Characterization of the canine mda-7 gene,transcripts and expression patterns

Human melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) displays potent growth suppressing and cell killing activity against a wide variety of human and rodent cancer cells. In this study, we identified a canine ortholog of the human mda-7/IL-24 gene located within a cluster of IL-10 family members on chromosome 7. The full-length mRNA sequence of canine mda-7 was determined, which encodes a 186-amino acid protein that has 66% similarity to human MDA-7/IL-24. Canine MDA-7 is constitutively expressed in cultured normal canine epidermal keratinocytes (NCEKs), and its expression levels are increased after lipopolysaccharide stimulation. In cultured NCEKs, the canine mda-7 pre-mRNA is differentially spliced, via exon skipping and alternate 5′-splice donor sites, to yield five splice variants (canine mda-7sv1, canine mda-7sv2, canine mda-7sv3, canine mda-7sv4 and canine mda-7sv5) that encode four protein isoforms of the canine MDA-7 protein. These protein isoforms have a conserved N-terminus (signal peptide sequence) and are dissimilar in amino acid sequences at their C-terminus. Canine MDA-7 is not expressed in primary canine tumor samples, and most tumor derived cancer cell lines tested, like its human counterpart. Unlike human MDA-7/IL-24, canine mda-7 mRNA is not expressed in unstimulated or lipopolysaccharide (LPS), concanavalin A (ConA) or phytohemagglutinin (PHA) stimulated canine peripheral blood mononuclear cells (PBMCs). Furthermore, in-silico analysis revealed that canonical canine MDA-7 has a potential 28 amino acid signal peptide sequence that can target it for active secretion. This data suggests that canine mda-7 is indeed an ortholog of human mda-7/IL-24, its protein product has high amino acid similarity to human MDA-7/IL-24 protein and it may possess similar biological properties to human MDA-7/IL-24, but its expression pattern is more restricted than its human ortholog.     

Gene expression signature of human HepG2 cell line

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is associated with various clinico-pathological characteristics such as genetic mutations and viral infections. Therefore, numerous laboratories look out for identifying always new putative markers for the improvement of HCC diagnosis/prognosis. Many molecular profiling studies investigated gene expression changes related to HCC. HepG2 represents a pure cell line of human liver carcinoma, often used as HCC model due to the absence of viral infection. In this study we compare gene expression profiles associated with HepG2 (as HCC model) and normal hepatocyte cells by microarray technology. Hierarchical cluster analysis of genes evidenced that 2646 genes significantly down-regulated in HepG2 cells compared to hepatocytes whereas a further 3586 genes significantly up-regulated. By using the Ingenuity Pathway Analysis (IPA) program, we have classified the genes that were differently expressed and studied the functional networks correlating these genes in the complete human interactome. Moreover, to confirm the differentially expressed genes as well as the reliability of our microarray data, we performed a quantitative Real time RT-PCR analysis on 9 up-regulated and 11 down-regulated genes, respectively. In conclusion this work i) provides a gene signature of human hepatoma cells showing genes that change their expression as a consequence of liver cancer in the absence of any genetic mutations or viral infection, ii) evidences new differently expressed genes found in our signature compared to previous published studies and iii) suggests some genes on which to focus future studies to understand if they can be used to improve the HCC prognosis/diagnosis.     

Distinct roles of the R3H and RRM domains in poly(A)-specific ribonuclease structural integrity and catalysis

