Yeast Cells Expressing the Human Mitochondrial DNA Polymerase Reveal Correlations between Polymerase Fidelity and Human Disease Progression

Mutations in the human mitochondrial polymerase (polymerase-γ (Pol-γ)) are associated with various mitochondrial disorders, including mitochondrial DNA (mtDNA) depletion syndrome, Alpers syndrome, and progressive external opthamalplegia. To correlate biochemically quantifiable defects resulting from point mutations in Pol-γ with their physiological consequences, we created “humanized” yeast, replacing the yeast mtDNA polymerase (MIP1) with human Pol-γ. Despite differences in the replication and repair mechanism, we show that the human polymerase efficiently complements the yeast mip1 knockouts, suggesting common fundamental mechanisms of replication and conserved interactions between the human polymerase and other components of the replisome. We also examined the effects of four disease-related point mutations (S305R, H932Y, Y951N, and Y955C) and an exonuclease-deficient mutant (D198A/E200A). In haploid cells, each mutant results in rapid mtDNA depletion, increased mutation frequency, and mitochondrial dysfunction. Mutation frequencies measured in vivo equal those measured with purified enzyme in vitro. In heterozygous diploid cells, wild-type Pol-γ suppresses mutation-associated growth defects, but continuous growth eventually leads to aerobic respiration defects, reduced mtDNA content, and depolarized mitochondrial membranes. The severity of the Pol-γ mutant phenotype in heterozygous diploid humanized yeast correlates with the approximate age of disease onset and the severity of symptoms observed in humans.     

Nitrosothiol Formation and Protection against Fenton Chemistry by Nitric Oxide-induced Dinitrosyliron Complex Formation from Anoxia-initiated Cellular Chelatable Iron Increase

Dinitrosyliron complexes (DNIC) have been found in a variety of pathological settings associated with ^•NO. However, the iron source of cellular DNIC is unknown. Previous studies on this question using prolonged ^•NO exposure could be misleading due to the movement of intracellular iron among different sources. We here report that brief ^•NO exposure results in only barely detectable DNIC, but levels increase dramatically after 1–2 h of anoxia. This increase is similar quantitatively and temporally with increases in the chelatable iron, and brief ^•NO treatment prevents detection of this anoxia-induced increased chelatable iron by deferoxamine. DNIC formation is so rapid that it is limited by the availability of ^•NO and chelatable iron. We utilize this ability to selectively manipulate cellular chelatable iron levels and provide evidence for two cellular functions of endogenous DNIC formation, protection against anoxia-induced reactive oxygen chemistry from the Fenton reaction and formation by transnitrosation of protein nitrosothiols (RSNO). The levels of RSNO under these high chelatable iron levels are comparable with DNIC levels and suggest that under these conditions, both DNIC and RSNO are the most abundant cellular adducts of ^•NO.     

Focal adhesion kinase maintains,but not increases the adhesion of dental pulp cells

Focal adhesion kinase (FAK) functions as a key enzyme in the integrin-mediated adhesion-signalling pathway. Here, we aimed to investigate the effects of FAK on adhesion of human dental pulp (HDP) cells. We transfected lentiviral vectors to silence or overexpress FAK in HDP cells ex vivo. Early cell adhesion, cell survival and focal contacts (FCs)-related proteins (FAK and paxillin) were examined. By using immunofluorescence, the formation of FCs and cytoskeleton was detected, respectively. We found that both adhesion and survival of HDP cells were suppressed by FAK inhibition. However, FAK overexpression slightly inhibited cell adhesion and exhibited no change in cell survival compared with the control. A thick rim of cytoskeleton accumulated and smaller dot-shaped FCs appeared in FAK knockdown cells. Phosphorylation of paxillin (p-paxillin) was inhibited in FAK knockdown cells, verifying that the adhesion was inhibited. Less cytoskeleton and elongated FCs were observed in FAK-overexpressed cells. However, p-paxillin had no significant difference compared with the control. In conclusion, the data suggest that FAK maintains cell adhesion, survival and cytoskeleton formation, but excessive FAK has no positive effects on these aspects.     

