[A 10-year community intervention for disability prevention and changes in physical, nutritional, psychological and social functions among community-dwelling older adults in Kusatsu, Gunma Prefecture, Japan].

OBJECTIVES: We reported previously that a 10-year community intervention for disability prevention successfully extended healthy life expectancy at 70 years and decreased the enrollment rate of the Long-Term Care Insurance in Kusatsu, Gunma Prefecture, Japan. In order to clarify functional factors that contributed to healthy aging, this study examined changes in physical, nutritional, psychological and social functions in older adults who participated in annual health checkups over the period. METHODS: Data sources were participants in annual health checkups conducted from 2002 to 2012 and respondents to biannual monitoring surveys conducted from 2003 to 2011. The target population was all older adults aged 70 years and over living in Kusatsu. The average participation rate over the period was 34.7% for the annual health checkups and 95.0% for the monitoring surveys. First, we examined the representativeness of the participants in annual health checkups by comparing them with the responders to monitoring surveys in terms of their higher-level functional capacity, as measured by the Tokyo Metropolitan Institute of Gerontology Index of Competence (TMIG-IC) (Analysis 1). Second, we examined changes in the physical (4 measures), nutritional (3 measures), and psychological and social (4 measures) functions of participants in annual health checkups over the period. In this analysis, we standardized the data for each year on 11 measures to a mean of 0 and a standard deviation of 1.0 using the 2002 data as the standard, and conducted statistical tests for the slopes of the linear approximate equation (intercept=0) (Analysis 2). RESULTS: In Analysis 1, the TMIG-IC scores for participants in the annual health checkups were significantly higher in both sexes than were those for responders to the monitoring surveys. However, there were no significant year×group interactions in the scores. The difference in scores between the two groups was small for participants in their seventies, but large for participants in their eighties or over. Analysis 2 showed that all physical functions improved significantly over the period in both sexes, and the slopes of the linear approximate equation were steeper for maximal and usual gait speeds (slope=0.050 and 0.048, respectively, in men; 0.067 and 0.060, respectively, in women) than for other measures. In women, in addition to physical function, scores on the Mini-Mental State Examination (slope=0.053), Geriatric Depression Scale (slope=0.027), and Social Roll Scale (slope=0.019) also increased significantly. CONCLUSION: Although participants in annual health checkups were biased toward better functioning, the degree of the bias did not change significantly over the period. During the same period, physical function for both sexes, and psychological and social functions for women, improved significantly. It may be concluded that functional improvement in older adults contributed to the healthy longevity in Kusatsu.

CLE-CLAVATA1 peptide-receptor signaling module regulates the expansion of plant root systems in a nitrogen-dependent manner.

Morphological plasticity of root systems is critically important for plant survival because it allows plants to optimize their capacity to take up water and nutrients from the soil environment. Here we show that a signaling module composed of nitrogen (N)-responsive CLE (CLAVATA3/ESR-related) peptides and the CLAVATA1 (CLV1) leucine-rich repeat receptor-like kinase is expressed in the root vasculature in Arabidopsis thaliana and plays a crucial role in regulating the expansion of the root system under N-deficient conditions. CLE1, -3, -4, and -7 were induced by N deficiency in roots, predominantly expressed in root pericycle cells, and their overexpression repressed the growth of lateral root primordia and their emergence from the primary root. In contrast, clv1 mutants showed progressive outgrowth of lateral root primordia into lateral roots under N-deficient conditions. The clv1 phenotype was reverted by introducing a CLV1 promoter-driven CLV1:GFP construct producing CLV1:GFP fusion proteins in phloem companion cells of roots. The overaccumulation of CLE2, -3, -4, and -7 in clv1 mutants suggested the amplitude of the CLE peptide signals being feedback-regulated by CLV1. When CLE3 was overexpressed under its own promoter in wild-type plants, the length of lateral roots was negatively correlated with increasing CLE3 mRNA levels; however, this inhibitory action of CLE3 was abrogated in the clv1 mutant background. Our findings identify the N-responsive CLE-CLV1 signaling module as an essential mechanism restrictively controlling the expansion of the lateral root system in N-deficient environments.

The use of cationic nanogels to deliver proteins to myeloma cells and primary T lymphocytes that poorly express heparan sulfate.

