Activation of TAK1 by Sef-S induces apoptosis in 293T cells.

Sef (similar expression to fgf genes, also named IL-17RD) was identified as a negative regulator of fibroblast growth factor signaling. Sef-S, an alternative splice isoform of Sef, inhibits FGF-induced NIH3T3 cell proliferation. Here we report that Sef-S physically interacts with TAK1, induces Lys63-linked TAK1 polyubiquitination on lysine 209 and TAK1-mediated JNK and p38 activation. Co-overexpression of TAK1 WT, K34R, K150R, K158R mutants with Sef-S induces Lys63-linked TAK1 polyubiquitination whereas TAK1 K63R and K209R mutants fail. Furthermore, co-overexpression of Sef-S and TAK1 induce 293T cells apoptosis. These results reveal Sef-S actives Lys63-linked TAK1 polyubiquitination on lysine 209, induces TAK1-mediated JNK and p38 activation and also results apoptosis in 293T cells.

Conformation-dependent scFv antibodies specifically recognize the oligomers assembled from various amyloids and show colocalization of amyloid fibrils with oligomers in patients with amyloidoses.

Increasing evidence indicates that amyloid aggregates, including oligomers, protofibrils or fibrils, are pivotal toxins in the pathogenesis of many amyloidoses such as Alzheimer's disease (AD), Parkinson's disease, Huntington's disease, prion-related diseases, type 2 diabetes and hereditary renal amyloidosis. Various oligomers assembled from different amyloid proteins share common structures and epitopes. Here we present data indicating that two oligomer-specific single chain variable fragment (scFv) antibodies isolated from a naïve human scFv library could conformation-dependently recognize oligomers assembled from α-synuclein, amylin, insulin, Aß1-40, prion peptide 106-126 and lysozyme, and fibrils from lysozyme. Further investigation showed that both scFvs inhibited the fibrillization of α-synuclein, amylin, insulin, Aß1-40 and prion peptide 106-126, and disaggregated their preformed fibrils. However, they both promoted the aggregation of lysozyme. Nevertheless, the two scFv antibodies could attenuate the cytotoxicity of all amyloids tested. Moreover, the scFvs recognized the amyloid oligomers in all types of plaques, Lewy bodies and amylin deposits in the brain tissues of AD and PD patients and the pancreas of type 2 diabetes patients respectively, and showed that most amyloid fibril deposits were colocalized with oligomers in the tissues. Such conformation-dependent scFv antibodies may have potential application in the investigation of aggregate structures, the mechanisms of aggregation and cytotoxicity of various amyloids, and in the development of diagnostic and therapeutic reagents for many amyloidoses.

GABARAPL1 negatively regulates Wnt/ß-catenin signaling by mediating Dvl2 degradation through the autophagy pathway.

Wnt signaling is critical for many biological processes and is tightly regulated. In this study, we found that GABARAPL1 (GABA(A) receptor-associated protein like 1, GABARAPL1) interacts with Dvl2 by both yeast two-hybrid screening and immunoprecipitation experiments. Furthermore, we observed that p62 is required for the interaction of Dvl2 and GABARAPL1. Luciferase assays indicated that GABARAPL1 represses Wnt/ß-catenin signaling stimulated by Wnt1, Dvl2 and ß-catenin. We further demonstrated that GABARAPL1 mediates degradation of Dvl2 and the effect is blocked by addition of 3-MA, a specific inhibitor of autophagy. Finally, we provided evidence that over-expression of GABARAPL1 inhibits proliferation and tumor growth of MCF7 cells in vitro and in nude mice. Taken together, our results suggested that GABARAPL1 as a tumor repressor inhibits Wnt signaling via mediating Dvl2 degradation through the autophagy pathway.

[Eukaryotic expression of human IL17-RD-ECD and generation of its monoclonal antibody].

IL-17 Receptor D (IL-17 RD) is a cytokine receptor that mediates IL-17 signaling and plays an important role in responding to the invasion of extracellular pathogens and many inflammatory and autoimmune diseases such as rheumatoid arthritis. In this study we report the generation of a mouse monoclonal antibody against human IL-17 RD. The recombinant human IL-17RD extracellular domain (hIL-17RD-ECD) was produced in the baculovirus expression system and purified from culture medium of sf9 insect cells. The purified protein was used as a T-dependent antigen to immune Balb/C mice. B cells from the spleen of immunized mice were fused with murine cell SP2/0. Hybridoma cell lines were screened for the production of the monoclonal antibody against hIL-17-RD-ECD using ELISA. A hybridoma cell line 1F8 was found to have a high production of the antibody, which was further confirmed for the specificity by both western blot and ELISA analyses. The monoclonal antibody obtained from hybridoma 1F8 was characterized to be IgG1+Kappa subclass. This study provided a base for the further therapeutic application of the antibody on the autoimmune disease including rheumatoid arthritis.

