ANKRD49 promotes the invasion and metastasis of lung adenocarcinoma via a P38/ATF-2 signalling pathway.

Lung adenocarcinoma (LUAD) is the most challenging neoplasm to treat in clinical practice. Ankyrin repeat domain 49 protein (ANKRD49) is highly expressed in several carcinomas; however, its pattern of expression and role in LUAD are not known. Tissue microarrays, immunohistochemistry, χ2 test, Spearman correlation analysis, Kaplan-Meier, log-rank test, and Cox's proportional hazard model were used to analyse the clinical cases. The effect of ANKRD49 on the LUAD was investigated using CCK-8, clonal formation, would healing, transwell assays, and nude mice experiment. Expressions of ANKRD49 and its associated downstream protein molecules were verified by real-time PCR, Western blot, immunohistochemistry, and/or immunofluorescence analyses. ANKRD49 expression was highly elevated in LUAD. The survival rate and Cox's modelling analysis indicated that there may be an independent prognostic indicator for LUAD patients. We also found that ANKRD49 promoted the invasion and migration in both in in vitro and in vivo assays, through upregulating matrix metalloproteinase (MMP)-2 and MMP-9 activities via the P38/ATF-2 signalling pathway Our findings suggest that ANKRD49 is a latent biomarker for evaluating LUAD prognosis and promotes the metastasis of A549 cells via upregulation of MMP-2 and MMP-9 in a P38/ATF-2 pathway-dependent manner.

A Microarray Study on the Expression of ANKRD49 in Lung Squamous Cell Carcinoma and Its Clinicopathologic Significance.

Lung squamous cell carcinoma (LUSC) is associated with poor clinical outcomes and identifying novel biomarkers that are involved in the progression of LUSC is important for prognosis and targeted treatment. Herein, ankyrin repeat domain 49 (ANKRD49) protein in LUSC versus paired noncancerous lung tissues was tested and its clinical significance was evaluated through χ 2 test, log-rank test, and Cox proportional hazards model. The results showed the ANKRD49 protein in LUSC was elevated and correlated with the tumor-node-metastasis stage, lymph node metastasis, distal metastasis, and differentiation. Patients with higher ANKRD49 had lower overall survival rate and higher ANKRD49 expression in lung tissues may be used as an independent prognostic marker for LUSC patients.

A sensitive fluorescence biosensor based on metal ion-mediated DNAzyme activity for amplified detection of acetylcholinesterase.

In this paper, we developed an amplified fluorescence biosensor for acetylcholinesterase (AChE) activity detection by taking advantage of the mercury ion-mediated Mgzyme (Mg2+-dependent DNAzyme) activity. The catalytic activity of Mgzyme can be inhibited by the formation of T-Hg2+-T base pairs between the Mgzyme and mercury ions. Therefore, the Mgzyme-Hg2+ complex has no activity on a molecular beacon (MB) substrate, which afforded a very weak fluorescence background for this biosensor. After the addition of acetylcholinesterase (AChE), the substrate acetylthiocholine could be hydrolyzed to thiocholine, which has a stronger binding power with mercury ions than T-Hg2+-T base pairs. Therefore, the Mgzyme activity was recovered. The activated Mgzyme could hybridize with the MB substrate and undergo many cleavage cycles, resulting in a significant increase of fluorescence intensity. This biosensor displayed high sensitivity with the detection limit as low as 0.01 mU mL-1. Moreover, this design did not require complex composition and sequence design; thus it is simple and convenient. This biosensor was also applied for the determination of AChE in human blood and showed satisfactory results.

ANKRD49 inhibits etoposide-induced intrinsic apoptosis of GC-1 cells by modulating NF-κB signaling.

Spermatogenesis is a complicated process that is tightly regulated by the well-coordinated expression of a series of genes in the testes. Ankyrin repeat domain-containing protein 49 (ANKRD49), an evolutionarily conserved protein highly expressed in the testes, is mainly found in spermatogonia, spermatocytes, and round spermatids. However, the exact function of ANKRD49 in spermatogenesis has remained elusive. In this study, we sought to investigate the role of ANKRD49 in apoptosis and determine the mechanism underlying this process in male germ cell-derived GC-1 cells. Nuclear staining with Hoechst 33258 and annexin V-FITC/PI, as well as analysis of caspase 3 activity, mitochondrial membrane potential, and apoptotic protein expression, showed that etoposide-induced apoptosis was attenuated by ANKRD49 overexpression but promoted by RNA interference-induced ANKRD49 knockdown. Furthermore, assessment of the levels of caspase 9, caspase 8, and proteins of the Bcl-2 family revealed ANKRD49 to be involved in an intrinsic apoptosis pathway. Examination of the subcellular distribution of the NF-κB p65 subunit after treatment with an NF-κB signaling inhibitor or p65 small interfering RNA demonstrated that ANKRD49 modulated etoposide-induced GC-1 cell apoptosis via the NF-κB pathway. Taken together, these results suggest that ANKRD49 plays an important role in reducing intrinsic apoptosis of GC-1 cells by modulating the NF-κB signaling pathway.