Deadenylases specifically catalyze the degradation of eukaryotic mRNA poly(A) tail in the 3′- to 5′-end direction with the release of 5′-AMP as the product. Among the deadenylase family, poly(A)-specific ribonuclease (PARN) is unique in its domain composition, which contains three potential RNA-binding domains: the catalytic nuclease domain, the R3H domain and the RRM domain. In this research, we investigated the roles of these RNA-binding domains by comparing the structural features and enzymatic properties of mutants lacking either one or two of the three RNA-binding domains. The results showed that the R3H domain had the ability to bind various oligonucleotides at the micromolar level with no oligo(A) specificity. The removal of the R3H domain dissociated PARN into monomers, which still possessed the RNA-binding ability and catalytic functions. Unlike the critical role of the RRM domain in PARN processivity, the removal of the R3H domain did not affect the catalytic pattern of PARN. Our results suggested that both R3H and RRM domains were essential for the high affinity of long poly(A) substrate, but the R3H domain did not contribute to the substrate recognition of PARN. Compared to the RRM domain, the R3H domain played a more important role in the structural integrity of the dimeric PARN. The multiple RNA-binding domain architecture endows PARN the property of highly efficient catalysis in a highly processive mode.     

Excision of the oxidatively formed 5-hydroxyhydantoin and 5-hydroxy-5-methylhydantoin pyrimidine lesions by Escherichia coli and Saccharomyces cerevisiae DNA N-glycosylases

Background

(5R?) and (5S?) diastereomers of 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd) are major oxidation products of 2′-deoxycytidine and thymidine respectively. If not repaired, when present in cellular DNA, these base lesions may be processed by DNA polymerases that induce mutagenic and cell lethality processes.

Methods

Synthetic oligonucleotides that contained a unique 5-hydroxyhydantoin (5-OH-Hyd) or 5-hydroxy-5-methylhydantoin (5-OH-5-Me-Hyd) nucleobase were used as probes for repair studies involving several E. coli, yeast and human purified DNA N-glycosylases. Enzymatic reaction mixtures were analyzed by denaturing polyacrylamide gel electrophoresis after radiolabeling of DNA oligomers or by MALDI-TOF mass spectrometry measurements.

Results

In vitro DNA excision experiments carried out with endo III, endo VIII, Fpg, Ntg1 and Ntg2, show that both base lesions are substrates for these DNA N-glycosylases. The yeast and human Ogg1 proteins (yOgg1 and hOgg1 respectively) and E. coli AlkA were unable to cleave the N-glycosidic bond of the 5-OH-Hyd and 5-OH-5-Me-Hyd lesions. Comparison of the k_cat/K_m ratio reveals that 8-oxo-7,8-dihydroguanine is only a slightly better substrate than 5-OH-Hyd and 5-OH-5-Me-Hyd. The kinetic results obtained with endo III indicate that 5-OH-Hyd and 5-OH-5-Me-Hyd are much better substrates than 5-hydroxycytosine, a well known oxidized pyrimidine substrate for this DNA N-glycosylase.

Conclusions

The present study supports a biological relevance of the base excision repair processes toward the hydantoin lesions, while the removal by the Fpg and endo III proteins are effected at better or comparable rates to that of the removal of 8-oxoGua and 5-OH-Cyt, two established cellular substrates.

General significance

The study provides new insights into the substrate specificity of DNA N-glycosylases involved in the base excision repair of oxidized bases, together with complementary information on the biological role of hydantoin type lesions.     

Structure and conformation of the disulfide bond in dimeric lung surfactant peptides SP-B1-25 and SP-B8-25

