CD4+/CD8+ Ratio and Growth Differentiation Factor 8 Levels in Peripheral Blood of Large Canine Males Are Useful Parameters to Build an Age Prediction Model.

PURPOSE: To build an age prediction model, we measured CD4+ and CD8+ cells, and humoral components in canine peripheral blood. MATERIALS AND METHODS: Large Belgian Malinois (BGM) and German Shepherd Dog (GSD) breeds (n=27), aged from 1 to 12 years, were used for this study. Peripheral bloods were obtained by venepuncture, then plasma and peripheral blood mononuclear cells (PBMCs) were separated immediately. Six myokines, including interleukin (IL)-6, IL-8, IL-15, leukemia inhibitory factor (LIF), growth differentiation factor 8 (GDF8), and GDF11 were measured from plasma and CD4+/CD8+ T-lymphocytes ratio were measured from PBMC. These parameters were then tested with age prediction models to find the best fit model. RESULTS: We found that the T-lymphocyte ratio (CD4+/CD8+) was significantly correlated with age (r=0.46, p=0.016). Among the six myokines, only GDF8 showed a significant correlation with age (r=0.52, p=0.005). Interestingly, these two markers showed better correlations in male dogs than females, and BGM breed than GSD. Using these two age biomarkers, we could obtain the best fit in a quadratic linear mixed model (r=0.77, p=3×10-6). CONCLUSIONS: Age prediction is a challenging task because of complication with biological age. Our quadratic linear mixed model using CD4+/CD8+ ratio and GDF8 level showed a meaningful age prediction.

Growth signaling and longevity in mouse models.

Reduction of insulin/insulin-like growth factor 1 (IGF1) signaling (IIS) extends the lifespan of various species. So far, several longevity mouse models have been developed containing mutations related to growth signaling deficiency by targeting growth hormone (GH), IGF1, IGF1 receptor, insulin receptor, and insulin receptor substrate. In addition, p70 ribosomal protein S6 kinase 1 (S6K1) knockout leads to lifespan extension. S6K1 encodes an important kinase in the regulation of cell growth. S6K1 is regulated by mechanistic target of rapamycin (mTOR) complex 1. The v-myc myelocytomatosis viral oncogene homolog (MYC)-deficient mice also exhibits a longevity phenotype. The gene expression profiles of these mice models have been measured to identify their longevity mechanisms. Here, we summarize our knowledge of long-lived mouse models related to growth and discuss phenotypic characteristics, including organ-specific gene expression patterns. [BMB Reports 2019; 52(1): 70-85].

Caloric Restriction-Induced Extension of Chronological Lifespan Requires Intact Respiration in Budding Yeast.

Caloric restriction (CR) has been shown to extend lifespan and prevent cellular senescence in various species ranging from yeast to humans. Many effects of CR may contribute to extend lifespan. Specifically, CR prevents oxidative damage from reactive oxygen species (ROS) by enhancing mitochondrial function. In this study, we characterized 33 single electron transport chain (ETC) gene-deletion strains to identify CR-induced chronological lifespan (CLS) extension mechanisms. Interestingly, defects in 17 of these 33 ETC gene-deleted strains showed loss of both respiratory function and CR-induced CLS extension. On the contrary, the other 16 respiration-capable mutants showed increased CLS upon CR along with increased mitochondrial membrane potential (MMP) and intracellular adenosine triphosphate (ATP) levels, with decreased mitochondrial superoxide generation. We measured the same parameters in the 17 non-respiratory mutants upon CR. CR simultaneously increased MMP and mitochondrial superoxide generation without altering intracellular ATP levels. In conclusion, respiration is essential for CLS extension by CR and is important for balancing MMP, ROS, and ATP levels.

Correlation between Antioxidant Enzyme Activity, Free Iron Content and Lipid Oxidation in Four Lines of Korean Native Chicken Meat.