Fusion proteins containing protein transduction domain (PTD) are widely used for intracellular delivery of exogenous proteins. PTD-mediated delivery requires expression of heparan sulfate on the surface of the target cells. However, some of metastatic tumor cells and primary lymphocytes poorly express heparan sulfate. Here we demonstrate that proteins complexed with nanosize hydrogels formed by cationic cholesteryl group-bearing pullulans (cCHP) are efficiently delivered to myeloma cells and primary CD4(+) T lymphocytes probably by induction of macropinocytosis, although these cells are resistant to PTD-mediated protein delivery as a consequence of poor heparan sulfate expression. The anti-apoptotic protein Bcl-xL delivered by cCHP nanogels efficiently blocked apoptosis of these cells, establishing functional regulation of cells by proteins delivered by cCHP nanogels. Thus, cCHP nanogel is a useful tool to deliver proteins for development of new cancer therapy and immune regulation.

Metabolomics data reveal a crucial role of cytosolic glutamine synthetase 1;1 in coordinating metabolic balance in rice.

Rice plants grown in paddy fields preferentially use ammonium as a source of inorganic nitrogen. Glutamine synthetase (GS) catalyses the conversion of ammonium to glutamine. Of the three genes encoding cytosolic GS in rice, OsGS1;1 is critical for normal growth and grain filling. However, the basis of its physiological function that may alter the rate of nitrogen assimilation and carbon metabolism within the context of metabolic networks remains unclear. To address this issue, we carried out quantitative comparative analyses between the metabolite profiles of a rice mutant lacking OsGS1;1 and its background wild type (WT). The mutant plants exhibited severe retardation of shoot growth in the presence of ammonium compared with the WT. Overaccumulation of free ammonium in the leaf sheath and roots of the mutant indicated the importance of OsGS1;1 for ammonium assimilation in both organs. The metabolite profiles of the mutant line revealed: (i) an imbalance in levels of sugars, amino acids and metabolites in the tricarboxylic acid cycle, and (ii) overaccumulation of secondary metabolites, particularly in the roots under a continuous supply of ammonium. Metabolite-to-metabolite correlation analysis revealed the presence of mutant-specific networks between tryptamine and other primary metabolites in the roots. These results demonstrated a crucial function of OsGS1;1 in coordinating the global metabolic network in rice plants grown using ammonium as the nitrogen source.

Development of new HLA-B*3505 genotyping method using Invader assay.

Several pharmacogenetic studies have revealed strong associations between specific human leukocyte antigen (HLA) alleles and the susceptibility to drug hypersensitivity. Recently, we reported HLA-B*3505 as a strong genetic biomarker for the nevirapine-induced skin rash in Thai population. Here, we developed a new HLA-B*3505 genotyping method by a combination of the Universal Invader assay and sequence-specific primer PCR. From the sequence alignment of 68 HLA-B alleles in the Thai population, we selected the two most discriminative SNPs (rs1140412 and rs4997052) as target SNP sites. When we carried out the assay using 324 Thai individuals, fluorescence intensities of HLA-B*3505-positive and HLA-B*3505-negative samples were apparently discriminated at the endpoint of the reaction. Our results were 100% concordant with those obtained by a sequence-based typing method. As our assay is simple and rapid, we believe our method will be a useful tool for pharmacogenetic testing of the nevirapine-induced skin rash.

[Research on screening for frailty: development of "the Kaigo-Yobo Checklist"].

PURPOSE: Early detection of older persons at high-risk with an effective screening tool is a prerequisite for strategies to optimize care among the community-dwelling elderly. We have focused on risks regarding the likelihood of developing homeboundedness, falls, and poor nutrition, and proposed an original 18 item-scale called "the Kaigo-Yobo Checklist" as a questionnaire-based screening tool. This study examined the reliability and validity of this scale using a longitudinal cohort of community-dwelling elderly. METHODS: Among 1039 older persons aged 70 years and over living in Kusatsu Town, Gunma Prefecture in 2001, 916 persons (88.2%) responded to the baseline interview survey including assessment with the 18-items of "the Kaigo-Yobo Checklist". Using these data, we performed the following analyses: (1) elimination of improper items according to pass and non-response rates; (2) internal reliability analysis based on Cronbach's alpha and Good-Poor approaches; (3) partial correlation analysis with IADL scores (Instrumental Self-Maintenance subscale of TMIG-Index of Competence) treated as external criteria. Further, we collected individual records under the long-term care insurance system in Kusatsu Town from April 2000 to November 2005, and determined the predictive value of the checklist for onset of certification of long-term care insurance over 4 years (2001-2005) with the trend test and logistic regression models. RESULTS: (1) Three among 18 items did not show pass rates of 75% to 95% with a non-response rate under 1%. Thus we excluded these three items from the original checklist, yielding a 15 item-scale with 15 points for full marks, i.e., a modified version of "the Kaigo-Yobo Checklist". (2) The Cronbach's alpha was 0.79, and Good-Poor analysis determined that the high-score group (> or =2 points) had a higher mean score for all items of the checklist compared to the low-score group ( < or =1 point) (P < 0.001). (3) The partial correlation coefficient between the checklist score and the IADL score was -0.64 (P < 0.001). (4) The baseline checklist score was positively and linearly associated with the risk of developing a state in need of care during the 4-year follow-up; the adjusted odds ratio for the increment of 1 point was 1.21 (95% confidence interval, 1.10-1.33) [1.24 (1.11-1.38) when deceased or moving-out cases were excluded from the analysis]. CONCLUSION: The modified version of "the Kaigo-Yobo Checklist" had concurrent and predictive validity, and good reliability as a questionnaire-based scale for screening high-risk older persons.