Construction of a cDNA library and preliminary analysis of the expressed sequence tags of the earthworm Eisenia fetida (Savigny, 1826).

Earthworms are useful indicator organisms of soil health and Eisenia fetida have been extensively used as test organisms in ecotoxicological studies. In order to gain insight into the gene expression profiles associated with physiological functions of earthworms, a full­length enriched cDNA library of the Eisenia fetida genome was successfully constructed using Switching Mechanism at 5'End of RNA Template technology. Construction of a cDNA library and analysis of Expressed Sequence Tags (ESTs) are efficient approaches for collecting genomic information and identifying genes important for a given biological process. Furthermore, analysis of the expression abundance of ESTs was performed with the aim of providing genetic and transcriptomic information on the development and regenerative process of earthworms. Phrep and Crossmatch were used to process EST data and a total of 1,140 high­quality EST sequences were determined by sequencing random cDNA clones from the library. Clustering analysis of sequences revealed a total of 593 unique sequences including 225 contiguous and 368 singleton sequences. Basic Local Alignment Search Tool analysis against the Kyoto Encyclopedia of Genes and Genomes database resulted in 593 significant hits (P­value <1x10­8), of which 168 were annotated through Gene Ontology analysis. The STRING database was used to determine relationships among the 168 ESTs, identifying associated genes involved in protein­protein interactions and gene expression regulation. Based on nucleic acid and protein sequence homology, the mutual relationships between 287 genes could be obtained, which identified a portion of the ESTs as known genes. The present study reports on the construction of a high­quality cDNA library representative of adult earthworms, on a preliminary analysis of ESTs and on a putative functional analysis of ESTs. The present study is expected to enhance our understanding of the molecular basis underlying the biological development of earthworms.

Silver-ion-mediated Mg2+-dependent DNAzyme activity for amplified fluorescence detection of cysteine.

In this paper, by taking advantage of the fact that silver ions could mediate the Mg2+-dependent DNAzyme (Mgzyme) activity, we for the first time developed a turn-on fluorescent biosensor for amplified cysteine (Cys) detection. Because Mgzyme can interact with the silver ion and form cytosine-Ag+-cytosine (C-Ag+-C) base pairs, the conformation of its catalytic core was changed. As a result, the catalytic activity of Mgzyme was suppressed and the Mgzyme-Ag+ complex could not initiate the cleavage reaction. Therefore, the background fluorescence of the biosensor was very low. In the presence of Cys, Cys can bind tightly to the silver ion and disrupt the C-Ag+-C base pairs in the Mgzyme-Ag+ complex, leading to the restoration of Mgzyme activity. The activated Mgzyme could hybridize with the MB substrate and undergo many cleavage cycles, resulting in a significant increase of fluorescence intensity. This designed strategy provided amplified fluorescence detection of cysteine, with a detection limit of 2 nM. Moreover, the strong binding between Cys and Ag+ ensured that the biosensor had a desirable selectivity for Cys. This sensing system was also used to detect Cys in human urine samples and displayed satisfying results.

Recombinant club cell protein 16 (CC16) ameliorates cigarette smoke­induced lung inflammation in a murine disease model of COPD.

