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1.  Osteoinduction on Acid and Heat Treated Porous Ti Metal Samples in Canine Muscle 
PLoS ONE  2014;9(2):e88366.
Samples of porous Ti metal were subjected to different acid and heat treatments. Ectopic bone formation on specimens embedded in dog muscle was compared with the surface characteristics of the specimen. Treatment of the specimens by H2SO4/HCl and heating at 600°C produced micrometer-scale roughness with surface layers composed of rutile phase of titanium dioxide. The acid- and heat-treated specimens induced ectopic bone formation within 6 months of implantation. A specimen treated using NaOH followed by HCl acid and then heat treatment produced nanometer-scale surface roughness with a surface layer composed of both rutile and anatase phases of titanium dioxide. These specimens also induced bone formation after 6 months of implantation. Both these specimens featured positive surface charge and good apatite-forming abilities in a simulated body fluid. The amount of the bone induced in the porous structure increased with apatite-forming ability and higher positive surface charge. Untreated porous Ti metal samples showed no bone formation even after 12 months. Specimens that were only heat treated featured a smooth surface composed of rutile. A mixed acid treatment produced specimens with micrometer-scale rough surfaces composed of titanium hydride. Both of them also showed no bone formation after 12 months. The specimens that showed no bone formation also featured almost zero surface charge and no apatite-forming ability. These results indicate that osteoinduction of these porous Ti metal samples is directly related to positive surface charge that facilitates formation of apatite on the metal surfaces in vitro.
doi:10.1371/journal.pone.0088366
PMCID: PMC3919776  PMID: 24520375
2.  DdrA, DdrD, and PprA: Components of UV and Mitomycin C Resistance in Deinococcus radiodurans R1 
PLoS ONE  2013;8(7):e69007.
Mutants created by deleting the ddrA, ddrB, ddrC, ddrD, and pprA loci of Deinococcus radiodurans R1alone and in all possible combinations of pairs revealed that the encoded gene products contribute to this species’ resistance to UV light and/or mitomycin C. Deleting pprA from an otherwise wild type cell sensitizes the resulting strain to UV irradiation, reducing viability by as much as eight fold relative to R1. If this deletion is introduced into a ΔddrA or ΔddrD background, the resulting strains become profoundly sensitive to the lethal effects of UV light. At a fluence of 1000 Jm-2, the ΔddrA ΔpprA and ΔddrD ΔpprA strains are 100- and 1000-fold more sensitive to UV relative to the strain that has only lost pprA. Deletion of ddrA results in a 100 fold increase in strain sensitivity to mitomycin C, but in backgrounds that combine a deletion of ddrA with deletions of either ddrC or ddrD, mitomycin resistance is restored to wild type levels. Inactivation of ddrB also increases D. radiodurans sensitivity to mitomycin, but unlike the ddrA mutant deleting ddrC or ddrD from a ΔddrB background further increases that sensitivity. Despite the effect that loss of these gene products has on DNA damage resistance, none appear to directly affect either excision repair or homologous recombination suggesting that they participate in novel processes that facilitate tolerance to UV light and interstrand crosslinks in this species.
doi:10.1371/journal.pone.0069007
PMCID: PMC3698191  PMID: 23840905
3.  An Alternative Model for the Early Peopling of Southern South America Revealed by Analyses of Three Mitochondrial DNA Haplogroups 
PLoS ONE  2012;7(9):e43486.
After several years of research, there is now a consensus that America was populated from Asia through Beringia, probably at the end of the Pleistocene. But many details such as the timing, route(s), and origin of the first settlers remain uncertain. In the last decade genetic evidence has taken on a major role in elucidating the peopling of the Americas. To study the early peopling of South America, we sequenced the control region of mitochondrial DNA from 300 individuals belonging to indigenous populations of Chile and Argentina, and also obtained seven complete mitochondrial DNA sequences. We identified two novel mtDNA monophyletic clades, preliminarily designated B2l and C1b13, which together with the recently described D1g sub-haplogroup have locally high frequencies and are basically restricted to populations from the extreme south of South America. The estimated ages of D1g and B2l, about ∼15,000 years BP, together with their similar population dynamics and the high haplotype diversity shown by the networks, suggests that they probably appeared soon after the arrival of the first settlers and agrees with the dating of the earliest archaeological sites in South America (Monte Verde, Chile, 14,500 BP). One further sub-haplogroup, D4h3a5, appears to be restricted to Fuegian-Patagonian populations and reinforces our hypothesis of the continuity of the current Patagonian populations with the initial founders. Our results indicate that the extant native populations inhabiting South Chile and Argentina are a group which had a common origin, and suggest a population break between the extreme south of South America and the more northern part of the continent. Thus the early colonization process was not just an expansion from north to south, but also included movements across the Andes.
doi:10.1371/journal.pone.0043486
PMCID: PMC3438176  PMID: 22970129
4.  Genes down-regulated in spaceflight are involved in the control of longevity in Caenorhabditis elegans 
Scientific Reports  2012;2:487.
