Caregiving centenarians: Cross-national comparison in Caregiver-Burden between the United States and Japan.

Background and Objectives: The personal distress associated with caring for a family member has been well documented; however, questions about the burden of caregiving for centenarians and cross-national differences in the caregiving context, remain unanswered.Research Design and Methods: This study includes reports by caregivers of 538 near-centenarians and centenarians in the U.S. and Japan: 234 from the Georgia Centenarian Study and 304 from the Tokyo Centenarian Study. Basic descriptive and multivariate regression analyses were conducted. Mean levels of caregiver burden and near-centenarian and centenarians' characteristics (as predictors) for caregiver burden were compared between the U.S. and Japan. The near-centenarian and centenarians' functional capacity and personality were assessed as predictors.Results: Differential predictive patterns in caregiver burden were found in the two groups. In the U.S., near-centenarian and centenarians' agreeableness and conscientiousness were negatively associated with caregiver burden; whereas the near-centenarian and centenarians' neuroticism and number of diseases were positively associated with caregiver burden. In Japan, the near-centenarian and centenarians' activities of daily living, openness, and agreeableness were negatively associated with caregiving burden. Interaction effects between functional capacity and personality, on caregiver burden were observed only in the U.S. In the U.S., higher levels of agreeableness and openness significantly changed the level of caregiver burden associated with vision problems and a greater number of diseases.Discussion and Implications: Cross-national comparative predictors of caregiving burden between the two countries emphasized that caring for centenarians should be understood in the caregiving context, as well as the social context.

Beneficial effects of Lactobacillus casei strain Shirota on academic stress-induced sleep disturbance in healthy adults: a double-blind, randomised, placebo-controlled trial.

The present study examined whether Lactobacillus casei strain Shirota (LcS) improves sleep quality under psychological stress. A double-blind, placebo-controlled trial was conducted in healthy 4th year medical students exposed to academic examination stress. The trial was repeated over two consecutive years in different groups of students, and the data were pooled. For 8 weeks prior to and 3 weeks after a national standardised examination, a total of 48 and 46 subjects received a daily dose of 100 ml of LcS-fermented milk or non-fermented placebo milk, respectively. Study measures included subjective anxiety, overnight single-channel electroencephalography (EEG) recordings, and the Oguri-Shirakawa-Azumi (OSA) sleep inventory scores of subjective sleep quality. Total OSA scores were significantly lower than baseline on the day before the exam and recovered after the exam, indicating a stress-induced decline in sleep quality. There was a significant positive effect of LcS treatment on OSA factors for sleepiness on rising and sleep length. Sleep latency measured by EEG lengthened as the exam approached in the placebo group but was significantly suppressed in the LcS group. The percentage of stage 3 non-REM (N3) sleep decreased in the placebo group as the exam approached, whereas it was maintained in the LcS group throughout the trial. Delta power during the first sleep cycle, measured as an index of sleep intensity, increased as the exam approached in the LcS group and was significantly higher than in the placebo group. These findings suggest that daily consumption of LcS may help to maintain sleep quality during a period of increasing stress. The observed retention of N3 sleep and increased delta power in the LcS group may have contributed to higher perceived sleep satisfaction.

Longitudinal study of factors affecting taste sense decline in old-old individuals.

The sense of taste plays a pivotal role for personal assessment of the nutritional value, safety and quality of foods. Although it is commonly recognised that taste sensitivity decreases with age, alterations in that sensitivity over time in an old-old population have not been previously reported. Furthermore, no known studies utilised comprehensive variables regarding taste changes and related factors for assessments. Here, we report novel findings from a 3-year longitudinal study model aimed to elucidate taste sensitivity decline and its related factors in old-old individuals. We utilised 621 subjects aged 79-81 years who participated in the Septuagenarians, Octogenarians, Nonagenarians Investigation with Centenarians Study for baseline assessments performed in 2011 and 2012, and then conducted follow-up assessments 3 years later in 328 of those. Assessment of general health, an oral examination and determination of taste sensitivity were performed for each. We also evaluated cognitive function using Montreal Cognitive Assessment findings, then excluded from analysis those with a score lower than 20 in order to secure the validity and reliability of the subjects' answers. Contributing variables were selected using univariate analysis, then analysed with multivariate logistic regression analysis. We found that males showed significantly greater declines in taste sensitivity for sweet and sour tastes than females. Additionally, subjects with lower cognitive scores showed a significantly greater taste decrease for salty in multivariate analysis. In conclusion, our longitudinal study revealed that gender and cognitive status are major factors affecting taste sensitivity in geriatric individuals.