This study was conducted to observe the association between antioxidant enzyme activity, free iron content and lipid oxidation of Korean native chicken (KNC) meat during refrigerated storage. Four lines of KNC (Yeonsan ogye, Hyunin black, Hoengseong yakdak and Hwangbong) were raised under similar conditions. A total of 16 roosters were randomly sampled and slaughtered at the age of 12 mon. The breast and thigh meats were stored aerobically for 10 d at 4℃. Although thigh meat had higher antioxidant enzyme activity, it was more susceptible to lipid oxidation and released more iron during storage than breast meat. Aerobic refrigerated storage for 10 d significantly decreased the activity of antioxidant enzymes and increased the amount of free iron and malondialdehyde. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were negatively correlated with lipid oxidation, whereas that of catalase was not. The amount of free iron was positively associated with lipid oxidation. We concluded that chicken line did not affect strongly on antioxidant enzyme activity and lipid oxidation in breast meat of KNC. However, the thigh meat of Hwangbong and Hyunin black had higher SOD and GSH-Px activity, respectively, and lower malondialdehyde contents than that of other chickens. SOD, GSH-Px and free iron play significant roles in meat lipid oxidation during refrigerated storage.

Whole-transcriptome analysis of mouse adipose tissue in response to short-term caloric restriction.

Caloric restriction (CR) has been shown to extend the lifespan of many species by improving cellular function and organismal health. Additionally, fat reduction by CR may play an important role in lengthening lifespan and preventing severe age-related diseases. Interestingly, CR induced the greatest transcriptome change in the epididymal fat of mice in our study. In this transcriptome analysis, we identified and categorized 446 genes that correlated with CR level. We observed down-regulation of several signaling pathways, including insulin/insulin-like growth factor 1 (insulin/IGF-1), epidermal growth factor (EGF), transforming growth factor beta (TGF-ß), and canonical wingless-type mouse mammary tumor virus integration site (Wnt). Many genes related to structural features, including extracellular matrix structure, cell adhesion, and the cytoskeleton, were down-regulated, with a strong correlation to the degree of CR. Furthermore, genes related to the cell cycle and adipogenesis were down-regulated. These biological processes are well-identified targets of insulin/IGF-1, EGF, TGF-ß, and Wnt signaling. In contrast, genes involved in specific metabolic processes, including the tricarboxylic acid cycle and the electron transport chain were up-regulated. We performed in silico analysis of the promoter sequences of CR-responsive genes and identified two associated transcription factors, Paired-like homeodomain 2 (Pitx2) and Paired box gene 6 (Pax6). Our results suggest that strict regulation of signaling pathways is critical for creating the optimal energy homeostasis to extend lifespan.

Mitochondrial Efficiency-Dependent Viability of Saccharomyces cerevisiae Mutants Carrying Individual Electron Transport Chain Component Deletions.

Mitochondria play a crucial role in eukaryotic cells; the mitochondrial electron transport chain (ETC) generates adenosine triphosphate (ATP), which serves as an energy source for numerous critical cellular activities. However, the ETC also generates deleterious reactive oxygen species (ROS) as a natural byproduct of oxidative phosphorylation. ROS are considered the major cause of aging because they damage proteins, lipids, and DNA by oxidation. We analyzed the chronological life span, growth phenotype, mitochondrial membrane potential (MMP), and intracellular ATP and mitochondrial superoxide levels of 33 single ETC component-deleted strains during the chronological aging process. Among the ETC mutant strains, 14 (sdh1Δ, sdh2Δ, sdh4Δ, cor1Δ, cyt1Δ, qcr7Δ, qcr8Δ, rip1Δ, cox6Δ, cox7Δ, cox9Δ, atp4Δ, atp7Δ, and atp17Δ) showed a significantly shorter life span. The deleted genes encode important elements of the ETC components succinate dehydrogenase (complex II) and cytochrome c oxidase (complex IV), and some of the deletions lead to structural instability of the membrane-F1F0-ATP synthase due to mutations in the stator stalk (complex V). These short-lived strains generated higher superoxide levels and produced lower ATP levels without alteration of MMP. In summary, ETC mutations decreased the life span of yeast due to impaired mitochondrial efficiency.

Enhancement of mitochondrial function correlates with the extension of lifespan by caloric restriction and caloric restriction mimetics in yeast.