How microgravitational space environments affect aging is not well understood. We observed that, in Caenorhabditis elegans, spaceflight suppressed the formation of transgenically expressed polyglutamine aggregates, which normally accumulate with increasing age. Moreover, the inactivation of each of seven genes that were down-regulated in space extended lifespan on the ground. These genes encode proteins that are likely related to neuronal or endocrine signaling: acetylcholine receptor, acetylcholine transporter, choline acetyltransferase, rhodopsin-like receptor, glutamate-gated chloride channel, shaker family of potassium channel, and insulin-like peptide. Most of them mediated lifespan control through the key longevity-regulating transcription factors DAF-16 or SKN-1 or through dietary-restriction signaling, singly or in combination. These results suggest that aging in C. elegans is slowed through neuronal and endocrine response to space environmental cues.
doi:10.1038/srep00487
PMCID: PMC3390002  PMID: 22768380
5.  FTO genotype and adiposity in children: physical activity levels influence the effect of the risk genotype in adolescent males 
European Journal of Human Genetics  2010;18(12):1339-1343.
Studies of the fat mass and obesity-associated (FTO) gene provide compelling evidence of genetic variation in the general population that influences fat levels and obesity risk. Studies of the interaction between genetic and environmental factors such as physical activity (PA) will promote the understanding of how lifestyle can modulate genetic contributions to obesity. In this study, we investigated the effect of FTO genotype, and interactions with PA or energy intake, in young children and adolescents. In all, 1–5-year-old children from the Growth, Exercise and Nutrition Epidemiological Study in preSchoolers (GENESIS) study (N=1980) and 11–18-year-old Greek adolescents (N=949) were measured for adiposity-related phenotypes and genotyped at the FTO single-nucleotide polymorphism (SNP) marker, rs17817449. Adolescents were classified as physically active or inactive based on self-reported levels of PA. In adolescents, FTO genotype influenced weight (P=0.001) and BMI (P=0.007). There was also a significant SNP*PA*gender interaction (P=0.028) on BMI, which reflected the association between FTO genotype and BMI in males (P=0.016), but not females (P=0.15), and significant SNP*PA interaction in males (P=0.007), but not females (P=0.74). The FTO genotype effect was more pronounced in inactive than active males. Inactive males homozygous for the G allele had a mean BMI 3 kg/m2 higher than T carriers (P=0.008). In the GENESIS study, no significant association between FTO genotype and adiposity was found. The present findings highlight PA as an important factor modifying the effect of FTO genotype.
doi:10.1038/ejhg.2010.131
PMCID: PMC3002848  PMID: 20717169
obesity; FTO; physical activity
6.  Lifespan-Extending Effects of Royal Jelly and Its Related Substances on the Nematode Caenorhabditis elegans 
PLoS ONE  2011;6(8):e23527.
Background
One of the most important challenges in the study of aging is to discover compounds with longevity-promoting activities and to unravel their underlying mechanisms. Royal jelly (RJ) has been reported to possess diverse beneficial properties. Furthermore, protease-treated RJ (pRJ) has additional pharmacological activities. Exactly how RJ and pRJ exert these effects and which of their components are responsible for these effects are largely unknown. The evolutionarily conserved mechanisms that control longevity have been indicated. The purpose of the present study was to determine whether RJ and its related substances exert a lifespan-extending function in the nematode Caenorhabditis elegans and to gain insights into the active agents in RJ and their mechanism of action.
Principal Findings
We found that both RJ and pRJ extended the lifespan of C. elegans. The lifespan-extending activity of pRJ was enhanced by Octadecyl-silica column chromatography (pRJ-Fraction 5). pRJ-Fr.5 increased the animals' lifespan in part by acting through the FOXO transcription factor DAF-16, the activation of which is known to promote longevity in C. elegans by reducing insulin/IGF-1 signaling (IIS). pRJ-Fr.5 reduced the expression of ins-9, one of the insulin-like peptide genes. Moreover, pRJ-Fr.5 and reduced IIS shared some common features in terms of their effects on gene expression, such as the up-regulation of dod-3 and the down-regulation of dod-19, dao-4 and fkb-4. 10-Hydroxy-2-decenoic acid (10-HDA), which was present at high concentrations in pRJ-Fr.5, increased lifespan independently of DAF-16 activity.
Conclusions/Significance
These results demonstrate that RJ and its related substances extend lifespan in C. elegans, suggesting that RJ may contain longevity-promoting factors. Further analysis and characterization of the lifespan-extending agents in RJ and pRJ may broaden our understanding of the gene network involved in longevity regulation in diverse species and may lead to the development of nutraceutical interventions in the aging process.
doi:10.1371/journal.pone.0023527
PMCID: PMC3153499  PMID: 21858156
7.  Stability of Mitochondrial Membrane Proteins in Terrestrial Vertebrates Predicts Aerobic Capacity and Longevity 
Genome Biology and Evolution  2011;3:1233-1244.
