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Leptin actions at the pituitary level have been extensively investigated in mammalian species, but remain insufficiently characterized in lower vertebrates, especially in teleost fish. Prolactin (PRL) is a pituitary hormone of central importance to osmoregulation in fish. Using goldfish as a model, we examined the global and brain-pituitary distribution of a leptin receptor (lepR) and examined the relationship between expression of lepR and major pituitary hormones in different pituitary regions. The effects of recombinant goldfish leptin-AI and leptin-AII on PRL mRNA expression in the pituitary were further analysed, and the mechanisms underlying signal transduction for leptin-induced PRL expression were determined by pharmacological approaches. Our results showed that goldfish lepR is abundantly expressed in the brain-pituitary regions, with highly overlapping PRL transcripts within the pituitary. Recombinant goldfish leptin-AI and leptin-AII proteins could stimulate PRL mRNA expression in dose- and time-dependent manners in the goldfish pituitary, by both intraperitoneal injection and primary cell incubation approaches. Moreover, the PI3K/Akt/mTOR, MKK3/6/p38MAPK, and MEK1/2/ERK1/2--but not JAK2/STAT 1, 3 and 5 cascades--were involved in leptin-induced PRL mRNA expression in the goldfish pituitary. [ABSTRACT FROM AUTHOR]

Nlrp2 is a maternal effect gene specifically expressed by mouse ovaries; deletion of this gene from zygotes is known to result in early embryonic arrest. In the present study, we identified FAF1 protein as a specific binding partner of the NLRP2 protein in both mouse oocytes and preimplantation embryos. In addition to early embryos, both Faf1 mRNA and protein were detected in multiple tissues. NLRP2 and FAF1 proteins were co-localized to both the cytoplasm and nucleus during the development of oocytes and preimplantation embryos. Co-immunoprecipitation assays were used to confirm the specific interaction between NLRP2 and FAF1 proteins. Knockdown of the Nlrp2 or Faf1 gene in zygotes interfered with the formation of a NLRP2-FAF1 complex and led to developmental arrest during early embryogenesis. We therefore conclude that NLRP2 interacts with FAF1 under normal physiological conditions and that this interaction is probably essential for the successful development of cleavage-stage mouse embryos. Our data therefore indicated a potential role for NLRP2 in regulating early embryo development in the mouse. [ABSTRACT FROM AUTHOR]

Objective: The purpose of this study was to evaluate the frequency of RASSF1A hypermethylation in patients with acute myeloid leukemia (AML), in an attempt to modify the current molecular model for disease prognosis. Materials and methods: Aberrant RASSF1A promoter methylation levels were assessed in 226 newly diagnosed non-M3 AML patients and 30 apparently healthy controls, by quantitative methylation-specific polymerase chain reaction. Meanwhile, RASSF1A mRNA levels were detected by real-time quantitative polymerase chain reaction. Furthermore, hematological characteristics, cytogenetic abnormalities, and genetic aberrations were assessed. Finally, associations of RASSF1A hypermethylation with clinical outcomes were evaluated. Results: RASSF1A hypermethylation was observed in 23.0% of patients with non-M3 AML (52/226), but not in controls. Meanwhile, hypermethylation of the RASSF1A promoter was significantly associated with ASXL1 mutation. Furthermore, the log-rank test revealed that RASSF1A hypermethylation indicated decreased relapse-free survival (RFS) and overall survival (OS) in patients with non-M3 AML (P=0.012 and P=0.014, respectively). In multivariate analysis, RASSF1A hypermethylation was an independent prognostic factor for RFS (P=0.040), but not for OS (P=0.060). Conclusion: Hypermethylation of the RASSF1A promoter is associated with ASXL1 mutation in non-M3 AML patients, likely indicating poor outcome. These findings provide a molecular basis for stratified diagnosis and prognostic evaluation. [ABSTRACT FROM AUTHOR]