CYP2D6 genotyping for functional-gene dosage analysis by allele copy number detection.

BACKGROUND: Cytochrome P450 2D6 (CYP2D6), one of the most important drug-metabolizing enzymes, has been reported to possess variation in the encoding CYP2D6 gene (cytochrome P450, family 2, subfamily D, polypeptide 6) that affects enzymatic activity. For the pharmacogenetic study of CYP2D6, accurate measurement of the dosage of the functional gene is essential; however, current genotyping techniques are insufficient because of their inability to provide the exact copy number of functional CYP2D6 genes. METHODS: We developed 3 quantitative real-time PCR (qPCR) assays for estimating the total copy number of the CYP2D6 gene, as well as 24-multiplex PCR-based real-time Invader assays (mPCR-RETINAs) for estimating the allele ratio at each variation locus. After determining the allele copy number at each locus, we estimated the frequencies of CYP2D6 alleles in a population and the diplotype in each individual by a CNVphaser (copy number variation phaser). The qPCR assays and RETINAs used for HapMap Japanese and Chinese samples were applied to 455 Japanese individuals. RESULTS: Forty-two individuals (9.2%) had one CYP2D6 gene copy, 207 (45.5%) had 2 copies, 161 (35.4%) had 3 copies, 40 (8.8%) had 4 copies, and 5 (1.1%) had 5 copies of the CYP2D6 gene. We found 16 different CYP2D6 alleles, with frequencies similar to those described in previous reports. In the diplotype analysis, we observed that CYP2D6*1/*1 and *1/*10-*36 were the most common diplotypes (approximately 20%) in our population. CONCLUSIONS: Our method is the first to determine the exact number of functional CYP2D6 gene copies. We believe our method will facilitate and accelerate the detailed pharmacogenetic analysis of CYP2D6.

AtAMT1;4, a pollen-specific high-affinity ammonium transporter of the plasma membrane in Arabidopsis.

Pollen represents an important nitrogen sink in flowers to ensure pollen viability. Since pollen cells are symplasmically isolated during maturation and germination, membrane transporters are required for nitrogen import across the pollen plasma membrane. This study describes the characterization of the ammonium transporter AtAMT1;4, a so far uncharacterized member of the Arabidopsis AMT1 family, which is suggested to be involved in transporting ammonium into pollen. The AtAMT1;4 gene encodes a functional ammonium transporter when heterologously expressed in yeast or when overexpressed in Arabidopsis roots. Concentration-dependent analysis of (15)N-labeled ammonium influx into roots of AtAMT1;4-transformed plants allowed characterization of AtAMT1;4 as a high-affinity transporter with a K(m) of 17 microM. RNA and protein gel blot analysis showed expression of AtAMT1;4 in flowers, and promoter-gene fusions to the green fluorescent protein (GFP) further defined its exclusive expression in pollen grains and pollen tubes. The AtAMT1;4 protein appeared to be localized to the plasma membrane as indicated by protein gel blot analysis of plasma membrane-enriched membrane fractions and by visualization of GFP-tagged AtAMT1;4 protein in pollen grains and pollen tubes. However, no phenotype related to pollen function could be observed in a transposon-tagged line, in which AtAMT1;4 expression is disrupted. These results suggest that AtAMT1;4 mediates ammonium uptake across the plasma membrane of pollen to contribute to nitrogen nutrition of pollen via ammonium uptake or retrieval.

Sulphur starvation induces the expression of microRNA-395 and one of its target genes but in different cell types.