The cellular energy produced by mitochondria is a fundamental currency of life. However, the extent to which mitochondrial (mt) performance (power and endurance) is adapted to habitats and life strategies of vertebrates is not well understood. A global analysis of mt genomes revealed that hydrophobicity (HYD) of mt membrane proteins (MMPs) is much lower in terrestrial vertebrates than in fishes and shows a strong negative correlation with serine/threonine composition (STC). Here, we present evidence that this systematic feature of MMPs was crucial for the evolution of large terrestrial vertebrates with high aerobic capacity. An Arrhenius-type equation gave positive correlations between STC and maximum life span (MLS) in terrestrial vertebrates (with a few exceptions relating to the lifestyle of small animals with a high resting metabolic rate [RMR]) and negative correlations in secondary marine vertebrates, such as cetaceans and alligators (which returned from land to water, utilizing buoyancy with increased body size). In particular, marked STC increases in primates (especially hominoids) among placentals were associated with very high MLS values. We connected these STC increases in MMPs with greater stability of respiratory complexes by estimating the degradation of the Arrhenius plot given by accelerating mtRMR up to mt maximum metabolic rate. Both mtRMR and HYD in terrestrial vertebrates decreased with increasing body mass. Decreases in mtRMR raise MMP stability when high mobility is not required, whereas decreased HYD may weaken this stability under the hydrophobic environment of lipid bilayer. High maximal metabolic rates (5–10 RMR), which we postulate require high MMP mobility, presumably render MMPs more unstable. A marked rise in STC may therefore be essential to stabilize MMPs, perhaps as dynamic supercomplexes, via hydrogen bonds associated with serine/threonine motifs.
doi:10.1093/gbe/evr079
PMCID: PMC3227407  PMID: 21824868
vertebrate evolution; mitochondrial membrane protein stability; hydrophobicity; serine/threonine composition; aerobic capacity; longevity
8.  Enhanced Autophagy and Reduced Expression of Cathepsin D Are Related to Autophagic Cell Death in Epstein-Barr Virus-Associated Nasal Natural Killer/T-Cell Lymphomas: An Immunohistochemical Analysis of Beclin-1, LC3, Mitochondria (AE-1), and Cathepsin D in Nasopharyngeal Lymphomas 
Acta Histochemica et Cytochemica  2011;44(3):119-131.
This study investigated autophagy in 37 cases of nasopharyngeal lymphomas including 23 nasal natural killer (NK)/T-cell lymphomas (NKTCL), 3 cytotoxic T-cell lymphomas (cytotoxic-TML) and 9 B-cell lymphomas (BML) by means of antigen-retrieval immunohistochemistry of beclin-1, LC3, mitochondria (AE-1) and cathepsin D. Peculiar necrosis was noted in EBV+ lymphomas comprising 21 NKTCL, 2 cytotoxic-TML and 1 BML. Lymphomas without peculiar necrosis showed high expression of beclin-1, macrogranular cytoplasmal stain of LC3 with sporadic nuclear stain, a hallmark of autophagic cell death (ACD), some aggregated mitochondria and high expression of cathepsin D, suggesting a state of growth with enhanced autophagy with sporadic ACD. EBV+ NKTCL with the peculiar necrosis, showed significantly low level of macrogranular staining of LC3, aggregated mitochondria and low expression of cathepsin D in the cellular areas when degenerative lymphoma cells showed decreased beclin-1, significantly advanced LC3-labeled autophagy, residual aggregated mitochondria and significantly reduced expression of cathepsin D, suggesting advanced autophagy with regional ACD. Consequently it was suggested that enhanced autophagy and reduced expression of lysosomal enzymes induced regional ACD under EBV infection in NKTCL.