Oligomers of homomeric voltage-gated potassium channels associate early in biogenesis as the nascent proteins emerge from the polysome. Less is known about howproteins emerging fromdifferent polysomes associate to form hetero-oligomeric channels. Here, we report that alternate mRNA transcripts encoding human ether-à-go-go-related gene (hERG) 1a and 1b subunits, which assemble to produce ion channels mediating cardiac repolarization, are physically associated during translation. We show that shRNA specifically targeting either hERG 1a or 1b transcripts reduced levels of both transcripts, but only when they were coexpressed heterologously. Both transcripts could be copurified with an Ab against the nascent hERG 1a N terminus. This interaction occurred even when translation of 1b was prevented, indicating the transcripts associate independent of their encoded proteins. The association was also demonstrated in cardiomyocytes, where levels of both hERG transcripts were reduced by either 1a or 1b shRNA, but native KCNE1 and ryanodine receptor 2 (RYR2) transcripts were unaffected. Changes in protein levels and membrane currents mirrored changes in transcript levels, indicating the targeted transcripts were undergoing translation. The physical association of transcripts encoding different subunits provides the spatial proximity required for nascent proteins to interact during biogenesis, and may represent a general mechanism facilitating assembly of heteromeric protein complexes involved in a range of biological processes. [ABSTRACT FROM AUTHOR]

This study aimed to explore whether antenatal supplement of taurine can improve the brain development of fetal rats with intrauterine growth restriction (IUGR) via protein kinase A (PKA)-Ca2+/calmodulin-dependent protein kinase II (CaMKII)/c-fos pathway. A total of 30 pregnant rats were randomly divided into the following three groups: control, IUGR, and IUGR with antenatal taurine supplementation groups. The expression of PKA, CaMKII, and c-fos mRNA and proteins in fetal rat brain tissues were detected by quantitative real time (qRT)-polymerase chain reaction (PCR) and Western blot, respectively; and the number of PKA-, CaMKII- and c-fos-positive cells was measured by immunohistochemistry. Western blot and qRT-PCR revealed that antenatal taurine supplementation significantly increased PKA, CaMKII, and c-fos protein and mRNA levels in fetal rats brain with IUGR (p < 0.05). Immunohistochemistry results showed that the numbers of PKA-, CaMKII-, and c-fos-positive cells were significantly decreased in the IUGR groups compared with the controls; while antenatal taurine could significantly increase the positive cell numbers (p < 0.05). So, we conclude that antenatal taurine improves IUGR fetal brain development which is associated with increasing the activity of PKA-CaMKII/c-fos signal pathway. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) infection is one of the major causes of hepatocellular carcinoma (HCC). Kinesin family member 4A (KIF4A) is a microtubule-based motor protein, which is upregulated in cervical and lung cancer. However, the expression of KIF4A in HBV-associated HCC, and the effect of HBV on the expression of KIF4A remain to be elucidated. In the present study, the expression profiles of KIF4A were examined in cancerous tissues and paracancerous tissues from patients with HCC, who presented with histories of chronic HBV infection, and the role of HBV in the induction of the expression of KIF4A was investigated. HepG2 cells were transfected with the pHBV1.3, HBV infectious clone and a construct, which contained the luciferase gene under the control of the KIF4A gene promoter. The results demonstrated that the expression of KIF4A was significantly higher in the HCC tissues than in the paracancerous tissues. HBV activated the KIF4A gene promoter and upregulated the mRNA and protein expression of KIF4A. Furthermore, activation of the gene expression of KIF4A increased in a pHBV1.3 concentration-dependent manner. These results provide novel insights into the understanding of HCC oncogenesis caused by HBV. [ABSTRACT FROM AUTHOR]

Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related mortality despite the fact that great advances have been made in therapeutic treatment methods. Therefore, in the present study, the role of adenovirus- mediated retinoblastoma 94 (Ad-RB94) gene therapy in NSCLC was investigated. Following treatment with Ad-RB94, the proportion of A549 cells in the G2/M phase was increased. In the mouse xenograft model, the overexpression of RB94 inhibited the tumor growth compared with the control group and the Ad-LacZ- treated group. In the transplanted tumors, the overexpression of RB94 induced the apoptosis of tumors as well as an increase in the mRNA levels of cyclinB1. In conclusion, the results of the present study suggested that RB94 may effectively inhibit NSCLC tumor cell growth by inducing G2/M cell cycle arrest and apoptosis, indicating that RB94 may be a promising candidate for adjuvant therapy with radiation or chemotherapy in NSCLC. [ABSTRACT FROM AUTHOR]

Pholiota adiposa is a kind of edible mushroom which has long been known for its health care applications. To reveal the exact mechanism of its protective functions in humans, in this study we isolated and identified the active compound PB3 of P. adiposa for the first time by a combination of chromatography techniques, including NKA macroporous resin and Sephadex G-15. PB3, with molecular mass of 267.2 Da and molecular formula of C10H13N5O4 discovered by mass spectrum (MS) was identified to be adenosine. Mice were injected intraperitoneally with purified fraction PB3. Seven days after injection, we found a 1.5-fold increase of IL10 at the mRNA level, while a down regulated expression of IL-2, IL-6 and IFN-γ to 49.0%, 56.9% and 73.4%, respectively, was detected in spleen by real-time quantitative PCR. What's more, SOD expression level was significantly increased by 1.6-fold compared to control. Fraction PB3 displayed anti-inflammatory potency and heightened SOD activity on the transcriptional level, which could be considered of further pharmaceutical or medication value. [ABSTRACT FROM AUTHOR]

Objectives: Endothelial progenitor cells (EPCs) can be used to repair tissues after myocardial infarction (MI) but EPC activators have adverse reactions. Rehmannia glutinosa is a herb used in traditional Chinese medicine, which can promote bone-marrow proliferation and protect the ischemic myocardium. We investigated the effects of Rehmannia glutinosa extract (RGE) on EPCs in a rat model of MI. Methods: A total of 120 male Wistar rats were randomized to 2 groups (n = 60 each) for treatment: high-dose RGE (1.5 g⋅kg-1⋅day-1 orally) for 8 weeks, then left anterior descending coronary artery ligation, mock surgery or no treatment, then RGE orally for 4 weeks; or normal saline (NS) as the above protocol. The infarct region of the left ventricle was assessed by serial sectioning and morphology. EPCs were evaluated by number and function. Protein and mRNA levels of CD133, vascular endothelial growth factor receptor 2 (VEGFR2), chemokine C-X-C motif receptor 4 (CXCR4), stromal cell-derived factor-1α (SDF-1α) were measured by immunohistochemistry, Western blot and quantitative PCR analysis. Results:RGE significantly improved left ventricular function, decreased the ischemic area and the apoptotic index in the infarct myocardium, also decreased the concentration of serum cardiac troponin T and brain natriuretic peptide at the chronic stage after MI (from week 2 to week 4). RGE increased EPC number, proliferation, migration and tube-formation capacity. It was able to up-regulate the expression of angiogenesis-associated ligand/receptor, including CD133, VEGFR2 and SDF-1α/CXCR4. In vitro, the effect of RGE on SDF-1a/CXCR4 cascade was reversed by the CXCR4 specific antagonist AMD3100. Conclusion: RGE may enhance the mobilization, migration and therapeutic angiogenesis of EPCs after MI by activating the SDF-1α/CXCR4 cascade. [ABSTRACT FROM AUTHOR]