Dietary Intake Is Associated with Occlusal Force Rather Than Number of Teeth in 80-y-Old Japanese.

There has been a growing interest in the association between the number of teeth and dietary intake in older populations. However, people around the age of 80 y have frequently lost most of their teeth, and dental prostheses replacing the missing teeth play an important role in masticatory function. Therefore, masticatory function cannot be evaluated by the number of teeth alone. The occlusal force of the complete dental arches is an index of masticatory function, reflecting not only the number of teeth, but the effect of removable dentures. The purpose of this cross-sectional study was to determine the relative importance of the number of teeth and occlusal force in association with dietary intake in 80-y-old Japanese people. This study included 760 community-dwelling Japanese people aged 79 y to 81 y. The authors measured bilateral maximal occlusal force in the intercuspal position using pressure-sensitive sheets. Removable denture wearers kept their dentures in place during the measurements. Energy-adjusted food groups and nutrient intake during the preceding month were assessed by a brief self-administered diet history questionnaire. The authors assessed linear trends in food and nutrient intake in relation to the number of teeth and occlusal force after adjusting for gender and socioeconomic status (education level, financial status, family structure, resident area and BMI). P values of < 0.05 were considered to be statistically significant. The authors found that the number of teeth was not associated with the energy-adjusted intake of any food group examined. In contrast, a decline in occlusal force was significantly associated with a lower intake of vegetables, fish and shellfish, protein, polyunsaturated fatty acids, dietary fiber and most vitamins and minerals ( P for trend < 0.05). We conclude that food and nutrient intake was more closely associated with occlusal force than the number of teeth in community-dwelling Japanese people aged 79 y to 81 y. Knowledge Transfer Statement: This cross-sectional study of older Japanese people showed that, after controlling for considerable covariates, occlusal force rather than the number of teeth is positively associated with energy-adjusted intake of vegetables, fish and shellfish, protein, polyunsaturated fatty acids, dietary fiber and most of vitamins and minerals. This means that reduced occlusal force may unconsciously lead older people toward a habitual unhealthy dietary intake. Older people have frequently lost most of their teeth and require prosthetics to restore masticatory function. Bilateral occlusal force is therefore a better measure of masticatory function than the number of remaining teeth. Our findings suggest that prosthetic rehabilitation is a significant factor in the prevention and management of chronic diseases and frailty through better dietary intake in older populations.

Factors related to taste sensitivity in elderly: cross-sectional findings from SONIC study.

The sense of taste is important, as it allows for assessment of nutritional value, as well as safety and quality of foods, with several factors suggested to be associated with taste sensitivity. However, comprehensive variables regarding taste and related factors have not been utilised in previous studies for assessments of sensitivity. In the present study, we performed cross-sectional analyses of taste sensitivity and related factors in geriatric individuals who participated in the SONIC Study. We analysed 2 groups divided by age, 69-71 years (young-old, n = 687) and 79-81 years (old-old, n = 621), and performed a general health assessment, an oral examination and determination of taste sensitivity. Contributing variables were selected by univariate analysis and then subjected to multivariate logistic regression analysis. In both groups, females showed significantly better sensitivity for bitter and sour tastes. Additionally, higher cognitive scores for subjects with a fine taste for salty were commonly seen in both groups, while smoking, drinking, hypertension, number of teeth, stimulated salivary flow salt intake and years of education were also shown to be associated with taste sensitivity. We found gender and cognitive status to be major factors affecting taste sensitivity in geriatric individuals.