doi:10.1267/ahc.10024
PMCID: PMC3130144  PMID: 21753858
nasopharyngeal lymphoma; NKT-cell lymphoma nasal type; Epstein-Barr virus (EBV); autophagy; autophagic cell death; antigen retrieval immunohistochemistry (beclin-1; LC3; mitochondria (AE-1) and cathepsin D)
9.  Association of a polymorphism of BTN2A1 with dyslipidemia in East Asian populations 
We previously identified rs6929846 of the butyrophilin, subfamily 2, member A1 gene (BTN2A1) as a susceptibility locus for myocardial infarction in Japanese individuals by a genome-wide association study. The aim of the present study was to examine the relation of the rs6929846 polymorphism of BTN2A1 to dyslipidemia in Japanese and Korean populations, given that dyslipidemia is an important risk factor for myocardial infarction. A total of 10,953 individuals from three independent subject panels were examined. The relations of the rs6929846 polymorphism of BTN2A1 to serum concentrations of triglycerides, high-density lipoprotein (HDL)-cholesterol and low-density lipoprotein (LDL)-cholesterol were examined in each subject panel. The C→T polymorphism (rs6929846) of BTN2A1 was significantly associated with serum concentrations of triglycerides in Japanese subject panels A (P=0.0004) and B (P=0.0010), and in the Korean population (P=0.0095), with the minor T allele being related to an increased serum concentration of triglycerides. The rs6929846 was associated with serum concentrations of HDL-cholesterol in Japanese subject panels A (P=0.0047) and B (P=0.0015), with the T allele being related to a decreased serum concentration of HDL-cholesterol, but not in the Korean population. This polymorphism was associated with the serum concentration of LDL-cholesterol only in Japanese subject panel B (P=0.0059), with the T allele being related to an increased serum concentration of LDL-cholesterol. The results suggest that BTN2A1 may be a susceptibility gene for hypertriglyceridemia in East Asian populations and for low serum HDL-cholesterol in the Japanese population.
doi:10.3892/etm.2011.266
PMCID: PMC3440727  PMID: 22977569
genetics; polymorphism; dyslipidemia; hyperlipidemia hypercholesterolemia
10.  Association of a polymorphism of BTN2A1 with chronic kidney disease in individuals with or without hypertension or diabetes mellitus 
Hypertension and diabetes mellitus are important risk factors for chronic kidney disease (CKD). We previously showed that the C→T polymorphism (rs6929846) of BTN2A1 was significantly associated with myocardial infarction. The purpose of the present study was to examine an association of rs6929846 of BTN2A1 with CKD in individuals with or without hypertension or diabetes mellitus, thereby contributing to the personalized prevention of CKD in such individuals separately. The study population comprised 7,542 unrelated individuals, including 2,289 subjects with CKD [estimated glomerular filtration rate (eGFR) <60 ml/min/1.73 m2] and 5,253 controls (eGFR ≥60 ml/min/1.73 m2) with or without hypertension or diabetes mellitus. The Chi-square test, a multivariable logistic regression analysis with adjustment for covariates, as well as a stepwise forward selection procedure revealed that the C→T polymorphism (rs6929846) of BTN2A1 was significantly associated with CKD in normotensive individuals, in diabetic individuals and in individuals with hypertension and diabetes mellitus, or without either condition, with the T allele representing a risk factor for CKD. Stratification of subjects based on hypertension or diabetes mellitus may thus be important in order to achieve personalized prevention of CKD with the use of genetic information.
doi:10.3892/etm.2011.191
PMCID: PMC3440629  PMID: 22977505
genetics; polymorphism; chronic kidney disease; diabetes mellitus; hypertension
11.  SIRT1 Regulates Thyroid-Stimulating Hormone Release by Enhancing PIP5Kγ Activity through Deacetylation of Specific Lysine Residues in Mammals 
PLoS ONE  2010;5(7):e11755.
Background
SIRT1, a NAD-dependent deacetylase, has diverse roles in a variety of organs such as regulation of endocrine function and metabolism. However, it remains to be addressed how it regulates hormone release there.
Methodology/Principal Findings
Here, we report that SIRT1 is abundantly expressed in pituitary thyrotropes and regulates thyroid hormone secretion. Manipulation of SIRT1 level revealed that SIRT1 positively regulated the exocytosis of TSH-containing granules. Using LC/MS-based interactomics, phosphatidylinositol-4-phosphate 5-kinase (PIP5K)γ was identified as a SIRT1 binding partner and deacetylation substrate. SIRT1 deacetylated two specific lysine residues (K265/K268) in PIP5Kγ and enhanced PIP5Kγ enzyme activity. SIRT1-mediated TSH secretion was abolished by PIP5Kγ knockdown. SIRT1 knockdown decreased the levels of deacetylated PIP5Kγ, PI(4,5)P2, and reduced the secretion of TSH from pituitary cells. These results were also observed in SIRT1-knockout mice.
Conclusions/Significance
Our findings indicated that the control of TSH release by the SIRT1-PIP5Kγ pathway is important for regulating the metabolism of the whole body.
doi:10.1371/journal.pone.0011755
PMCID: PMC2909264  PMID: 20668706
12.  Adaptive Threonine Increase in Transmembrane Regions of Mitochondrial Proteins in Higher Primates 
PLoS ONE  2008;3(10):e3343.
Background
The mitochondrial (mt) gene tree of placental mammals reveals a very strong acceleration of the amino acid (AA) replacement rate and a change in AA compositional bias in the lineage leading to the higher primates (simians), in contrast to the nuclear gene tree. Whether this acceleration and compositional bias were caused by adaptive evolution at the AA level or directional mutation pressure at the DNA level has been vigorously debated.
