Improvement of Physical Decline Through Combined Effects of Muscle Enhancement and Mitochondrial Activation by a Gastric Hormone Ghrelin in Male 5/6Nx CKD Model Mice

Because a physical decline correlates with an increased risk of a wide range of disease and morbidity, an improvement of physical performance is expected to bring significant clinical benefits. The primary cause of physical decline in 5/6 nephrectomized (5/6Nx) chronic kidney disease model mice has been regarded as a decrease in muscle mass; however, our recent study showed that a decrease in muscle mitochondria plays a critical role. In the present study, we examined the effects of a gastric hormone ghrelin, which has been reported to promote muscle mitochondrial oxidation, on the physical decline in the chronic kidney disease model mice, focusing on the epigenetic modulations of a mitochondrial activator gene, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). Ghrelin treatment improved a decline in exercise endurance of 5/6Nx mice, associated with an increase in both of the muscle mass and mitochondrial amount. The expression level of PGC-1α was decreased in the skeletal muscle of 5/6Nx mice, which was associated with an increase in the methylation ratio of the cytosine residue at 260 base pairs upstream of the initiation point. Conversely, ghrelin treatment de-methylated the cytosine residue and increased the expression of PGC-1α. A representative muscle anabolic factor, IGF-1, did not affect the expression of PGC-1α and muscle mitochondrial amount, although it increased muscle mass. As a result, IGF-1 treatment in 5/6Nx mice did not increase the decreased exercise endurance as effectively as ghrelin treatment did. These findings indicate an advantage of ghrelin treatment for a recovery of physical decline.

Semenogelin 1 Expression in Myeloma Cells: Interaction between DNA Methylation, MeCP2 Protein and Specific Cytokines.

Semenogelin (SEMG) 1 is a protein of semen coagulum with limited expression in normal tissues. It plays an important role in sperm clotting and is normally degraded into smaller fragments by prostate-specific antigen. The gene encoding SEMG 1 has been localized to the long arm of chromosome 20, a region of chromosome 20 that is frequently deleted in myeloproliferative diseases and myelodysplastic syndrome. We previously found SEMG 1 to be aberrantly expressed by tumor cells of hematologic malignancies, including multiple myeloma (MM). The aberrant expression of SEMG 1 in tumor cells of hematologic malignancies is associated in vivo with the generation of high titers IgG directed at SEMG 1 protein, suggesting the immunogenicity of the protein in the cancer-bearing patients. The combination of being immunogenic in cancer patients and limited expression in normal tissue expression makes SEMG 1 a potential candidate protein for tumor vaccines. In this study, we have set out to determine the molecular mechanisms associated with SEMG 1 expression in MM. Treatment of SEMG 1-positive MM cells with IL-4 and IL-6 resulted in the upregulation of SEMG 1 expression. In SEMG 1-negative MM cells, SEMG 1 expression could only be upregulated by IL-4 and IL-6 after pre-treatment with 5-azacytidine, suggesting that DNA methylation is likely the primary regulatory mechanism for SEMG 1 expression. Treatment of SEMG 1-negative MM cells induced SEMG 1 gene and protein expression. SEMG 1 promoter only has one CpG dinucleotide, located at position -11 of the gene. Bisulfite conversion and nucleotide sequencing was carried out on the genomic DNA from MM cells. MM cells that did not express SEMG 1 were 100% methylated. In contrast, 100% of the sequences obtained from SEMG 1-positive MM cells were unmethylated at the cytosine residue of the CpG dinucleotide. Induction of SEMG 1 expression by 5-azacytidine was associated with a decrease in the % of methylation of this cytosine residue, from 100% to 20%. These results, therefore, further implicate the role of DNA methylation in the primary regulation of SEMG 1 expression. Applying antibodies directed at MeCP2 in chromatin immunoprecipiation, MeCP2 protein binding to the SEMG 1 promoter sequence of MM cell lines and fresh MM cells was correlated to SEMG 1 gene silencing, suggesting the likely role of the MeCP2 protein in SEMG 1 gene repression. Further analysis by promoter truncation studies indicated the dependence of the promoter function on the sequence spanning the two putative GATA-1 binding sites within the gene. Using a reporter gene expression system, both IL-4 and IL-6 were found to upregulate SEMG 1 via their effect on the hypomethylated promoter gene. The effects of IL-4 and IL-6 on the function of the SEMG 1 promoter were dose dependent. In conclusion, the present study demonstrates that SEMG 1 expression in MM cells is regulated through the interaction between primary regulatory effect of promoter methylation, MeCP2 protein binding and the secondary effect of specific cytokines. Our findings provide insight into the molecular mechanisms affecting SEMG 1 expression and suggest the possible use of hypomethylating agents to upregulate SEMG 1 expression in tumor cells. Obviously, it remains to be determined whether or not there is a differential dose response among the different normal tissues in their sensitivity to the antigen-inducing effect of these agents.