Caloric restriction mimetics (CRMs) have been developed to mimic the effects of caloric restriction (CR). However, research reports for the effects of CRMs are often times inconsistent across different research groups. Therefore, in this study, we compared seven identified CRMs which extend the lifespans of various organisms including caffeine, curcumin, dapsone, metformin, rapamycin, resveratrol, and spermidine to CR for mitochondrial function in a single model, Saccharomyces cerevisiae. In this organism, rapamycin extended chronological lifespan (CLS), but other CRMs failed to extend CLS. Rapamycin enhanced mitochondrial function like CR did, but other CRMs did not. Both CR and rapamycin worked on mitochondrial function, but they worked at different windows of time during the chronological aging process.

Maintenance of cellular ATP level by caloric restriction correlates chronological survival of budding yeast.

The free radical theory of aging emphasizes cumulative oxidative damage in the genome and intracellular proteins due to reactive oxygen species (ROS), which is a major cause for aging. Caloric restriction (CR) has been known as a representative treatment that prevents aging; however, its mechanism of action remains elusive. Here, we show that CR extends the chronological lifespan (CLS) of budding yeast by maintaining cellular energy levels. CR reduced the generation of total ROS and mitochondrial superoxide; however, CR did not reduce the oxidative damage in proteins and DNA. Subsequently, calorie-restricted yeast had higher mitochondrial membrane potential (MMP), and it sustained consistent ATP levels during the process of chronological aging. Our results suggest that CR extends the survival of the chronologically aged cells by improving the efficiency of energy metabolism for the maintenance of the ATP level rather than reducing the global oxidative damage of proteins and DNA.

Effects of oxypeucedanin on global gene expression and MAPK signaling pathway in mouse neuroblastoma Neuro-2A cells.

Oxypeucedanin is a major coumarin aglycone that can be extracted from Ostericum koreanum. Coumarin aglycones have demonstrated various pharmacological effects, including anti-proliferation, anti-inflammation, and anti-pain. In this study, in order to understand the pharmacological properties of oxypeucedanin, we investigated global gene expression alteration in mouse neuroblastoma Neuro-2A cells. Results from the MTT assay indicated no decrease of cell viability up to 100 µM for 24 h. We measured gene expression profiles in Neuro-2A cells treated with either 10 µM or no oxypeucedanin for 24 h. We selected 128 differentially expressed genes (DEGs) for comparison of gene expression profiles by Bonferroni-adjusted p values (p < 0.1). Analysis of Gene Ontology (GO) biological process terms using the DEGs demonstrated the importance of protein metabolism, particularly ribosomal protein synthesis and protein degradation, intramembrane protein trafficking, and electron transport. Treatment with oxypeucedanin resulted in the downregulation of most DEGs for ribosomal protein synthesis and the electron transport chain (ETC). In contrast, most DEGs for protein degradation and cellular trafficking systems were upregulated. In addition, we found five upregulated DEGs for core and regulatory proteins involved in the mitogen-activated protein kinase (MAPK) signaling pathway. Independent translational validation of DEGs for MAPK signaling by immunoblot analysis showed consistent agreement with microarray data. Overall protein levels of Erk2 and p38MAPK were elevated, and their phosphorylated forms were also increased. These functional categories, based on transcriptional alteration and complicated modulation of MAPK signaling, might be underlying mechanisms responsible for the various pharmacological effects of oxypeucedanin.

Time-dependently expressed markers and the characterization for premature senescence induced by ionizing radiation in MCF7.

Senescence has been suggested as a defense mechanism to block sporadic induction of cancer cells. Radiation treatment induces proliferating cancer cells to turn into non-proliferating senescent cells in vitro. To characterize transcriptional reprogramming after radiation treatment, we measured the gene expression profiles of MCF7 at different time points after treatment. In these experiments, we found that IR induced premature senescence in MCF7 cells, and IR treatment resulted in significant changes in the expression of 305 marker genes (<1% FDR), which were strongly correlated (|r|>0.9) with IR treatment in a time-dependent manner. Functional analysis of these markers indicated that the dynamics of cytoskeletal structure and lysosomal activity gradually increased. The expression of maker genes for modulator proteins, that were responsible for the dynamics of actin stress fibers and focal adhesion, displayed a particularly strong positive correlation with senescence-associated (SA) morphological changes through time. We also observed a strong induction of genes related to lysosomal metabolic activity, which was accompanied by an increase in the number of SA-beta-Gal positive cells. However, the expression of genes for cell cycle progression, post-transcription and translation activities gradually decreased after radiation treatment. Especially, we observed clear cell cycle arrest specifically at the S and G2/M phases with consistent down-regulation of genes for microtubule assembly/disassembly or spindle biogenesis.