Probiotic Lactobacillus casei strain Shirota relieves stress-associated symptoms by modulating the gut-brain interaction in human and animal models.

BACKGROUND: This study aimed to examine the effects of Lactobacillus casei strain Shirota (LcS) on gut-brain interactions under stressful conditions. METHODS: Three double-blind, placebo-controlled trials were conducted to examine the effects of LcS on psychological and physiological stress responses in healthy medical students under academic examination stress. Subjects received LcS-fermented milk or placebo daily for 8 weeks prior to taking a national standardized examination. Subjective anxiety scores, salivary cortisol levels, and the presence of physical symptoms during the intervention were pooled and analyzed. In the animal study, rats were given feed with or without LcS for 2 weeks, then submitted to water avoidance stress (WAS). Plasma corticosterone concentration and the expression of cFos and corticotropin releasing factor (CRF) in the paraventricular nucleus (PVN) were measured immediately after WAS. In an electrophysiological study, gastric vagal afferent nerve activity was monitored after intragastric administration of LcS to urethane-anesthetized rats. KEY RESULTS: Academic stress-induced increases in salivary cortisol levels and the incidence rate of physical symptoms were significantly suppressed in the LcS group compared with the placebo group. In rats pretreated with LcS, WAS-induced increases in plasma corticosterone were significantly suppressed, and the number of CRF-expressing cells in the PVN was reduced. Intragastric administration of LcS stimulated gastric vagal afferent activity in a dose-dependent manner. CONCLUSIONS & INFERENCES: These findings suggest that LcS may prevent hypersecretion of cortisol and physical symptoms under stressful conditions, possibly through vagal afferent signaling to the brain and reduced stress reactivity in the PVN.

Association of Occlusal Force with Cognition in Independent Older Japanese People.

Recent longitudinal studies have shown the influence of multiple tooth loss on cognitive impairment, and earlier studies suggested that periodontal disease was related to cognitive decline. Tooth loss is associated with reduced masticatory function, which may affect stimulation of the central nervous system and dietary intake. Although some studies have reported a relationship between chewing ability and cognitive function, no studies have examined this area in terms of objective oral function. The aim of this study was to examine the association of occlusal force with cognitive decline in the preclinical stage among older people with higher-level functional capacity. This cross-sectional study for community-dwelling older people living in urban and rural areas in Japan examined 994 persons in the 70-y group (age range, 69-71 y) and 968 persons in the 80-y group (age range, 79-81 y). Retention of higher-level competence was defined according to the Tokyo Metropolitan Institute of Gerontology Index of Competence. Cognitive function was measured with the Japanese version of the Montreal Cognitive Assessment (MoCA-J). Oral status and function were assessed by the number of remaining teeth, periodontal pocket depth, and maximal occlusal force. Associations between the MoCA-J score and occlusal force were examined by bivariate and multivariate analysis. Approximately one-half of the participants retained higher-level functional capacity and were included in the analysis. Multiple regression analysis showed that occlusal force was significantly related to cognitive function after controlling for possible predictors including age, sex, socioeconomic status, medical condition, and handgrip strength. The number of remaining teeth and periodontal pocket depth were not significantly associated with cognitive function. Among community-dwelling older people with retained competence, maximal occlusal force was positively associated with their cognitive function. These results suggest that oral function might be a predictor for preclinical cognitive decline. Knowledge Transfer Statement: Multiple regression analysis showed that occlusal force was significantly related to cognition after controlling for possible predictors including handgrip strength as an indicator of general muscle strength, suggesting the independence of oral function. The number of remaining teeth did not have this association. The majority of older people have lost teeth and have received prosthodontic treatment, and their occlusal force is determined not only by the number of remaining teeth but also by prosthetic rehabilitation. These results can be used by clinicians focusing on prevention of tooth loss among the entire population, as well as to encourage partially edentulous and fully edentulous patients to restore their oral function with prostheses in order to eliminate a possible risk factor for cognitive impairment.