DNA methyltransferases from Neisseria meningitidis and Neisseria gonorrhoeae FA1090 associated with mismatch nicking endonucleases

The genes encoding the DNA methyltransferases M.NmeDI and M.NmeAI from Neisseria meningitidis associated with the genes encoding putative Vsr endonucleases were overexpressed in Escherichia coli. The enzymes were purified to apparent homogeneity on Ni-NTA agarose columns, yielding proteins of 49±1 kDa and 39·6±1 kDa, respectively, under denaturing conditions. M.NmeDI recognizes the degenerate sequence 5′-RCCGGB-3′. It methylates the first 5′ cytosine residue on both strands within the core sequence CCGG. The enzyme shows higher affinity with the hemimethylated degenerate sequence than with the unmethylated degenerate sequence. Comparison of the amino acid sequence of the target-recognizing domain of M.NmeDI with the closest neighbours recognizing the sequence 5′-RCCGGY-3′ showed the presence of the homologous domain and an additional domain that may be responsible for recognizing the degenerate sequence. M.NmeAI recognizes the sequence 5′-CCGG-3′ and methylates the second 5′ cytosine residue on both DNA strands. In Neisseria gonorrhoeae strain FA1090 the homologues of these ORFs are truncated due to a variety of mutations.

A thermodynamic analysis of the sequence-specific binding of RNA by bacteriophage MS2 coat protein

Most mutations in the sequence of the RNA hairpin that specifically binds MS2 coat protein either reduce the binding affinity or have no effect. However, one RNA mutation, a uracil to cytosine change in the loop, has the unusual property of increasing the binding affinity to the protein by nearly 100-fold. Guided by the structure of the protein–RNA complex, we used a series of protein mutations and RNA modifications to evaluate the thermodynamic basis for the improved affinity: The tight binding of the cytosine mutation is due to (i) the amino group of the cytosine residue making an intra-RNA hydrogen bond that increases the propensity of the free RNA to adopt the structure seen in the complex and (ii) the increased affinity of hydrogen bonds between the protein and a phosphate two bases away from the cytosine residue. The data are in good agreement with a recent comparison of the cocrystal structures of the two complexes, where small differences in the two structures are seen at the thermodynamically important sites.

Isolation and characterization of the M.EaeI modification methylase

The modification enzyme (M.EaeI) corresponding to the restriction endonuclease EaeI was partially purified from Enterobacter aerogenes PW201. The M.EaeI enzyme methylates the innermost cytosine residue in each strand of the family of related sequences that constitute the EaeI recognition site to give: 5′-Y-G-G-5mC-C-R-3′ where 5mC is 5-methylcytosine. M.EaeI protects these sites against cleavage by HaeIII, and protects overlapping 5′-C-C-G-G-3′ sites against cleavage by both HpaII and MspI.

Studies related to antitumor antibiotics. Part IX. Reactions of carzinophillin with DNA assayed by ethidium fluorescence

The reactions of the antitumor antibiotic carzinophillin (CZ) with native DNAs and synthetic polynucleotides have been examined by an ethidium fluorescence assay. CZ rapidly produces covalent linkage of the complementary strands of a variety of DNAs without activation. This process is accompanied by extensive alkylation, as detected by reduced fluorescence due to destruction of potential intercalation sites for ethidium. These processes which occur without loss of purine or pyrimidine bases show a preference for bonding to guanine groups (but not at the N-7 position). Examination of the reversibility of the cross-links suggests they involve one 'permanent' link to guanine and a second weaker linkage, possibly to a cytosine residue. Both cross-linking and alkylation show strong pH dependence and are favored at lower pH, suggesting that reactive sites on the antibiotic are basic. The addition of intercalating agents to DNA before treatment with CZ inhibits the cross-linking.

ULTRAVIOLET MUTAGENESIS AND ITS REPAIR IN AN ESCHERICHIA COLI STRAIN CONTAINING A NONSENSE CODON

ABSTRACT Ultraviolet mutagenesis and its repair were studied mainly in WU36-10-89, a uvr - strain of Escherichia coli containing a UAG mutation in a gene for leucine biosynthesis. Following ultraviolet (UV) irradiation revertants appearing with or without direct photoreactivation (PR) were classified according to the presence and type of suppressor they contained. We find UV mutation production to be quite specific. An analysis of revertants produced by UV indicates they are formed mainly from GC → AT and that the miscoding is due to a cytosine residue at the site of mutation in a cytosine-thymine (CT) dimer. We propose that the dimer serves as template during some aspects of repair replication and at the time of replication the C in the dimer directs the insertion of A in the complementary strand. We also note that C → A and T →G changes caused by a CT dimer occur much less frequently.