Raman spectroscopy was used to determine the conformation of the disulfide linkage between cysteine residues in the homodimeric construct of the N-terminal alpha helical domain of surfactant protein B (dSP-B_1-25). The conformation of the disulfide bond between cysteine residues in position 8 of the homodimer of dSP-B_1-25 was compared with that of a truncated homodimer (dSP-B_8-25) of the peptide having a disulfide linkage at the same position in the alpha helix. Temperature-dependent Raman spectra of the S-S stretching region centered at ∼ 500 cm^− 1 indicated a stable, although highly strained disulfide conformation with a χ(CS-SC) dihedral angle of ± 10° for the dSP-B_1-25 dimer. In contrast, the truncated dimer dSP-B_8-25 exhibited a series of disulfide conformations with the χ(CS-SC) dihedral angle taking on values of either ± 30° or 85± 20°. For conformations with χ(CS-SC) close to the ± 90° value, the Raman spectra of the 8-25 truncated dimers exhibited χ(SS-CC) dihedral angles of 90/180° and 20-30°. In the presence of a lipid mixture, both constructs showed a ν(S-S) band at ∼ 488 cm^− 1, corresponding to a χ(CS-SC) dihedral angle of ± 10°. Polarized infrared spectroscopy was also used to determine the orientation of the helix and β-sheet portion of both synthetic peptides. These calculations indicated that the helix was oriented primarily in the plane of the surface, at an angle of ∼ 60-70° to the surface normal, while the β structure had ∼ 40° tilt. This orientation direction did not change in the presence of a lipid mixture or with temperature. These observations suggest that: (i) the conformational flexibility of the disulfide linkage is dependent on the amino acid residues that flank the cysteine disulfide bond, and (ii) in both constructs, the presence of a lipid matrix locks the disulfide bond into a preferred conformation.     

The RNA expression signature of the HepG2 cell line as determined by the integrated analysis of miRNA and mRNA expression profiles

Understanding miRNAs' regulatory networks and target genes could facilitate the development of therapies for human diseases such as cancer. Although much useful gene expression profiling data for tumor cell lines is available, microarray data for miRNAs and mRNAs in the human HepG2 cell line have only been compared with that of other cell lines separately. The relationship between miRNAs and mRNAs in integrated expression profiles for HepG2 cells is still unknown. To explore the miRNA–mRNA correlations in hepatocellular carcinoma (HCC) cells, we performed miRNA and mRNA expression profiling in HepG2 cells and normal liver HL-7702 cells at the genome scale using next-generation sequencing technology. We identified 193 miRNAs that are differentially expressed in these two cell lines. Of these, 89 miRNAs were down-regulated in HepG2 cells compared with HL-7702 cells, while 104 miRNAs were up-regulated. We also observed 3035 mRNAs that are significantly dys-regulated in HepG2 cells. We then performed an integrated analysis of the expression data for differentially expressed miRNAs and mRNAs and found several miRNA–mRNA pairs that are significantly correlated in HepG2 cells. Further analysis suggested that these differentially expressed genes were enriched in four tumorigenesis-related signaling pathways, namely, ErbB, JAK–STAT, mTOR, and WNT, which until now had not been fully reported. Our results could be helpful in understanding the mechanisms of HCC occurrence and development.     

Expression,SNV identification,linkage disequilibrium,and combined genotype association analysis of the muscle-specific gene CSRP3 in Chinese cattle

The cysteine and glycine-rich protein 3 (CSRP3) plays an important role in the myofiber differentiation. Here, we identified five SNVs in all exon and intron regions of the CSRP3 gene using DNA sequencing, PCR-RFLP and forced-PCR-RFLP methods in 554 cattle. Four of the five SNVs were significantly associated with growth performance and carcass traits of the cattle. In addition, we evaluated haplotype frequency and linkage disequilibrium coefficient of five sequence variants. The result of haplotype analysis demonstrated 28 haplotypes present in Qinchuan and two haplotypes in Chinese Holstein. Only haplotypes 1 and 8 were being shared by two populations, haplotype 14 had the highest haplotype frequency in Qinchuan (17.4%) and haplotype 8 had the highest haplotype frequency in Chinese Holstein (94.4%). Statistical analyses of combined genotypes indicated that some combined genotypes were significantly or highly significantly associated with growth and carcass traits in the Qinchuan cattle population. qPCR analyses also showed that bovine CSRP3 gene was exclusively expressed in longissimus dorsi muscle and heart tissues. The data support the high potential of the CSRP3 as a marker gene for the improvement of growth performance and carcass traits in selection programs.     