Methodology/Principal Findings
Our phylogenetic analysis indicates that the rate acceleration in the simian lineage is accompanied by a marked increase in threonine (Thr) residues in the transmembrane helix regions of mt DNA-encoded proteins. This Thr increase involved the replacement of hydrophobic AAs in the membrane interior. Even after accounting for lack of independence due to phylogeny, a regression analysis reveals a statistical significant positive correlation between Thr composition and longevity in primates.
Conclusion/Significance
Because crucial roles of Thr and Ser in membrane proteins have been proposed to be the formation of hydrogen bonds enhancing helix-helix interactions, the Thr increase detected in the higher primates might be adaptive by serving to reinforce stability of mt proteins in the inner membrane. The correlation between Thr composition in the membrane interior and the longevity of animals is striking, especially because some mt functions are thought to be involved in aging.
doi:10.1371/journal.pone.0003343
PMCID: PMC2553178  PMID: 18836526
13.  Distilling Artificial Recombinants from Large Sets of Complete mtDNA Genomes 
PLoS ONE  2008;3(8):e3016.
Background
Large-scale genome sequencing poses enormous problems to the logistics of laboratory work and data handling. When numerous fragments of different genomes are PCR amplified and sequenced in a laboratory, there is a high immanent risk of sample confusion. For genetic markers, such as mitochondrial DNA (mtDNA), which are free of natural recombination, single instances of sample mix-up involving different branches of the mtDNA phylogeny would give rise to reticulate patterns and should therefore be detectable.
Methodology/Principal Findings
We have developed a strategy for comparing new complete mtDNA genomes, one by one, to a current skeleton of the worldwide mtDNA phylogeny. The mutations distinguishing the reference sequence from a putative recombinant sequence can then be allocated to two or more different branches of this phylogenetic skeleton. Thus, one would search for two (or three) near-matches in the total mtDNA database that together best explain the variation seen in the recombinants. The evolutionary pathway from the mtDNA tree connecting this pair together with the recombinant then generate a grid-like median network, from which one can read off the exchanged segments.
Conclusions
We have applied this procedure to a large collection of complete human mtDNA sequences, where several recombinants could be distilled by our method. All these recombinant sequences were subsequently corrected by de novo experiments – fully concordant with the predictions from our data-analytical approach.
doi:10.1371/journal.pone.0003016
PMCID: PMC2515346  PMID: 18714389
14.  Mitochondrial DNA Haplogroup D4a Is a Marker for Extreme Longevity in Japan 
PLoS ONE  2008;3(6):e2421.
We report results from the analysis of complete mitochondrial DNA (mtDNA) sequences from 112 Japanese semi-supercentenarians (aged above 105 years) combined with previously published data from 96 patients in each of three non-disease phenotypes: centenarians (99–105 years of age), healthy non-obese males, obese young males and four disease phenotypes, diabetics with and without angiopathy, and Alzheimer's and Parkinson's disease patients. We analyze the correlation between mitochondrial polymorphisms and the longevity phenotype using two different methods. We first use an exhaustive algorithm to identify all maximal patterns of polymorphisms shared by at least five individuals and define a significance score for enrichment of the patterns in each phenotype relative to healthy normals. Our study confirms the correlations observed in a previous study showing enrichment of a hierarchy of haplogroups in the D clade for longevity. For the extreme longevity phenotype we see a single statistically significant signal: a progressive enrichment of certain “beneficial” patterns in centenarians and semi-supercentenarians in the D4a haplogroup. We then use Principal Component Spectral Analysis of the SNP-SNP Covariance Matrix to compare the measured eigenvalues to a Null distribution of eigenvalues on Gaussian datasets to determine whether the correlations in the data (due to longevity) arises from some property of the mutations themselves or whether they are due to population structure. The conclusion is that the correlations are entirely due to population structure (phylogenetic tree). We find no signal for a functional mtDNA SNP correlated with longevity. The fact that the correlations are from the population structure suggests that hitch-hiking on autosomal events is a possible explanation for the observed correlations.
doi:10.1371/journal.pone.0002421
PMCID: PMC2408726  PMID: 18545700
15.  Establishment of a new method for precisely determining the functions of individual mitochondrial genes, using Dictyostelium cells 
BMC Genetics  2008;9:25.
Background
Disruption of mitochondrial genes may become a powerful tool for elucidating precisely the functions of individual mitochondrial genes. However, it is generally difficult to manipulate genetically mitochondrial genes, because 1) a mitochondrion is surrounded by inner and outer membranes, and 2) there are a large number of mtDNA copies in a single cell. This is the reason why we tried to establish a novel method for disrupting a certain mitochondrial gene (rps4), using Dictyostelium cells.