A lactose-specific lectin (named Arachis hypogaea L Lectin (AHL)) was purified from the seed of the peanut Arachis hypogaea L. by single-step affinity chromatography using lactose-coupled agarose. AHL showed strong hemagglutinating activity to human A/B/O erythrocytes (RBC) treated with neuraminidase. The results of matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry (MALDI-TOF-TOF MS) analysis indicated that the protein AHL shared high homology with the peanut lectin chain A protein (gi|1942899). The AHL has binding activity to some Gram-negative bacteria and fungi spores which caused disease in peanut. Transcripts of AHL were detected in different peanut tissues, and the expression profile of the AHL gene changed in different parts of the plants. The variation in the mRNA transcript level in seeds was quantitated by real-time polymerase chain reaction during different growth periods. The expression profile of the AHL gene changed greatly in samples collected in different days after pegging. We explored the variation in AHL mRNA expression in six cultivars of peanut; the expression level observed in Lu Hua 12 was the highest. The data we described can provide us with clues to know more properties about peanut seed lectins at the biology chemistry and molecular biology level. [ABSTRACT FROM AUTHOR]

Purpose: To explore the effects of Icaritin on chronic myeloid leukemia (CML) cells and underlying mechanisms. Method: CML cells were incubated with various concentration of Icaritin for 48 hours, the cell proliferation was analyzed by MTT and the apoptosis was assessed with Annexin V and Hoechst 33258 staining. Cell hemoglobinization was determined. Western blotting was used to evaluate the expressions of MAPK/ERK/JNK signal pathway and Jak-2/Phorpho-Stat3/Phorsph- Akt network-related protein. NOD-SCID nude mice were applied to demonstrate the anti-leukemia effect of Icaritin in vivo. Results: Icaritin potently inhibited proliferation of K562 cells (IC50 was 8 &mgr;M) and primary CML cells (IC50 was 13.4 &mgr;M for CML-CP and 18 &mgr;M for CML-BC), induced CML cells apoptosis and promoted the erythroid differentiation of K562 cells with time-dependent manner. Furthermore, Icaritin was able to suppress the growth of primary CD34+ leukemia cells (CML) and Imatinib-resistant cells, and to induce apoptosis. In mouse leukemia model, Icaritin could prolong lifespan of NOD-SCID nude mice inoculated with K562 cells as effective as Imatinib without suppression of bone marrow. Icaritin could upregulate phospho-JNK or phospho-C-Jun and down-regulate phospho-ERK, phospho-P-38, Jak-2, phospho-Stat3 and phospho-Akt expression with dose- or time-dependent manner. Icaritin had no influence both on c-Abl and phospho-c-Abl protein expression and mRNA levels of Bcr/Abl. [ABSTRACT FROM AUTHOR]

Bacillus amyloliquefaciens C06, a potential agent in biological preservation of post-harvest fruit, was found to secrete extra-cellular γ-polyglutamic acid (γ-PGA) in liquid culture. In this work, M306, a transposon mutant of B. amyloliquefaciens C06, defective in forming structured colony and displaying enhanced ability of producing γ-PGA, was obtained. Inverse PCR and quantitative reverse transcription PCR (qRT-PCR) analysis demonstrated that the defective phenotype in M306 was associated with an ORF showing high similarity to RBAM_034550 from B. amyloliquefaciens FZB42. In this paper, the ORF was designated pbrA, standing for γ-PGA production and biofilm formation regulatory factor. qRT-PCR analysis also indicated that pbrA down-regulated mRNA expression of epsD and yqxM, the crucial genes involved in biofilm formation, but affected little on expression of ywtB, the gene directing γ-PGA synthesis. Evaluations in γ-PGA productivity of wild-type C06 and its mutants C06Δ epsA and C06Δ tasA, respectively, deficient in producing exopolysaccharides (EPS) and TasA, revealed that γ-PGA overproduction in M306 was probably due to the redistributed metabolic flux caused by defective production of EPS. [ABSTRACT FROM AUTHOR]