Club cell protein (CC16) is expressed primarily by club cells possesses anti­inflammatory properties and is located in the bronchiolar epithelium. Previous studies have demonstrated that CC16 deficiency is associated with the progression of chronic obstructive pulmonary disease (COPD). In the present study, the therapeutic effects of recombinant rat CC16 protein in mice with COPD were examined and the underlying mechanisms investigated. A total of 30 adult male C57/BL6 mice were randomly divided into three groups (10 mice/group). A mouse COPD model was generated by exposing 20 mice to cigarette smoke (CS) for 24 weeks. A total of 10 mice were treated intranasally with rCC16 (2.5 µg/g body weight) and control mice were exposed to normal room air. Results indicated that rCC16 treatment ameliorated pathological damage in the lungs and reduced the production of tumor necrosis factor (TNF)­α, interleukin (IL)­6 and IL­8, which were induced by CS exposure. After rCC16 administration, endogenous CC16 was upregulated and the body weight of COPD mice was increased, whereas the opposite was observed in CS­exposed mice. Additionally, rCC16 treatment inhibited the DNA binding of NF­κB/p65 in lung tissues and reduced nuclear translocation of NF­κB/p65 in BALF and epithelial cells. Moreover, rCC16 treatment lead to a decrease in the total number of BALF cells, including macrophages, which was elevated in COPD mice. In conclusion, the present results demonstrate that rCC16 has therapeutic effects on COPD by downregulating pro­inflammatory factors via the NF­κB pathway.

Significance of 6-minute walk test for patients with heart failure / 心血管康复医学杂志

The present article addressed method, indications, contraindications of 6 min walk test (6MWT), and its correlation with plasma level of brain natriuretic peptide (BNP) and NYHA class. 6MWT can be used for rehabilitation guidance, improve exercise tolerance and assess their cardiac function and prognosis, etc. in patients with heart failure.

HSVtk/GCV system on hepatoma carcinoma cells: Construction of the plasmid pcDNA3.1­pAFP-TK and targeted killing effect.

Previous studies demonstrated that herpes simplex virus thymidine kinase (HSVtk) could phosphorylate non­toxic gancyclovir (GCV) efficiently to produce phosphorylated products that result in cell apoptosis, to kill tumor cells. The present study aimed to construct a plasmid vector, pcDNA3.1­pAFP­TK, carrying the suicide gene driven by the alpha­fetoprotein (AFP) promoter, to investigate the cytotoxicity of HSVtk/GCV suicide gene system on hepatoma carcinoma cells. Reverse transcription­polymerase chain reaction and western blotting results demonstrated that the HSVtk gene was effectively expressed in HepG2 hepatoma carcinoma cells transfected with pcDNA3.1­pAFP­TK plasmid, whereas HSVtk gene expression was not detected in normal HL­7702 liver cells. In addition, MTT assays indicated that cell viability of HepG2 cells with the plasmid pcDNA3.1­pAFP­TK decreased in a dose­dependent manner following treatment with GCV for 48 h. Flow cytometry also revealed that the cell apoptosis rate and mitochondrial membrane potential reduction rate in the HepG2 cells treated with HSVtk/GCV suicide gene system were significantly higher than in the control group. Apoptosis rates in the control group and the pcDNA3.1­pAFP­TK group were (1.00±0.62%) and (38.70±6.03%), respectively. Mitochondrial membrane potential reduction rates in the control group and the pcDNA3.1-pAFP-TK group were (0.57±0.11%) and (22.84±5.79%), respectively. Caspase­3 staining demonstrated that activated caspase­3 increased significantly in the HepG2 cells treated with HSVtk/GCV suicide gene system, whereas in the control group activated caspase­3 increase was not observed. The results of the present study, therefore, indicated that HSVtk suicide gene was obviously expressed in the HepG2 cells and that the HSVtk/GCV system was effective at killing HepG2 hepatoma carcinoma cells.

The role of intestinal endotoxemia in a rat model of aluminum neurotoxicity.

The present study aimed to investigate the effects of intestinal endotoxemia (IETM) in a rat model of aluminum neurotoxicity established by D-galactose and aluminum trichloride (AlCl3). Adult Wistar rats were administered D­galactose and AlCl3 to create the aluminum neurotoxicity model. The learning and memory abilities of the rats were subsequently observed using a Morris water maze test and the serum levels of lipopolysaccharide (LPS), tumor necrosis factor (TNF)­α, interleukin (IL)­1, diamine oxidase (DAO), glutamine (Gln) and glutaminase were measured. The expression of S­100ß in the serum was detected using an enzyme­linked immunosorbent assay. The expression levels of the amyloid ß­protein (Aß) precursor (APP), presenilin 1 (PS1), ß­site APP­cleaving enzyme (BACE), zona occludens protein (ZO)­1 and Aß 1­40 in the brain of rats were detected via reverse­transcription polymerase chain reaction, western blotting and immunohistochemistry. The levels of LPS, TNF­α, IL­1, DAO, Gln and S­100ß in serum and the mRNA and protein expression levels of APP, PS1, BACE and Aß1­40 in the brain were markedly increased in the model rats compared with controls. The level of glutaminase in the serum and the expression of ZO­1 in the brain were decreased in the model rats compared with controls. IETM was present in the rat model of aluminum neurotoxicity established by D­galactose and AlCl3 and may be important in the development of this neurotoxicity.