Association of periodontal status with occlusal force and food acceptability in 70-year-old adults: from SONIC Study.

This cross-sectional study aimed to investigate the association of periodontal status with occlusal force and food acceptability. We hypothesised that mastication deteriorated with reduced periodontal support, even when posterior occlusal contacts with natural teeth were maintained and the patients remained clinically asymptomatic. Participants were 482 independently living 69-71-year-olds, classified as Eichner's group A, having no mobile teeth and no periodontal symptoms. The periodontal probing depth (PPD) and restoration status of each tooth were examined. Occlusal force in the intercuspal position was measured with pressure-sensitive films. Food acceptability was evaluated from the difficulty experienced in chewing apples, grilled beef, and hard rice crackers. Multivariate regression analysis was performed to investigate the association of periodontal status with occlusal force and food acceptability. A P-value of <0.05 was considered statistically significant. Multiple linear regression analysis showed that occlusal force had significant negative associations with maximal PPD (standardised partial regression coefficient (ß) = -0.121) after controlling for gender, handgrip strength, number of teeth, and percentage of restored teeth. Approximately 15% of participants were included in the compromised food acceptability group. Logistic regression analyses showed that compromised food acceptability was significantly associated with PPD, after controlling for gender, number of teeth, and percentage of restored teeth. Periodontal probing depth (PPD) was significantly correlated with occlusal force and self-rated food acceptability after controlling for the possible confounding factors in septuagenarians, even those with complete posterior occlusal contacts and no tooth mobility.

Reptiles and mammals have differentially retained long conserved noncoding sequences from the amniote ancestor.

Many noncoding regions of genomes appear to be essential to genome function. Conservation of large numbers of noncoding sequences has been reported repeatedly among mammals but not thus far among birds and reptiles. By searching genomes of chicken (Gallus gallus), zebra finch (Taeniopygia guttata), and green anole (Anolis carolinensis), we quantified the conservation among birds and reptiles and across amniotes of long, conserved noncoding sequences (LCNS), which we define as sequences ≥500 bp in length and exhibiting ≥95% similarity between species. We found 4,294 LCNS shared between chicken and zebra finch and 574 LCNS shared by the two birds and Anolis. The percent of genomes comprised by LCNS in the two birds (0.0024%) is notably higher than the percent in mammals (<0.0003% to <0.001%), differences that we show may be explained in part by differences in genome-wide substitution rates. We reconstruct a large number of LCNS for the amniote ancestor (ca. 8,630) and hypothesize differential loss and substantial turnover of these sites in descendent lineages. By contrast, we estimated a small role for recruitment of LCNS via acquisition of novel functions over time. Across amniotes, LCNS are significantly enriched with transcription factor binding sites for many developmental genes, and 2.9% of LCNS shared between the two birds show evidence of expression in brain expressed sequence tag databases. These results show that the rate of retention of LCNS from the amniote ancestor differs between mammals and Reptilia (including birds) and that this may reflect differing roles and constraints in gene regulation.

Do personality characteristics predict longevity? Findings from the Tokyo Centenarian Study.

To explore whether personality influences longevity we examined the personality characteristics of centenarians. We developed a new method that compares an actual personality test score for centenarians with a predicted test score for a 100-year-old, calculated from younger controls. The participants consisted of 70 cognitively intact Japanese centenarians aged 100-106 years and 1812 elderly people aged 60-84 years, all residents of Tokyo. The NEO five factor inventory (NEO-FFI) was used to assess the "big five" personality traits: neuroticism, extraversion, openness, agreeableness, and conscientiousness. The results showed higher openness in both male and female centenarians, and higher conscientiousness and extraversion in female centenarians, as compared to controls. These results suggest that high scores in the specific personality traits conscientiousness, extraversion, and openness, are associated with longevity. We speculate that these personality traits contribute to longevity through health-related behavior, stress reduction, and adaptation to the challenging problems of the "oldest old".

Identification and characterization of the miniature pig Huntington's disease gene homolog: evidence for conservation and polymorphism in the CAG triplet repeat.