The aim of the study is to investigate the cholinergic deficit in Alzheimer's disease (AD) and identify candidate blood biomarkers for the diagnosis of the disease. Twenty-nine elderly Chinese diagnosed with AD and 33 age-matched controls were selected. The activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in plasma were detected by a spectrophotometric method, and the mRNA levels of α4 and β2 nicotinic acetylcholine receptor (nAChR) subunits in blood leukocytes were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that AChE activity in plasma was significantly lower in the AD group than in normal controls, while BuChE activity did not show any differences between AD and controls; mRNA levels of both α4 and β2 nAChR subunits in blood leukocytes were significantly lower in the AD group than in controls. The AChE activity and the mRNA levels of α4 and β2 nAChR subunits in the AD patients were also significantly correlated with cognitive test scores. No differences of AChE in plasma or α4 and β2 nAChR subunits in blood leukocytes were detected between smoking and non-smoking subjects. The results indicated that the decreases in the activity of AChE and in the mRNA levels of nAChR α4 and β2 subunits from the peripheral blood of patients with AD might serve as supplementary indicators for the clinical diagnosis of AD. [ABSTRACT FROM AUTHOR]

Messenger RNA (mRNA) turnover in eukaryotic cells begins with shortening of the poly (A) tail at the 3′ end, a process called deadenylation. In yeast, the deadenylation reaction is predominantly mediated by CCR4 and CCR4-associated factor 1 (CAF1), two components of the well-characterised protein complex named CCR4-NOT. We report here that AtCAF1a and AtCAF1b, putative Arabidopsis homologs of the yeast CAF1 gene, partially complement the growth defect of the yeast caf1 mutant in the presence of caffeine or at high temperatures. The expression of AtCAF1a and AtCAF1b is induced by multiple stress-related hormones and stimuli. Both AtCAF1a and AtCAF1b show deadenylation activity in vitro and point mutations in the predicted active sites disrupt this activity. T-DNA insertion mutants disrupting the expression of AtCAF1a and/or AtCAF1b are defective in deadenylation of stress-related mRNAs, indicating that the two AtCAF1 proteins are involved in regulated mRNA deadenylation in vivo. Interestingly, the single and double mutants of AtCAF1a and AtCAF1b show reduced expression of pathogenesis-related (PR) genes PR1 and PR2 and are more susceptible to Pseudomonas syringae pv tomato DC3000 (Pst DC3000) infection, whereas transgenic plants over-expressing AtCAF1a show elevated expression of PR1 and PR2 and increased resistance to the same pathogen. Our results suggest roles of the AtCAF1 proteins in regulated mRNA deadenylation and defence responses to pathogen infections.Cell Research (2009) 19:307–316. doi: 10.1038/cr.2008.317; published online 9 December 2008 [ABSTRACT FROM AUTHOR]

1. Paeoniflorin is one of the main effective components of the total glucosides of paeony (TGP) extracted from the root of Paeonia lactiflora which has been used for gynaecological problems and for cramp, pain and giddiness for over 1 500 years in Chinese medicine. Anti-inflammatory, antioxidative, antihepatic injury and immunoregulatory activities of TGP have been extensively proved in our laboratory for many years. Our present study investigates the effects and mechanisms of paeoniflorin on immunological liver injury in mice. 2. A model of immunological liver injury was induced by tail vein injection of bacillus Calmette-Guérin (BCG) and lipopolysaccharide (LPS) in mice. Activities of serum alanine aminotransferase (ALT) were measured by biochemical methods. Hepatic tissue sections were stained with haematoxylin and eosin and examined under a light microscope. Tumor necrosis factor (TNF)-a, interleukin (IL)-6, lipopolysaccharide binding protein (LBP) and CD14 mRNA (messenger ribonucleic acid) expression in mouse liver were determined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. 3. Immunological liver injury induced by BCG plus LPS was successfully duplicated. Serum ALT activities were significantly decreased by paeoniflorin. (25, 50, 100 mg/kg). Histological examination demonstrated that paeoniflorin could attenuate the area and extent of necrosis and reduce the immigration of inflammatory cells. The increase in TNF-a, LBP and CD14 mRNA expression in mouse liver after BCG and LPS injection was significantly decreased by paeoniflorin (100 mg/kg) and was changed by paeoniflorin (25, 50 mg/kg) at different time-point. The augmentation of IL-6 mRNA in mouse liver was markedly increased by paeoniflorin at 1 h and 3 h after LPS injection. 4. Paeoniflorin could significantly protect against immunological liver injury in mice. TNF-a, IL-6, LBP and CD14 mRNA expression in mouse liver may be involved in BCG plus LPS induced liver injury. The protective mechanism of paeoniflorin might be partially related to modulation of TNF-a, IL-6, LBP and CD14 mRNA expressions in mouse liver. [ABSTRACT FROM AUTHOR]