Results
Here, we have developed a new method for specifically disrupting a mitochondrial gene (rps4 ; ribosomal protein subunit S4), by a combination of homologous recombination and delivery of an appropriate restriction endonuclease (SfoI) into mitochondria. First, mitochondrially targeted SfoI whose expression is under control of the tetracycline (Tet)-regulated gene expression system was introduced into cells heteroplasmic with respect to the rps4 gene. Then, the heteroplasmic cells were produced by homologous recombination by use of the construct in which the unique SfoI site and the 5'-half of the rps4 coding region were deleted not to be digested by SfoI, and therefore their mitochondria have both the wild-type mtDNA and the mutant mtDNA with the disrupted rps4 gene. In response to removal of Tet from growth medium, SfoI was selectively delivered into mitochondria and digested only the wild-type mtDNA but not the mutated rps4. Thus one can gain rps4-null cells with only the mutated mtDNA, under the Tet-minus condition.
Conclusion
The mitochondrial gene-disruption method presented here must be widely useful for precisely determining the functions of individual mitochondrial genes. This is the first report to demonstrate complete and specific mitochondrial gene disruption.
doi:10.1186/1471-2156-9-25
PMCID: PMC2330148  PMID: 18366713
16.  Hyperthermic treatment of DMBA-induced rat mammary cancer using magnetic nanoparticles 
Background
We have developed magnetite cationic liposomes (MCLs) and applied them as a mediator of local hyperthermia. MCLs can generate heat under an alternating magnetic field (AMF). In this study, the in vivo effect of hyperthermia mediated by MCLs was examined using 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary cancer as a spontaneous cancer model.
Method
MCLs were injected into the mammary cancer and then subjected to an AMF.
Results
Four rats in 20 developed mammary tumors at more than 1 site in the body. The first-developed tumor in each of these 4 rats was selected and heated to over 43°C following administration of MCLs by an infusion pump. After a series of 3 hyperthermia treatments, treated tumors in 3 of the 4 rats were well controlled over a 30-day observation period. One of the 4 rats exhibited regrowth after 2 weeks. In this rat, there were 3 sites of tumor regrowth. Two of these regrowths were reduced in volume and regressed completely after 31 days, although the remaining one grew rapidly. These results indicated hyperthermia-induced immunological antitumor activity mediated by the MCLs.
Conclusion
Our results suggest that hyperthermic treatment using MCLs is effective in a spontaneous cancer model.
doi:10.1186/1477-044X-6-2
PMCID: PMC2266920  PMID: 18298831
18.  Identification of Mitochondrial DNA Polymorphisms That Alter Mitochondrial Matrix pH and Intracellular Calcium Dynamics 
PLoS Genetics  2006;2(8):e128.
Mitochondrial DNA (mtDNA) is highly polymorphic, and its variations in humans may contribute to individual differences in function as well as susceptibility to various diseases such as Parkinson disease, Alzheimer disease, bipolar disorder, and cancer. However, it is unclear whether and how mtDNA polymorphisms affect intracellular function, such as calcium signaling or pH regulation. Here we searched for mtDNA polymorphisms that have intracellular functional significance using transmitochondrial hybrid cells (cybrids) carrying ratiometric Pericam (RP), a fluorescent calcium indicator, targeted to the mitochondria and nucleus. By analyzing the entire mtDNA sequence in 35 cybrid lines, we found that two closely linked nonsynonymous polymorphisms, 8701A and 10398A, increased the basal fluorescence ratio of mitochondria-targeted RP. Mitochondrial matrix pH was lower in the cybrids with 8701A/10398A than it was in those with 8701G/10398G, suggesting that the difference observed by RP was mainly caused by alterations in mitochondrial calcium levels. Cytosolic calcium response to histamine also tended to be higher in the cybrids with 8701A/10398A. It has previously been reported that 10398A is associated with an increased risk of Parkinson disease, Alzheimer disease, bipolar disorder, and cancer, whereas 10398G associates with longevity. Our findings suggest that these mtDNA polymorphisms may play a role in the pathophysiology of these complex diseases by affecting mitochondrial matrix pH and intracellular calcium dynamics.
Synopsis
Mitochondria play important roles in energy production and regulation of intracellular calcium levels. Mitochondria have their own genetic material, mitochondrial DNA (mtDNA). In spite of its short length (16 kbp), mtDNA is highly variable among individuals and is thought to contribute to interindividual functional variability in energy-requiring activities such as intelligence and athletic performance. However, it is unclear whether mtDNA polymorphisms affect intracellular function and condition. Using transmitochondrial hybrid cells, the authors found two closely linked mtDNA polymorphisms, 10398A/G and 8701A/G, which cause alterations in mitochondrial pH and calcium concentration. Cytosolic calcium response to histamine tended to be different between transmitochondrial hybrid cells carrying these two mtDNA polymorphisms. It has been reported that the 10398A mtDNA polymorphism is a risk factor for Parkinson disease, Alzheimer disease, cancer, and bipolar disorder, whereas 10398G is associated with longevity. The present findings suggest that these mtDNA polymorphisms may play a role in the pathophysiology of these complex diseases by affecting mitochondrial matrix pH and intracellular calcium dynamics.
doi:10.1371/journal.pgen.0020128
PMCID: PMC1534079  PMID: 16895436
19.  Identification of Mitochondrial DNA Polymorphisms That Alter Mitochondrial Matrix pH and Intracellular Calcium Dynamics 
PLoS Genetics  2006;2(8):e128.