Label-free LC-MS/MS shotgun proteomics to investigate the anti-inflammatory effect of rCC16.

Clara cell protein (CC16) is an anti-inflammatory protein, which is expressed in the airway epithelium. It is involved in the development of airway inflammatory diseases, including chronic obstructive pulmonary disease and asthma. However, the exact molecular mechanism underlying its anti­inflammatory action remains to be fully elucidated. The aim of the present study was to define the protein profiles of the anti­inflammatory effect of CC16 in lipopolysaccharide (LPS)­treated rat tracheal epithelial (RTE) cells using shotgun proteomics. Protein extracts were obtained from control RTE cells, RTE cells treated with LPS and RTE cells treated with LPS and recombinant CC16 (rCC16). Subsequent label­free quantification and bioinformatics analyses identified 12 proteins that were differentially expressed in the three treatment groups as a cluster of five distinct groups according to their molecular functions. Five of the twelve proteins were revealed to be associated with the cytoskeleton: Matrix metalloproteinase­9, myosin heavy chain 10, actin­related protein­3 homolog, elongation factor 1­α­1 (EF­1­α­1), and acidic ribosomal phosphoprotein P0. Five of the twelve proteins were associated with cellular proliferation: DNA­dependent protein kinase catalytic subunit, EF­1­α­1, tyrosine 3­monooxygenase, caspase recruitment domain (CARD) protein 12 and adenosylhomocysteinase (SAHH) 3. Three proteins were associated with gene regulation: EF­1­α­1, SAHH 3 and acidic ribosomal phosphoprotein P0. Three proteins were associated with inflammation: Tyrosine 3­monooxygenase, CARD protein 12 and statin­related protein. ATPase (H+­transporting, V1 subunit A, isoform 1) was revealed to be associated with energy metabolism, and uridine diphosphate glycosyltransferase 1 family polypeptide A8 with drug metabolism and detoxification. The identified proteins were further validated using reverse transcription­quantitative polymerase chain reaction. These protein profiles, and their interacting protein network, may facilitate the elucidation of the molecular mechanisms underlying the anti­inflammatory effects of CC16.

The Ankyrin Repeat Domain 49 (ANKRD49) Augments Autophagy of Serum-Starved GC-1 Cells through the NF-κB Pathway.

The ankyrin repeat domain 49 (ANKRD49) is an evolutionarily conserved protein highly expressed in testes. However, the function of ANKRD49 in spermatogenesis is unknown. In this study, we found that ANKRD49 resides primarily in nucleus of spermatogonia, spermatocytes and round spermatids. ANKRD49 overexpression augments starvation-induced autophagy in male germ GC-1 cells whereas shRNA knockdown of ANKRD49 attenuates the autophagy. Inhibition of NF-κB pathway by its inhibitors or p65 siRNA prevents the ANKRD49-dependent autophagy augmentation, demonstrating that ANKRD49 enhances autophagy via NF-κB pathway. Our findings suggest that ANKRD49 plays an important role in spermatogenesis via promotion of autophagy-dependent survival.

Intranasal immunisation of the recombinant Toxoplasma gondii receptor for activated C kinase 1 partly protects mice against T. gondii infection.

Nasal vaccination is an effective therapeutic regimen for preventing certain infectious diseases. The mucosal immune response is important for resistance to Toxoplasma gondii infection. In this study, we evaluated the immune responses elicited in BALB/c mice by nasal immunisation with recombinant T. gondii receptor for activated C kinase 1 (rTgRACK1) and their protective efficacy against T. gondii RH strain during both chronic and lethal infections. Nasal vaccination with rTgRACK1 increased the level of secretory IgA in nasal, intestinal and vesical washes, and the level of IFN-γ and IL-2 in intestinal washes, indicating that rTgRACK1 vaccination promotes mucosal immune responses. The mice immunised with rTgRACK1 also displayed increased levels of rTgRACK1-specific IgA, total IgG, IgG1 and in particular IgG2a in their blood sera, increased production of IFN-γ, IL-2 and IL-4 but not IL-10 from their isolated spleen cells, and enhanced splenocyte proliferation in vitro. rTgRACK1-vaccinated mice were effectively protected against infection with T. gondii RH strain, showing over 50% reduction of tachyzoite burdens in their liver and brain tissues during a chronic infection, and also a 45% increase in their survivals during a lethal challenge. These results indicate that rTgRACK1 might represent an intriguing immunogen for developing a mucosal vaccine against toxoplasmosis.

