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 many faces of calmodulin in cell proliferation,programmed cell death,autophagy, and cancer

Calmodulin (CaM) is a ubiquitous Ca^2 + receptor protein mediating a large number of signaling processes in all eukaryotic cells. CaM plays a central role in regulating a myriad of cellular functions via interaction with multiple target proteins. This review focuses on the action of CaM and CaM-dependent signaling systems in the control of vertebrate cell proliferation, programmed cell death and autophagy. The significance of CaM and interconnected CaM-regulated systems for the physiology of cancer cells including tumor stem cells, and processes required for tumor progression such as growth, tumor-associated angiogenesis and metastasis are highlighted. Furthermore, the potential targeting of CaM-dependent signaling processes for therapeutic use is discussed.     

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.     

Independent tyrosyl contributions to the CD of Ff gene 5 protein and the distinctive effects of Y41H and Y41F mutants on protein-protein cooperative interactions

The gene 5 protein (g5p) of the Ff virus contains five Tyr, individual mutants of which have now all been characterized by CD spectroscopy. The protein has a dominant tyrosyl 229-nm L(a) CD band that is shown to be approximately the sum of the five individual Tyr contributions. Tyr41 is particularly important in contributing to the high cooperativity with which the g5p binds to ssDNA, and Y41F and Y41H mutants are known to differ in dimer-dimer packing interactions in crystal structures. We compared the solution structures and binding properties of the Y41F and Y41H mutants using CD spectroscopy. Secondary structures of the mutants were similar by CD analyses and close to those derived from the crystal structures. However, there were significant differences in the binding properties of the two mutant proteins. The Y41H protein had an especially low binding affinity and perturbed the spectrum of poly[d(A)] in 2 mM Na(+) much less than did Y41F and the wild-type gene 5 proteins. Moreover, a change in the Tyr 229 nm band, assigned to the perturbation of Tyr34 at the dimer-dimer interface, was absent in titrations with the Y41H mutant under low salt conditions. In contrast, titrations with the Y41H mutant in 50 mM Na(+) exhibited typical CD changes of both the nucleic acid and the Tyr 229-nm band. Thus, protein-protein and g5p-ssDNA interactions appeared to be mutually influenced by ionic strength, indicative of correlated changes in the ssDNA binding and cooperativity loops of the protein or of indirect structural constraints.     

Identification of the 5-HT1A Receptor Binding Subunit in Rat Brain Membranes Using the Photoaffinity Probe [3H]8-Methoxy-2-[N-n-Propyl, N-3-(2-Nitro-4-Azidophenyl)Aminopropyl]Aminotetralin

The synthesis of a tritiated derivative of the 5-HT1A photoaffinity probe 8-methoxy-2-[N-n-propyl, N-3-(2-nitro-4-azidophenyl)aminopropyl]aminotetralin ([3H]8-methoxy-3'-NAP-amino-PAT) allowed the use of this probe for attempting the irreversible labeling of specific binding sites in rat brain membranes. Sodium dodecyl-sulfate-polyacrylamide gel electrophoresis of proteins solubilized from hippocampal microsomal membranes that had been incubated with 20 nM [3H]8-methoxy-3'-NAP-amino-PAT under UV light revealed a marked incorporation of 3H label into a 63-kilodalton protein termed PI. As expected of a possible correspondence between PI and 5-HT1A receptor binding sites, 3H labeling by the photoaffinity probe could be prevented by selective 5-HT1A ligands such as 8-hydroxy-2-(di-n-propylamino)tetralin, ipsapirone, buspirone, and gepirone and by N-ethylmaleimide, but not by the 5-HT2 antagonist ketanserin, noradrenaline- and dopamine-related drugs, monoamine oxidase inhibitors, and chlorimipramine. Furthermore, the regional and subcellular distributions of PI were identical to those of specific 5-HT1A binding sites. These results indicated that the binding subunit of the 5-HT1A receptor is a 63-kilodalton protein with a functionally important sulfhydryl group(s).