Genetic and epigenetic silencing of the beclin 1 gene in sporadic breast tumors.

BACKGROUND: Beclin 1, an important autophagy-related protein in human cells, is involved in cell death and cell survival. Beclin 1 mapped to human chromosome 17q21. It is widely expressed in normal mammary epithelial cells. Although down-regulated expression with mono-allelic deletions of beclin 1 gene was frequently observed in breast tumors, whether there was other regulatory mechanism of beclin 1 was to be investigated. We studied the expression of beclin 1 and explored the possible regulatory mechanisms on its expression in breast tumors. METHODS: 20 pairs of tumors and adjacent normal tissues from patients with sporadic breast invasive ductal cancer (IDCs) were collected. The mRNA expression of beclin 1 was detected by real-time quantitative RT-PCR. Loss of heterozygosity (LOH) was determined by real-time quantitative PCR and microsatellite methods. The protein expression of beclin 1, p53, BRCA1 and BRCA2 was assessed by immunohistochemistry. CpG islands in 5' genomic region of beclin 1 gene were identified using MethylPrimer Program. Sodium bisulfite sequencing was used in examining the methylation status of each CpG island. RESULTS: Decreased beclin 1 mRNA expression was detected in 70% of the breast tumors, and the protein levels were co-related to the mRNA levels. Expression of beclin 1 mRNA was demonstrated to be much higher in the BRCA1 positive tumors than that in the BRCA1 negative ones. Loss of heterozygosity was detected in more than 45% of the breast tumors, and a dense cluster of CpG islands was found from the 5' end to the intron 2 of the beclin 1 gene. Methylation analysis showed that the promoter and the intron 2 of beclin 1 were aberrantly methylated in the tumors with decreased expression. CONCLUSIONS: These data indicated that LOH and aberrant DNA methylation might be the possible reasons of the decreased expression of beclin 1 in the breast tumors. The findings here shed some new light on the regulatory mechanisms of beclin 1 in breast cancer.

A new single nucleotide polymorphism genotyping method based on gap ligase chain reaction and a microsphere detection assay.

BACKGROUND: The establishment of new methods to detect human genetic variations, such as single nucleotide polymorphisms, is of importance in hereditary disease diagnosis and pharmacogenomics. Several single nucleotide polymorphism genotyping technologies have been presented in recent years. However, techniques which would allow accurate, fast and cheap allelic determination and multiple single nucleotide polymorphism detection in parallel are still in great need. METHODS: Here, we present a new genotyping technique based on gap ligase chain reaction and a fluorescent polystyrene microsphere measurement platform. We chose the human polymorphism rs3730386 as our candidate to establish this method. Probes for gap ligase chain reaction were designed to recognize the target alleles sensitively and specifically and to produce templates for amplifying the correspondent target fragments used in the following hybridization. The genotypes were finally determined by a hybridization process based on the Luminex fluorescent polystyrene microspheres measurement platform. RESULTS: This method was successfully applied for the detection of selected single nucleotide polymorphisms with high sensitivity and specificity. The genotypes were validated by DNA sequencing. CONCLUSIONS: The new genotyping method benefited from the high sensitivity and specificity of gap ligase chain reaction and the detection platform of Luminex. The method allows multiplex analysis of single nucleotide polymorphism, because 100 types of microspheres are available from Luminex.

Tim18, a component of the mitochondrial translocator, mediates yeast cell death induced by arsenic.

Evidence is presented that Tim18, a mitochondria translocase, plays a role in the previously described apoptosis induced by arsenite in Saccharomyces cerevisiae. Tim18 deletion mutant exhibited resistance to arsenite. After arsenite treatment, both the wild type and Tim18-deficient cells showed reactive oxygen species (ROS) production. Arsenite induced the higher expression of tim18 in wild type yeast cells. We found that the tim18 deletion mutant also exhibited resistance to other apoptotic stresses such as acetic acid, H2O2, and hyperosmotic stress. These results suggest that Tim18 is important for yeast cell death induced by arsenic, and it may act downstream of ROS production.

miRAS: a data processing system for miRNA expression profiling study.