The Ski protein has been proposed to serve as a corepressor for Smad4 to maintain a transforming growth factor-β (TGF-β)-responsive promoter at a repressed, basal level. However, there have been no reports so far that it indeed acts on a natural promoter. We have previously cloned the human Smad7 promoter and shown that it contains the 8-base pair palindromic Smad-binding element (SBE) necessary for TGF-β induction. In this report, we have characterized the negative regulation of Smad7 promoter basal activity by Ski. We show that Ski inhibits the Smad7 promoter basal activity in a SBE-de- pendent manner. Mutation of the SBE abrogates the inhibitory effect of Ski on the Smad7 promoter. Moreover, mutation of the SBE increases the Smad7 promoter basal activity. Using the chromatin immunoprecipitation assay, we further show that Ski together with Smad4 binds to the endogenous Smad7 promoter. Finally, we show that RNAi knockdown of Ski increases Smad7 reporter gene activity in transient transfection assays as well as elevating the endogenous level of Smad7 mRNA. Taken together, our results provide the first evidence that Ski is indeed a corepressor for Smad4, which can inhibit a natural TGF-β responsive gene at the basal state. [ABSTRACT FROM AUTHOR]

Background: A limiting factor of cDNA microarray technology is the need for a substantial amount of RNA per labeling reaction. Thus, 20-200 micro-grams total RNA or 0.5-2 micro-grams poly (A) RNA is typically required for monitoring gene expression. In addition, gene expression profiles from large, heterogeneous cell populations provide complex patterns from which biological data for the target cells may be difficult to extract. In this study, we chose to investigate a widely used mRNA amplification protocol that allows gene expression studies to be performed on samples with limited starting material. We present a quantitative study of the variation and noise present in our data set obtained from experiments with either amplified or non-amplified material. Results: Using analysis of variance (ANOVA) and multiple hypothesis testing, we estimated the impact of amplification on the preservation of gene expression ratios. Both methods showed that the gene expression ratios were not completely preserved between amplified and non-amplified material. We also compared the expression ratios between the two cell lines for the amplified material with expression ratios between the two cell lines for the non-amplified material for each gene. With the aid of multiple t-testing with a false discovery rate of 5%, we found that 10% of the genes investigated showed significantly different expression ratios. Conclusion: Although the ratios were not fully preserved, amplification may prove to be extremely useful with respect to characterizing low expressing genes. [ABSTRACT FROM AUTHOR]

γ-Aminobutyric acid (GABA), one of the most important neurotransmitters in the brain, is also found in the periphery. GABA[sub A] receptors are chloride channels opened by GABA whose presence in the rat superior cervical ganglion has been indicated by functional and binding measurements. We describe the first molecular data on the possible subunit composition of these receptors, detecting mRNAs for 12 subunits (α1–5, β1–3, γ1–3, δ) by reverse-transcriptase polymerase chain reaction. Preliminary quantitation gave highest levels (in descending order) for the γ2 (both short and long forms), β3, γ3, and α1 subunits. [ABSTRACT FROM AUTHOR]