Huntington's disease (HD) is associated with a significant expansion of a CAG trinucleotide repeat, which results in a lengthened polyglutamine tract in the single gene product, huntingtin, on human 4p16.3. We isolated cDNA clones that encompassed the entire coding sequence of the miniature pig HD gene (Sus HD) from two porcine testis cDNA libraries. The cDNA contig revealed a 12,749-nucleotide transcript coding for a 345-kDa protein (3139 amino acid residues), which exhibited 96% peptide sequence homology to human huntingtin. Northern blot analysis revealed that the Sus HD gene was ubiquitously expressed as two large transcripts of approximately 11 and 13 kb in size in all the tested tissues, much like the human HD gene. The CAG trinucleotide repeat was found to be interrupted by CAA triplets and to encode 17 or 18 consecutive glutamine residues. In our laboratory stock of miniature pig, three allotypes in the triplet repeat sequence were found. Thus, the Sus HD gene closely resembles its human counterpart in terms of sequence and expression pattern. In particular, human-miniature pig similarities in the normal length of the CAG triplet repeat as well as its repeat-number polymorphism may indicate that miniature pig would provide a good animal model for Huntington's disease.

The RS447 human megasatellite tandem repetitive sequence encodes a novel deubiquitinating enzyme with a functional promoter.

We have recently identified a tandem repetitive DNA sequence that we designated the RS447 megasatellite. In this study, we describe a functional novel deubiquitinating enzyme (USP17, 60 kDa) gene that is intronless and encoded by the RS447 repeating unit. Northern blot analysis in conjunction with 5' and 3' rapid amplification of cDNA ends confirmed the presence of poly(A)(+) containing RS447 RNA in normal cells. We also identified a functional promoter sequence as well as an open reading frame within every RS447 repeat. When USP17 was expressed in Escherichia coli, it exhibited deubiquitinating activity in vivo. An antibody against USP17 detected USP17 protein in human cells. Our results indicate that the RS447 repeating unit on this megasatellite repeat codes for and actively expresses a functional deubiquitinating enzyme. Although it is expressed ubiquitously in human tissues, USP17 exhibited a unique expression pattern in that its complementary strand is transcribed as an antisense transcript that may modulate the level of USP17 expression in the human brain.

Different mutation frequencies and spectra among organs by N-methyl-N-nitrosourea in rpsL (strA) transgenic mice.

The frequencies and spectra of N-methyl-N-nitrosourea (MNU)-induced in vivo somatic mutations were determined in rpsL (strA) transgenic mice. The wild-type rpsL gene, which exhibits a streptomycin-sensitive (Sm(S)) phenotype, was used as the rescue marker gene. Studies of mutation spectra among different organs and tissues were simplified using this system because of the short coding sequence (375 bp) of the rpsL gene. MNU administration to transgenic mice significantly elevated the mutation frequencies in various adult organs. Two distinctive patterns of mutation spectrum were observed, depending on the organs tested. Mutations derived from labile organs (spleen and thymus) were predominantly G:C to A:T transitions, as expected for MNU mutagenesis. Stable organs like the liver and brain, however, carried many fewer G:C to A:T transitions but significantly more single base deletions, of which the spectrum was very similar to that of background mutations in the rpsL transgenic mice. This spectrum difference among more and less proliferating organs was confirmed by the predominant occurrence of G:C to A:T transitions in fetal liver cells exposed to transplacental MNU treatment. In addition, most (approximately 90%) of the G:C to A:T transitions induced by MNU were detected in the first nucleotide of some 5'-G-(C or G)-3' sequences, many of which corresponded to the middle guanine residue of 5'-purine-G-(C or G)-3' sequences. It is thus suggested that at particular sites, the neighboring bases in both the 5' side and 3' side seem to influence either the susceptibility to DNA damage or the ability to repair MNU-induced lesions.

Studies of in vivo mutations in rpsL transgene in UVB-irradiated epidermis of XPA-deficient mice.