Plants play an important role in the global sulphur cycle because they assimilate sulphur from the environment and build it into methionine and cysteine. Several genes of the sulphur assimilation pathway are regulated by microRNA-395 (miR395) that is itself induced by a low-sulphur (-S) environment. Here, we show that the six Arabidopsis miR395 loci are induced differently. We find that MIR395 loci are expressed in the vascular system of roots and leaves and root tips. Induction of miR395 by a -S environment in both roots and leaves suggests that translocation of miR395 from leaves to roots through the phloem is not necessary for plants growing on -S soil/medium. We also demonstrate that induction of miR395 is controlled by SLIM1, a key transcription factor in the sulphur assimilation pathway. Unexpectedly, the mRNA level of a miR395 target gene, SULTR2;1, strongly increases during miR395 induction in roots. We show that the spatial expression pattern of MIR395 transcripts in the vascular system does not appear to overlap with the expression pattern previously reported for SULTR2;1 mRNA. These results illustrate that negative temporal correlation between the expression level of a miRNA and its target gene in a complex tissue cannot be a requirement for target gene validation.

Multiplex PCR-based real-time invader assay (mPCR-RETINA): a novel SNP-based method for detecting allelic asymmetries within copy number variation regions.

We report the development of a real-time Invader assay combined with multiplex PCR (mPCR-RETINA), an SNP-based approach that can measure the allelic ratio in copy number variation (CNV) regions of a genome. RETINA monitors the real-time fluorescence intensity of each allele during the Invader assay and detects allelic asymmetries caused by genomic duplication/multiplication in heterozygous individuals. By combining mPCR-RETINA and real-time quantitative PCR that detects total copy number, we can estimate the copy number of each allele in CNV regions, which should be useful for investigating the functional significance of allele copy number with disease susceptibilities and drug responses. Also, mPCR-RETINA can efficiently refine the detailed structures of CNV regions. Due to the combination of RETINA with multiplex PCR, mPCR-RETINA requires a very small amount of genomic DNA for analysis (0.1-0.38 ng/locus). Additionally, mPCR-RETINA has clear advantages in its simple protocol and target-specific reaction, even in nonunique regions. We believe mPCR-RETINA will provide a significant contribution to identifying functional alleles in CNV regions.

[Physical activity level and physical, mental and social factors in community-dwelling elderly people].

PURPOSE: This study was conducted to examine the relationship between physical activity level (PAL) and physical, mental and social factors in community-dwelling elderly people. METHODS: The subjects comprised 428 residents aged 70 or over living in Kusatsu-machi, Gunma, who took part in an interview and tests of physical fitness as part of the "NIKKORI-KENKOSODANJIGYO". Data for 330 out of the 428 residents were adopted for this study. The PAL was evaluated with a questionnaire developed by Naito et al. (2003). Subjects were interviewed on physical, mental and social functioning including the Tokyo Metropolitan Institute of Gerontology (TMIG) Index of Competence and cognitive function (Mini Mental State Examination: MMSE). Physical fitness tests included assessment of handgrip strength, usual and maximal walking speed, and one-leg standing balance with eyes open. RESULTS: Correlations between PAL and the TMIG Index of Competence, physical (e.g. physical fitness), mental (e.g. depression) and social (e.g. roles at home) factors, and smoking status were significant by ANCOVA adjusted for age and sex. Analysis using a general linear model indicated that smoking status, usual walking speed, depression, roles at home, frequency of going outdoors and visual impairment all together explained 13.5% of the PAL variance. CONCLUSION: The findings indicate that PAL in the community-dwelling elderly aged 70 years or over is associated with physical, mental and social factors.

[Longitudinal analysis of factors associated with participation in community-based mass screening for the frail elderly in need of care].

PURPOSE: To examine factors associated with participation in a community based comprehensive health check to screen frail elderly before they become in need of long-term care. METHODS: All residents aged 70 years and over living in Kusatsu, Gumma prefecture were surveyed for their sociodemographics, and physical, mental and social functioning through in-person interview in the years 2001 and 2003. In the following years--in 2002 and 2004, respectively--mass screenings were conducted to detect early signs of need for care among the elderly. Using the baseline interview information as explanatory variables, we performed multiple logistic regression analysis in order to examine the social determinants of participation in the mass-screening. RESULTS: For the first mass-screening, having high blood pressure and poor subjective health decreased the likelihood of attending the screening by 34% and 65% respectively. In contrast, one point increases in IADL, mobility and social-role score increased the probability of attendance by 27%, 26% and 26% respectively. After the screening was re-conducted, the factors influencing attendance appeared to have changed. With a third mass-screening, while the IADL and mobility score still had significant effects, elderly having excellent subjective health were less likely to go by some 48%. It was also found that not having visual impairment had a significantly positive effect on attendance. CONCLUSION: If local government conducts community-based mass screening aiming to screen the frail elderly in need of care, it is likely that there are high risk elderly among the non-participants. It is very important to follow up those non-participants using an attendance roll and provide appropriate advice. It is also worth discussing mass screening that focuses more on health promotion than on "screening" itself.