Hepatocyte gene transfer mediated by stable polyplexes based on MPP-containing DNA complexes.

BACKGROUND: In the field of gene therapy, viral vectors as delivery tools have a number of disadvantages for medical application. This study aimed to explore a novel nonviral vector as a vehicle for gene therapy. METHODS: Transvector-rpE-MPP and EGFP (enhanced green fluorescent protein) were used as the gene transfer carrier and the reporter gene, respectively. Polyplexes which integrate transvector-rpE-MPP, the object gene, and EGFP were formed. The optimal charge ratio, stability, and transduction capacity of the polyplexes in mouse hepatocytes in vitro and in mouse liver in vivo were investigated. The polyplexes of transvector-rpE-MPP and pcDNA(3)-EGFP, with charge ratios of 0, 0.25, 0.5, 0.75, 1 and 1.5 were compared to determine the optimal charge ratio. RESULTS: Polyplexes with charge ratios of 1:1 were most stable; pcDNA(3)-EGFP in these complexes resisted digestion by DNase I and blood plasma. On the other hand, pcDNA(3)-EGFP alone was digested. Fluorescence analysis indicated that transvector-rpE-MPP successfully delivered the reporter gene EGFP into hepatocytes and that EGFP expression was detected in hepatocyte cultures and in liver tissue. CONCLUSION: These results have laid a foundation for further study of a novel nonviral gene delivery system.

Construction of recombinant plasmid and prokaryotic expression in E. coli and biological activity analysis of human placenta arresten gene.

BACKGROUND: The proliferation and metastasis of cancers depend on angiogenesis. This property provides the feasibility for the treatment of cancer by inhibition of angiogenesis, and many angiogenic inhibitors have been demonstrated to effectively inhibit angiogenesis and consequently the growth of solid cancer. As for the newly identified angiogenesis inhibitor, arresten, some studies have found its high activity on restrainting tumor vessel. This study was to assess the anti-angiogenic activity of arresten. METHODS: The arresten gene was obtained from a healthy puerperal's placenta tissue by the reverse transcriptase-polymerase chain reaction (RT-PCR) method, and molecular cloning to prokaryotic expression plasmid pBV220 by recombination strategy. The prokaryotic expression plasmid pBV220/arr was identified by restriction enzyme digestion and sequenced. The pBV220/arr was transformed into E. coli JM109, DH5alpha, BL21 and BL21 (DE3) by the CaCl2 transformation method. The arresten expression level was detected by SDS-PAGE. The expressed product was purified, re-naturalized and detected for its biological activity of inhibiting the angiogenesis of chorioallantoic membrane (CAM). RESULTS: The arresten gene was cloned and pBV220/arr was constructed. The arresten expression level of protein was highly increased after pBV220/arr was transformed into E. coli BL21 (DE3). SDS-PAGE showed that the expressed arresten proteins were mainly inclusion bodies and had a molecular weight of 26 kDa. The expressed arresten protein showed evident biological activities. CONCLUSIONS: The successful construction of recombinant plasmid pBV220/arr and the effective expression in E.coli have laid a foundation for further study of its anti-angiogenic function and may pave the way for future anti-tumor application.

Protein transduction domain of membrane penetrating peptide can efficiently deliver DNA and protein into mouse liver for gene therapy.

BACKGROUND: The development of a harmless and efficient nonviral gene delivery system that can facilitate the penetration of nucleic acids through the plasma membrane is a key to successful gene therapy. The aim of this study was to test a nonviral gene transferring vector's function of delivering DNA into liver cells to provide an important clue for gene transfer in liver gene therapy. METHODS: The complex of DNA and DNA delivering protein was injected into mice through their tail veins. Then the mice were killed and their liver tissue was sectioned. The gene transferring results were detected using a confocal laser scanning microscope. RESULTS: Fluorescence analysis indicated that both DNA-membrane penetrating peptide (MPP) complex and DNA- hepatocyte specific receptor binding domain (HSRBD)-MPP complex could go into liver cells. The fluorescence value of liver cells in the DNA- HSRBD-MPP group was higher than that in the DNA-MPP group. CONCLUSIONS: MPP can successfully deliver DNA and protein into cells, and MPP with a HSRBD can specifically deliver DNA into liver cells. These have laid a foundation for further study on the nonviral liver cell gene delivering system.