We have established xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair (NER) deficiency, which rapidly developed skin tumors when exposed to a low dose of chronic UV like XP-A patients, confirming that the NER process plays an important role in preventing UVB-induced skin cancer. To examine the in vivo mutation in the UVB-irradiated epidermis, we established XPA (-/-), (+/-) and (+/+) mice carrying the Escherichia coli rpsL transgene with which the mutation frequencies and spectra in the UVB-irradiated epidermal tissue can be examined conveniently. The XPA (-/-) mice showed a higher frequency of UVB-induced mutation in the rpsL transgene with a low dose (150 J/m(2)) of UVB-irradiation than the XPA (+/-) and (+/+) mice, while, at a high dose (900 J/m(2)) they showed almost the same frequency of mutation as the XPA (+/-) and (+/+) mice, probably because of cell death in the epidermis of the XPA (-/-) mice. However, CC-->TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the XPA (-/-) mice than the XPA (+/-) and (+/+) mice at both doses of UVB. This rpsL/XPA mouse system will be useful for further analyzing the role of NER in the mutagenesis and carcinogenesis induced by various carcinogens.

Targeted deletion of the H-ras gene decreases tumor formation in mouse skin carcinogenesis.

To clarify the role of the H-Ras in vivo, we generated H-ras null mutant mice by gene targeting. In spite of the importance of the Ras in cell proliferation and differentiation, H-ras null mutant mice grew normally and were fertile. The oldest H-ras mutant mice grew to be more than 30 months old. We used the H-ras deficient mice to study the importance of the H-ras and other ras genes in the development of skin tumors induced by initiation with 7, 12-dimethylbenz(a)anthracene (DMBA) followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). We showed that H-ras null mutant mice develop approximately six times less papillomas compared with wild-type littermates after 20 weeks of TPA treatment. While all papillomas examined (17 out of 17) in wild-type mice have mutations of H-ras at codon 61, 13 (62%) out of 21 papillomas in H-ras null mutant mice have mutations of K-ras gene at codon 12, 13, or 61 and another eight (38%) papillomas have no mutations in these codons of K-ras or N-ras genes. This suggests that the activation of H-ras gene is critical in the wild-type mice, but the activation of K-ras gene can replace the H-ras activation in the initiation step of skin tumor development in the H-ras deficient mice. Oncogene (2000).

Radiation-induced mutations in the spleen and brain of lacZ transgenic mice.

PURPOSE: To study the dose-response and molecular nature of radiation-induced mutations in the spleen and brain of lacZ transgenic mice. MATERIALS AND METHODS: Line 60 transgenic mice containing the bacterial lacZ gene in a plasmid background were used. Mutants were selected using phenyl-beta-D-galactoside. The nature of mutants was determined by sequencing DNAs of mutant lacZ genes found in control and irradiated tissues. RESULTS: X-ray irradiation at 50 and 100 Gy showed linear dose-responses for mutation induction in both tissues. The slope, however, was about twice as steep in spleen than in brain. DNA sequence analyses showed that the predominant type of mutation induced by radiation in both tissues were large deletions. CONCLUSIONS: Radiation induces mutations in spleen and brain at different efficiencies but the molecular nature of the induced mutations are similar in the two issues.

The oocyte-specific methylated region of the U2afbp-rs/U2af1-rs1 gene is dispensable for its imprinted methylation.

Imprinted genes harbor discrete regions which are differentially methylated in gametes; usually the final differential methylation patterns in adults are established during embryogenesis through modifications of the initial methylation patterns in gametes. Previous reports have shown that a 200-bp region termed region II within the CpG island of the mouse imprinted U2afbp-rs gene is methylated in oocytes but not in sperm, suggesting that this region is a center for the propagation of methylated CpGs on the maternal allele and is also a candidate for an imprinting control element. To determine whether region II is required for the imprinted methylation of this gene at the endogenous locus, we generated mice carrying a deletion of this region. We herein show that parental methylation differences still exist in the CpG island on the region II-deleted allele. These findings suggest that region II is dispensable for the imprinted methylation of the U2afbp-rs gene.