BACKGROUND: The study of microRNAs (miRNAs) is attracting great considerations. Recent studies revealed that miRNAs play as important regulators of gene expression and some even as cancer players or inhibitors. Many studies try to discover new miRNAs and reveal the miRNA expression profile in cancer using a SAGE-based total RNA clone method. However, the data processing of this method is labor-intensive with several different biological databases and more than ten data processing steps involved. RESULTS: With miRAS, miRNAs and possible miRNA candidates contained in the submitted sequencing data were obtained together with their expression profile. The functions of known and predicted miRNAs were then analyzed by miRNA target prediction followed by target gene annotations. Finally, to extract the biological significance of the miRNAs in the samples, further annotations of the known miRNA and target genes were performed by collecting the public expression datasets of miRNA and target genes in normal and cancer tissues. CONCLUSION: We introduce a web-based analysis platform called miRNA Analysis System (miRAS), for processing and analyzing of the sequence data obtained from the total RNA clone method. The system was built on generalizing the study of a liver cancer cell line total RNA sequencing project. miRAS is freely available on the web.

Arsenic induces caspase- and mitochondria-mediated apoptosis in Saccharomyces cerevisiae.

In recent years, it has been shown that yeast, a unicellular organism, undergoes apoptosis in response to various factors. Here we demonstrate that the highly effective anticancer agent arsenic induces apoptotic process in yeast cells. Reactive oxygen species (ROS) production was observed in the process. Moreover, mitochondrial membrane potential decreased after arsenic treatment. Resistance of the rho(0) mutant strain (lacking mtDNA) to arsenic provides further evidence that this death process involves mitochondria. In addition, hypersensitivity of Deltasod1 to arsenic suggests the critical role of ROS. Cell death and DNA fragmentation decreased in a Deltayca1 deletion mutant, indicating the participation of yeast caspase-1 protein in apoptosis. The implications of these findings for arsenic-induced apoptosis are discussed.

Integrative analysis and validation of robust gene signature in lung cancer.

We studied robust gene signature (RGS) in lung cancer by using an approach of integrating a highly diverse collection of cancer genome-wide datasets, which were six public microarray datasets, one pair of Suppression Subtractive Hybridization EST library, one pair of Serial Analysis of Gene Expression (SAGE) experiments, and 191 Loss of Heterozygosity (LOH) reports obtained from 388 publications. Among the 109 RGS genes identified from our study, 14 of the 15 reported differentially expressed genes (DEGs) based on literature verification were consistent with our predictions. Out of the remaining 94 genes that were not reported as DEGs in lung cancer by any publication, we randomly picked eight and verified their expression in lung cancer versus normal tissues by semi-quantitative RT-PCR amplification, and all showed consistent expression pattern with our findings. System assessment analysis revealed that our integrative method had an accuracy of 95% and a correlation coefficients value of 0.92.

Remarkably high activities of testicular cytochrome c in destroying reactive oxygen species and in triggering apoptosis.

Hydrogen peroxide (H(2)O(2)) is the major reactive oxygen species (ROS) produced in sperm. High concentrations of H(2)O(2) in sperm induce nuclear DNA fragmentation and lipid peroxidation and result in cell death. The respiratory chain of the mitochondrion is one of the most productive ROS generating systems in sperm, and thus the destruction of ROS in mitochondria is critical for the cell. It was recently reported that H(2)O(2) generated by the respiratory chain of the mitochondrion can be efficiently destroyed by the cytochrome c-mediated electron-leak pathway where the electron of ferrocytochrome c migrates directly to H(2)O(2) instead of to cytochrome c oxidase. In our studies, we found that mouse testis-specific cytochrome c (T-Cc) can catalyze the reduction of H(2)O(2) three times faster than its counterpart in somatic cells (S-Cc) and that the T-Cc heme has the greater resistance to being degraded by H(2)O(2). Together, these findings strongly imply that T-Cc can protect sperm from the damages caused by H(2)O(2). Moreover, the apoptotic activity of T-Cc is three to five times greater than that of S-Cc in a well established apoptosis measurement system using Xenopus egg extract. The dramatically stronger apoptotic activity of T-Cc might be important for the suicide of male germ cells, considered a physiological mechanism that regulates the number of sperm produced and eliminates those with damaged DNA. Thus, it is very likely that T-Cc has evolved to guarantee the biological integrity of sperm produced in mammalian testis.

Integrated genome-wide gene expression map and high-resolution analysis of aberrant chromosomal regions in squamous cell lung cancer.

The recognition of recurrent aberrant regions in cancer is important to the discovery of candidate cancer related genes. Here we first constructed a genome-wide gene expression map of squamous lung carcinoma from the Stanford Microarray Database. High-resolution detection of aberrant chromosomal regions was performed by using moving-median method. 84% (27 of 32) of our results were consistent with the previous studies of comparative genomic hybridization or loss of heterozygosity. One overrepresented region in Xq28 was newly discovered to be related to squamous cell lung carcinoma. These observations could be of great interest for further studies.

Cardiovascular risk-associated allele frequencies for 15 genes in healthy elderly French and Chinese.