EPA,not DHA,prevents fibrosis in pressure overload-induced heart failure: potential role of free fatty acid receptor 4

Heart failure with preserved ejection fraction (HFpEF) is half of all HF, but standard HF therapies are ineffective. Diastolic dysfunction, often secondary to interstitial fibrosis, is common in HFpEF. Previously, we found that supra-physiologic levels of ω3-PUFAs produced by 12 weeks of ω3-dietary supplementation prevented fibrosis and contractile dysfunction following pressure overload [transverse aortic constriction (TAC)], a model that resembles aspects of remodeling in HFpEF. This raised several questions regarding ω3-concentration-dependent cardioprotection, the specific role of EPA and DHA, and the relationship between prevention of fibrosis and contractile dysfunction. To achieve more clinically relevant ω3-levels and test individual ω3-PUFAs, we shortened the ω3-diet regimen and used EPA- and DHA-specific diets to examine remodeling following TAC. The shorter diet regimen produced ω3-PUFA levels closer to Western clinics. Further, EPA, but not DHA, prevented fibrosis following TAC. However, neither ω3-PUFA prevented contractile dysfunction, perhaps due to reduced uptake of ω3-PUFA. Interestingly, EPA did not accumulate in cardiac fibroblasts. However, FFA receptor 4, a G protein-coupled receptor for ω3-PUFAs, was sufficient and required to block transforming growth factor β1-fibrotic signaling in cultured cardiac fibroblasts, suggesting a novel mechanism for EPA. In summary, EPA-mediated prevention of fibrosis could represent a novel therapy for HFpEF.     

Identification of potentially critical genes in the development of heart failure after ST-segment elevation myocardial infarction (STEMI)

Heart failure (HF) remains a common complication after acute ST-segment elevation myocardial infarction (STEMI). Here, we aim to identify critical genes related to the developed HF in patients with STEMI using bioinformatics analysis. The microarray data of GSE59867, including peripheral blood samples from nine patients with post-infarct HF and eight patients without post-infarct HF, were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) between HF and non-HF groups were screened by LIMMA package. Functional enrichment analyses of DEGs were conducted, followed by construction of a protein-protein interaction (PPI) network. The dynamic messenger RNA (mRNA) level of the hub genes during the follow-up was analyzed to further elucidate their role in HF development. A total of 58 upregulated and 75 downregulated DEGs were screen out. They were mainly enriched in biological processes about inflammatory response, extracellular matrix organization, response to cAMP, immune response, and positive regulation of cytosolic calcium ion concentration. Pathway analysis revealed that the DEGs were also involved in hematopoietic cell lineage, pathways in cancer, and extracellular matrix-receptor interaction. In the PPI network consisting of 58 nodes and 72 interactions, CXCL8 (degree = 15), THBS1 (degree = 8), FOS (degree = 7), and ITGA2B (degree = 6) were identified as the hub genes. In the comparison of patients with and without post-infarct HF, the mRNA level of these hub genes were all higher within 30 days but reached similar at 6 months after STEMI. In conclusion, CXCL8, THBS1, FOS, and ITGA2B may play important roles in the development of HF after acute STEMI.     

Quantitative structure-activity relationships of structurally similar odorless and odoriferous benzenoid musks

To reveal the difference of molecular property between structurallysimilar odorless and odoriferous musk compounds, 10 pairs ofbenzenoids (monocyclic-, dicyclic- and tricyclic-) were examined.Molecular structures of all compounds were optimized by MNDO(modified neglect of diatomic differential overlap) consideringconformation. Parameters effective in discriminating two groups,group A of 10 odorless compounds and group B of 10 musk odorcompounds, were searched from 34 candidate parameters by adaptiveleast squares. The best three parameters found were log P value(octanol/water partition coefficient), the longest side lengthof hexahedron circumscribing a molecule, and the parameter whichexpresses structural hindrance to the functional group whena molecule approaches the receptor site. The two groups of compoundswere completely discriminated using these three parameters.