Identification of two distinct transcripts for the neuronal apoptosis inhibitory protein gene.

The spinal muscular atrophies (SMA), characterized by motor neuron loss and progressive paralysis, are among the most common autosomal recessive disorders. Recently, two SMA candidate genes, NAIP (neuronal apoptosis inhibitory protein) and survival motor neuron (SMN), were reported and a 131-kb genomic sequence of 5q13.1 encompassing these two genes was determined. Based upon this genomic sequence, the original NAIP cDNA sequence published in 1995 was shown to contain foreign fragments. We therefore conducted an extensive cDNA cloning of NAIP from a human fetal brain library. Our studies confirmed that the cDNA sequence deduced from the 131-kb genomic sequence was the major transcript in the human fetal brain. In addition, a shorter and minor transcript was also newly identified. We thus designated the longer and shorter transcripts as NAIPl and NAIPs, respectively. The cDNA clones for NAIPl and NAIPs should facilitate the functional analysis of the NAIP gene and its association with neuronal apoptosis and SMA.

Inhibition of benzo[a]pyrene-induced mutagenesis by (-)-epigallocatechin gallate in the lung of rpsL transgenic mice.

Epigallocatechin gallate (EGCG) is a major water-soluble component of green tea. The antimutagenic activity of EGCG against benzo[a]pyrene (B[a]P)-induced mutations was assessed by using transgenic mice carrying the rpsL gene as a monitor of mutations. Seven-week-old male mice were given drinking water containing EGCG for 3 weeks. On day 7, mice were treated with a single i.p. injection of B[a]P (500 mg/kg body wt). Two weeks after the injection, the mutations in the rpsL gene were analyzed. B[a]P treatment resulted in an approximately 4-fold increase of mutation frequency at the rpsL gene in the lung. An approximately 60% reduction in the B[a]P-induced mutations in the lung was observed when mice were given EGCG at concentrations >0.005%. B[a]P-induced mutations mainly occurred at G:C basepairs in the several specific nucleotide sequences of the rpsL gene. These were AGG, CGG, CGT, TGG, TGC and GGT: all of them contained a guanine residue. Mutations seen similarly in the human Ki-ras codon 12 or p53 codons 157, 248, and 273 of lung tumor were also found in the rpsL gene, and the mutations were suppressed by the EGCG treatment. In conclusion, the antimutagenic effects of EGCG for B[a]P-induced mutagenesis in vivo suggest that drinking green tea may reduce the tumor-initiating potency of B[a]P in the lung.

Human megasatellite DNA RS447: copy-number polymorphisms and interspecies conservation.

We previously isolated a novel 4.7-kb RS447 sequence, which tandemly repeated approximately 50-70 copies and resided on human chromosome 4p15 (M. Kogi et al., 1997, Genomics 42: 278-283). Another tandem array (or arrays) of several RS447 copies was hereby identified on the distal part of chromosome 8p. To analyze copy-number polymorphisms of the RS447 repeats, genomic DNA samples of eight nonkindred Japanese were subjected to pulsed-field gel electrophoresis. The copy numbers of the RS447 tandem arrays on 4p15 varied drastically from allele to allele and ranged from approximately 34 to 94 copies. All eight Japanese subjects were apparently heterozygous for the RS447 copy number, and 12 copy-number-different alleles have been at least clearly distinguished. The RS447 tandem repeats were thus found to be hypervariable and highly polymorphic in a human population. The RS447 sequences, however, do not appear to be either "selfish" or "junk" DNA. The unit size and sequence of RS447 were found to be very similar between members in the human genome. The unit size of 4746 bp comprises a putative open reading frame of 1590 bp. The RS447 sequence was well conserved in all the tested mammalian species. The head-to-tail tandem repetitive structure in the RS447 homologs was also confirmed in those species. The RS447 sequence is, therefore, considered to consist of a new class of tandemly repeated satellite DNA elements in the mammalian genome, which may thus be called "megasatellite DNA."