Mitochondrial DNA (mtDNA) is highly polymorphic, and its variations in humans may contribute to individual differences in function as well as susceptibility to various diseases such as Parkinson disease, Alzheimer disease, bipolar disorder, and cancer. However, it is unclear whether and how mtDNA polymorphisms affect intracellular function, such as calcium signaling or pH regulation. Here we searched for mtDNA polymorphisms that have intracellular functional significance using transmitochondrial hybrid cells (cybrids) carrying ratiometric Pericam (RP), a fluorescent calcium indicator, targeted to the mitochondria and nucleus. By analyzing the entire mtDNA sequence in 35 cybrid lines, we found that two closely linked nonsynonymous polymorphisms, 8701A and 10398A, increased the basal fluorescence ratio of mitochondria-targeted RP. Mitochondrial matrix pH was lower in the cybrids with 8701A/10398A than it was in those with 8701G/10398G, suggesting that the difference observed by RP was mainly caused by alterations in mitochondrial calcium levels. Cytosolic calcium response to histamine also tended to be higher in the cybrids with 8701A/10398A. It has previously been reported that 10398A is associated with an increased risk of Parkinson disease, Alzheimer disease, bipolar disorder, and cancer, whereas 10398G associates with longevity. Our findings suggest that these mtDNA polymorphisms may play a role in the pathophysiology of these complex diseases by affecting mitochondrial matrix pH and intracellular calcium dynamics.
Synopsis
Mitochondria play important roles in energy production and regulation of intracellular calcium levels. Mitochondria have their own genetic material, mitochondrial DNA (mtDNA). In spite of its short length (16 kbp), mtDNA is highly variable among individuals and is thought to contribute to interindividual functional variability in energy-requiring activities such as intelligence and athletic performance. However, it is unclear whether mtDNA polymorphisms affect intracellular function and condition. Using transmitochondrial hybrid cells, the authors found two closely linked mtDNA polymorphisms, 10398A/G and 8701A/G, which cause alterations in mitochondrial pH and calcium concentration. Cytosolic calcium response to histamine tended to be different between transmitochondrial hybrid cells carrying these two mtDNA polymorphisms. It has been reported that the 10398A mtDNA polymorphism is a risk factor for Parkinson disease, Alzheimer disease, cancer, and bipolar disorder, whereas 10398G is associated with longevity. The present findings suggest that these mtDNA polymorphisms may play a role in the pathophysiology of these complex diseases by affecting mitochondrial matrix pH and intracellular calcium dynamics.
doi:10.1371/journal.pgen.0020128
PMCID: PMC1534079  PMID: 16895436
20.  Characterization of Pathways Dependent on the uvsE, uvrA1, or uvrA2 Gene Product for UV Resistance in Deinococcus radiodurans 
Journal of Bacteriology  2005;187(11):3693-3697.
The genome of a radiation-resistant bacterium, Deinococcus radiodurans, contains one uvsE gene and two uvrA genes, uvrA1 and uvrA2. Using a series of mutants lacking these genes, we determined the biological significance of these components to UV resistance. The UV damage endonuclease (UvsE)-dependent excision repair (UVER) pathway and UvrA1-dependent pathway show some redundancy in their function to counteract the lethal effects of UV. Loss of these pathways does not cause increased sensitivity to UV mutagenesis, suggesting either that these pathways play no function in inducing mutations or that there are mechanisms to prevent mutation other than these excision repair pathways. UVER efficiently removes both cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) from genomic DNA. In contrast, the UvrA1 pathway does not significantly contribute to the repair of CPDs but eliminates 6-4PPs. Inactivation of uvrA2 does not result in a deleterious effect on survival, mutagenesis, or the repair kinetics of CPDs and 6-4PPs, indicating a minor role in resistance to UV. Loss of uvsE, uvrA1, and uvrA2 reduces but does not completely abolish the ability to eliminate CPDs and 6-4PPs from genomic DNA. The result indicates the existence of a system that removes UV damage yet to be identified.
doi:10.1128/JB.187.11.3693-3697.2005
PMCID: PMC1112038  PMID: 15901692
21.  Cysteinyl-tRNACys Formation in Methanocaldococcus jannaschii: the Mechanism Is Still Unknown 
Journal of Bacteriology  2004;186(1):8-14.