In order to investigate possible ethnic differences in genetic and environmental determinants, we investigated several cardiovascular disease-associated genetic variations in successful ageing populations of France (Nancy) and China (Hong Kong). Allelic frequencies of these genetic variations were compared between healthy elderly Chinese (n=103) and French populations (n=100). A multi-locus assay was used to genotype 15 genes for 29 biallelic sites, genes implicated in lipid and homocysteine metabolism, thrombosis, leukocyte adhesion, and blood pressure regulation. For most of the candidate markers within lipid metabolism genes, the less frequent alleles were more common in the Chinese population compared with the French population, while the less frequent alleles of the majority of the other markers were detected only or more commonly in the French population. In conclusion, polymorphisms in 13 genes exhibited statistically significant differences in allelic frequencies between the two populations. Since the two populations were selected as examples of successful ageing, we could hypothesise that genetic factors that could play a role in a successful ageing process may be different between the two populations.

[Expression of apolipoprotein E in Alzheimer's disease and its significance].

OBJECTIVE: To study the association between Alzheimer' s disease (AD) and apolipoprotein E (apoE) polymorphism and apoE epsilon4 allele; and to investigate the role of apoE in senile plaque formation. METHODS: During the period from 1982 to 2003, 27 portmortem cases of AD from the archival files of Department of Pathology of Beijing Hospital, diagnosed according to the consortium to establish a registry for Alzheimer's disease (CERAD) criteria, were enrolled into this study. Among the 27 cases studied, there were 23 cases of definite AD and 4 cases of probable AD. Postmortem brain tissues from 67 neurologically unremarkable deceased were used as age-matched controls. Immunohistochemical study for beta-amyloid (Abeta) and Tau protein, as well as immunohistochemical study for Abeta/apoE, were performed in all AD cases using streptavidin-peroxidase (SP) and double immunostaining ( SP/ABC) methods, respectively. Senile plaques and neurofibrillary tangles in the 23 cases of definite AD were further quantified. The apoE genotypes in all cases were analyzed by polymerase chain reaction and restriction fragment length polymorphism technologies. RESULTS: Immunohistochemical study for Abeta distinguished 4 different types of senile plaques: diffuse non-neuritic plaques, diffuse neuritic plaques, dense-core neuritic plaques and dense-core non-neuritic plaques. Double immunohistochemistry for Abeta/apoE showed that some senile plaques were positive for both Abeta and apoE. The expression rates for Abeta and apoE in these 4 different types of senile plaques were 4. 28%, 84. 71%, 8.50% and 2.51%, respectively. The positivity rate for Abeta/apoE in diffuse neuritic plaques were significantly higher than those in other 3 types (P < 0.01). The frequency of occurrence of apoE epsilon4 allele in AD was significantly higher than that in the control group (P < 0.01). The numbers of senile plaques and neurofibrillary tangles in AD cases with apoE epsilon4 allele were also significantly higher than those in AD cases without apoE epsilon4 allele (P < 0.01). CONCLUSIONS: ApoE polymorphism is associated with AD. The presence of apoE epsilon4 allele carries a higher risk for the development of AD. ApoE may also play an important role in the transformation of diffuse non-neuritic plaques to diffuse neuritic plaques.

Effect of six candidate genes on early aging in a French population.

BACKGROUND AND AIMS: The objective of this study was to examine the association between an aging indicator previously defined from a nationwide population study, and lipids and apolipoproteins, angiotensin converting enzyme, paraoxonase activities, and six candidate genes related to the aging process. METHODS: Two hundred and fifty-six healthy Caucasian men (69.8 +/- 4.0 years) were included in the study. Total cholesterol, triglycerides, HDL-cholesterol, lipoprotein(a), apolipoprotein A1, B and E concentrations, and the activities of paraoxonase, arylesterase, and angiotensin-converting enzymes were determined by standardized laboratory methods. A multiplex assay was used to genotype the studied polymorphisms: apolipoprotein E, lipoprotein lipase, paraoxonase, methylenetetrahydrofolate reductase, cystathionine beta-synthase and angiotensin-converting enzymes. RESULTS: Paraoxonase polymorphism at codon 192 (Gln/Arg) was the only one significantly associated with the aging indicator, Gln homozygotes being more advanced in aging compared with Arg allele carriers. It was also observed that the aging indicator was positively correlated with serum concentrations of total cholesterol, triglycerides and apolipoprotein B, and negatively with the activities of basal and stimulated paraoxonase and arylesterase. Multiple regression analysis showed that triglycerides and basal paraoxonase activity explain 13.6% of the variance of the aging indicator. CONCLUSIONS: Triglyceride concentration and paraoxonase gene and activities may contribute to the aging process. Taking into account the smallness of the sample size, and the poor level of significance due to the im-plication of paraoxonase polymorphism at codon 192, these results need to be verified in further studies on a greater number of subjects.