Tight,cell type-specific control of LNX expression in the nervous system,at the level of transcription,translation and protein stability

LNX1 and LNX2 are E3 ubiquitin ligases that can interact with Numb — a key regulator of neurogenesis and neuronal differentiation. LNX1 can target Numb for proteasomal degradation, and Lnx mRNAs are prominently expressed in the nervous system, suggesting that LNX proteins play a role in neural development. This hypothesis remains unproven, however, largely because LNX proteins are present at very low levels in vivo. Here, we demonstrate expression of both LNX1 and LNX2 proteins in the brain for the first time. We clarify the cell-type specific expression of LNX isoforms in both the CNS and PNS, and identify a novel LNX1 isoform. Using luciferase reporter assays, we show that the 5′ untranslated region of the Lnx1_variant 2 mRNA, that generates the LNX1p70 isoform, strongly suppresses protein production. This effect is mediated in part by the presence of upstream open reading frames (uORFs), but also by a sequence element that decreases both mRNA levels and translational efficiency. By contrast, uORFs do not negatively regulate LNX1p80 or LNX2 expression. Instead, we find some evidence that protein turnover via proteasomal degradation may influence LNX1p80 levels in cells. These observations provide plausible explanations for the low levels of LNX1 proteins detected in vivo.     

Disruption of the novel gene fad104 causes rapid postnatal death and attenuation of cell proliferation, adhesion, spreading and migration

The molecular mechanisms at the beginning of adipogenesis remain unknown. Previously, we identified a novel gene, fad104 (factor for adipocyte differentiation 104), transiently expressed at the early stage of adipocyte differentiation. Since the knockdown of the expression of fad104 dramatically repressed adipogenesis, it is clear that fad104 plays important roles in adipocyte differentiation. However, the physiological roles of fad104 are still unknown. In this study, we generated fad104-deficient mice by gene targeting. Although the mice were born in the expected Mendelian ratios, all died within 1 day of birth, suggesting fad104 to be crucial for survival after birth. Furthermore, analyses of mouse embryonic fibroblasts (MEFs) prepared from fad104-deficient mice provided new insights into the functions of fad104. Disruption of fad104 inhibited adipocyte differentiation and cell proliferation. In addition, cell adhesion and wound healing assays using fad104-deficient MEFs revealed that loss of fad104 expression caused a reduction in stress fiber formation, and notably delayed cell adhesion, spreading and migration. These results indicate that fad104 is essential for the survival of newborns just after birth and important for cell proliferation, adhesion, spreading and migration.     

Calcium binding studies of peptides of human phospholipid scramblases 1 to 4 suggest that scramblases are new class of calcium binding proteins in the cell

Background

Phospholipid scramblases are a group of four homologous proteins conserved from C. elegans to human. In human, two members of the scramblase family, hPLSCR1 and hPLSCR3 are known to bring about Ca²⁺ dependent translocation of phosphatidylserine and cardiolipin respectively during apoptotic processes. However, affinities of Ca²⁺/Mg²⁺ binding to human scramblases and conformational changes taking place in them remains unknown.

Methods

In the present study, we analyzed the Ca²⁺ and Mg²⁺ binding to the calcium binding motifs of hPLSCR1–4 and hPLSCR1 by spectroscopic methods and isothermal titration calorimetry.

Results

The results in this study show that (i) affinities of the peptides are in the order hPLSCR1  > hPLSCR3 > hPLSCR2 > hPLSCR4 for Ca²⁺ and in the order hPLSCR1 > hPLSCR2 > hPLSCR3 > hPLSCR4 for Mg²⁺, (ii) binding of ions brings about conformational change in the secondary structure of the peptides. The affinity of Ca²⁺ and Mg²⁺ binding to protein hPLSCR1 was similar to that of the peptide I. A sequence comparison shows the existence of scramblase-like motifs among other protein families.

Conclusions

Based on the above results, we hypothesize that the Ca²⁺ binding motif of hPLSCR1 is a novel type of Ca²⁺ binding motif.

General significance

Our findings will be relevant in understanding the calcium dependent scrambling activity of hPLSCRs and their biological function.