Most organisms form Cys-tRNACys, an essential component for protein synthesis, through the action of cysteinyl-tRNA synthetase (CysRS). However, the genomes of Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, and Methanopyrus kandleri do not contain a recognizable cysS gene encoding CysRS. It was reported that M. jannaschii prolyl-tRNA synthetase (C. Stathopoulos, T. Li, R. Longman, U. C. Vothknecht, H. D. Becker, M. Ibba, and D. Söll, Science 287:479-482, 2000; R. S. Lipman, K. R. Sowers, and Y. M. Hou, Biochemistry 39:7792-7798, 2000) or the M. jannaschii MJ1477 protein (C. Fabrega, M. A. Farrow, B. Mukhopadhyay, V. de Crécy-Lagard, A. R. Ortiz, and P. Schimmel, Nature 411:110-114, 2001) provides the “missing” CysRS activity for in vivo Cys-tRNACys formation. These conclusions were supported by complementation of temperature-sensitive Escherichia coli cysS(Ts) strain UQ818 with archaeal proS genes (encoding prolyl-tRNA synthetase) or with the Deinococcus radiodurans DR0705 gene, the ortholog of the MJ1477 gene. Here we show that E. coli UQ818 harbors a mutation (V27E) in CysRS; the largest differences compared to the wild-type enzyme are a fourfold increase in the Km for cysteine and a ninefold reduction in the kcat for ATP. While transformants of E. coli UQ818 with archaeal and bacterial cysS genes grew at a nonpermissive temperature, growth was also supported by elevated intracellular cysteine levels, e.g., by transformation with an E. coli cysE allele (encoding serine acetyltransferase) or by the addition of cysteine to the culture medium. An E. coli cysS deletion strain permitted a stringent complementation test; growth could be supported only by archaeal or bacterial cysS genes and not by archaeal proS genes or the D. radiodurans DR0705 gene. Construction of a D. radiodurans DR0705 deletion strain showed this gene to be dispensable. However, attempts to delete D. radiodurans cysS failed, suggesting that this is an essential Deinococcus gene. These results imply that it is not established that proS or MJ1477 gene products catalyze Cys-tRNACys synthesis in M. jannaschii. Thus, the mechanism of Cys-tRNACys formation in M. jannaschii still remains to be discovered.
doi:10.1128/JB.186.1.8-14.2004
PMCID: PMC303452  PMID: 14679218
22.  Evidence of Horizontal Transfer of the EcoO109I Restriction-Modification Gene to Escherichia coli Chromosomal DNA 
Journal of Bacteriology  1999;181(21):6822-6827.
A DNA fragment carrying the genes coding for EcoO109I endonuclease and EcoO109I methylase, which recognize the nucleotide sequence 5′-(A/G)GGNCC(C/T)-3′, was cloned from the chromosomal DNA of Escherichia coli H709c. The EcoO109I restriction-modification (R-M) system was found to be inserted between the int and psu genes from satellite bacteriophage P4, which were lysogenized in the chromosome at the P4 phage attachment site of the corresponding leuX gene observed in E. coli K-12 chromosomal DNA. The sid gene of the prophage was inactivated by insertion of one copy of IS21. These findings may shed light on the horizontal transfer and stable maintenance of the R-M system.
PMCID: PMC94149  PMID: 10542186
23.  Preserving Genome Integrity: The DdrA Protein of Deinococcus radiodurans R1 
PLoS Biology  2004;2(10):e304.
The bacterium Deinococcus radiodurans can withstand extraordinary levels of ionizing radiation, reflecting an equally extraordinary capacity for DNA repair. The hypothetical gene product DR0423 has been implicated in the recovery of this organism from DNA damage, indicating that this protein is a novel component of the D. radiodurans DNA repair system. DR0423 is a homologue of the eukaryotic Rad52 protein. Following exposure to ionizing radiation, DR0423 expression is induced relative to an untreated control, and strains carrying a deletion of the DR0423 gene exhibit increased sensitivity to ionizing radiation. When recovering from ionizing-radiation-induced DNA damage in the absence of nutrients, wild-type D. radiodurans reassembles its genome while the mutant lacking DR0423 function does not. In vitro, the purified DR0423 protein binds to single-stranded DNA with an apparent affinity for 3′ ends, and protects those ends from nuclease degradation. We propose that DR0423 is part of a DNA end-protection system that helps to preserve genome integrity following exposure to ionizing radiation. We designate the DR0423 protein as DNA damage response A protein.
Deinococcus radiodurans is able to repair radiation induced genome damage by virtue of a homologue of the eukaryotic Rad52 protein, which preserves genome integrity by protecting DNA ends
doi:10.1371/journal.pbio.0020304
PMCID: PMC515370  PMID: 15361932

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