Transcriptional alteration of p53 related processes as a key factor for skeletal muscle characteristics in Sus scrofa.

The pig could be a useful model to characterize molecular aspects determining several delicate phenotypes because they have been bred for those characteristics. The Korean native pig (KNP) is a regional breed in Korea that was characterized by relatively high intramuscular fat content and reddish meat color compared to other western breeds such as Yorkshire (YS). YS grew faster and contained more lean muscle than KNP. We compared the KNP to Yorksire to find molecular clues determining muscle characteristics. The comparison of skeletal gene expression profiles between these two breeds showed molecular differences in muscle. We found 82 differentially expressed genes (DEGs) defined by fold change (more than 1.5 fold difference) and statistical significance (within 5% of false discovery rate). Functional analyses of these DEGs indicated up-regulation of most genes involved in cell cycle arrest, down-regulation of most genes involved in cellular differentiation and its inhibition, down-regulation of most genes encoding component of muscular-structural system, and up-regulation of most genes involved in diverse metabolism in KNP. Especially, DEGs in above-mentioned categories included a large number of genes encoding proteins directly or indirectly involved in p53 pathway. Our results indicated a possible role of p53 to determine muscle characteristics between these two breeds.

The impact of alpha-lipoic acid, coenzyme Q10 and caloric restriction on life span and gene expression patterns in mice.

We evaluated the efficacy of three dietary interventions started at middle age (14 months) to retard the aging process in mice. These were supplemental alpha-lipoic acid (LA) or coenzyme Q(10) (CQ) and caloric restriction (CR, a positive control). LA and CQ had no impact on longevity or tumor patterns compared with control mice fed the same number of calories, whereas CR increased maximum life span by 13% (p <.0001) and reduced tumor incidence. To evaluate these interventions at the molecular level, we used microarrays to monitor the expression of 9977 genes in hearts from young (5 months) and old (30 months) mice. LA, CQ, and CR inhibited age-related alterations in the expression of genes involved in the extracellular matrix, cellular structure, and protein turnover. However, unlike CR, LA and CQ did not prevent age-related transcriptional alterations associated with energy metabolism. LA supplementation lowered the expression of genes encoding major histocompatibility complex components and of genes involved in protein turnover and folding. CQ increased expression of genes involved in oxidative phosphorylation and reduced expression of genes involved in the complement pathway and several aspects of protein function. Our observations suggest that supplementation with LA or CQ results in transcriptional alterations consistent with a state of reduced oxidative stress in the heart, but that these dietary interventions are not as effective as CR in inhibiting the aging process in the heart.

Classification of multiple cancer types by multicategory support vector machines using gene expression data.

MOTIVATION: High-density DNA microarray measures the activities of several thousand genes simultaneously and the gene expression profiles have been used for the cancer classification recently. This new approach promises to give better therapeutic measurements to cancer patients by diagnosing cancer types with improved accuracy. The Support Vector Machine (SVM) is one of the classification methods successfully applied to the cancer diagnosis problems. However, its optimal extension to more than two classes was not obvious, which might impose limitations in its application to multiple tumor types. We briefly introduce the Multicategory SVM, which is a recently proposed extension of the binary SVM, and apply it to multiclass cancer diagnosis problems. RESULTS: Its applicability is demonstrated on the leukemia data (Golub et al., 1999) and the small round blue cell tumors of childhood data (Khan et al., 2001). Comparable classification accuracy shown in the applications and its flexibility render the MSVM a viable alternative to other classification methods. SUPPLEMENTARY INFORMATION: http://www.stat.ohio-state.edu/~yklee/msvm.htm