP, phosphorate-Akt (p-Akt) and total Akt protein were detected by Western blot. The results indicated that AG490 inhibited the proliferation of K562 cells in concentration-and time-dependent manners, with the IC(50) 338.0 µmol/L in 48 h. AG490 100 µmol/L also induced apoptosis of K562 cells in a time-dependent manner. AG490 100 µmol/L time-dependently down-regulated the protein expression of p-Akt and PHLPP, but without significant effect on expression of total Akt. It is concluded that AG490 can inhibit proliferation and induce apoptosis of K562 cells through down-regulation of p-Akt expression, but inhibiting efficacy of AG490 on K562 proliferation also may be limited due to the down-regulation of p-Akt regulatory protein PHLPP expression.