Non-invasive genotyping of transgenic animals using fecal DNA.

For genotyping of transgenic animals, many IACUC guidelines recommend the use of fecal DNA when possible because this approach is non-invasive. Existing methods for extracting fecal DNA may be costly or involve the use of toxic organic solvents. Furthermore, feces contain an abundance of PCR inhibitors that may hinder DNA amplification when they are co-purified with fecal DNA. Here the authors describe a cost-effective, non-toxic method for genotyping transgenic animals by using the reagent AquaStool to extract fecal DNA and remove PCR inhibitors. Genotyping results obtained from fecal DNA samples extracted using AquaStool were reliably accurate when compared with results obtained from tail DNA samples. Because it is non-invasive, the authors believe that use of this method for genotyping transgenic animals using fecal DNA samples may improve animal welfare.

Impact of physical activity and performance on medical care costs among the Japanese elderly.

AIM: Physical activity (PA) is known to be inversely associated with medical care costs. The amount of PA is strongly associated with the level of physical performance among the elderly population. Therefore, it is possible that known relation between PA and medical care merely shows the relation between physical performance and medical care. To know whether PA itself relates to medical care, considering physical performance is necessary. The aim of this study was to ascertain the impact of PA on medical care expenditure by considering the physical performance in an elderly community-dwelling population. METHODS: We investigated 483 subjects who did not have any history of diseases relating to limited PA and who completed both a self-administered questionnaire including questions on PA and underwent a physical performance measurement. We ascertained the total medical care costs through a computerized linkage with claims lodged between August 2002 and March 2008 with the Miyagi National Health Insurance Association. RESULTS: The physical performance was positively associated with their level of PA. After multivariate adjustment for covariables including the levels of physical performance, the per capita medical care costs were found to be $US 827.3 (598.0-1056.7) (mean, 95% confidence interval), $US 711.1 (476.4-945.8) and $US 702.0 (461.6-942.4) (P for linear trend = 0.02) per month for those who had the lowest, average and the highest level of PA, respectively. CONCLUSION: This prospective study indicates that a higher level of PA is associated with lower medical care costs among the Japanese elderly irrespective of physical performance.

Archiving and distributing mouse lines by sperm cryopreservation, IVF, and embryo transfer.

The number of genetically modified mouse lines has been increasing exponentially in the past few decades. In order to safeguard them from accidental loss and genetic drifting, to reduce animal housing cost, and to efficiently distribute them around the world, it is important to cryopreserve these valuable genetic resources. Preimplantation-stage embryos from thousands of mouse lines have been cryopreserved during the past two to three decades. Although reliable, this method requires several hundreds of embryos, which demands a sizable breeding colony, to safely preserve each line. This requirement imposes significant delay and financial burden for the archiving effort. Sperm cryopreservation is now emerging as the leading method for storing and distributing mouse lines, largely due to the recent finding that addition of a reducing agent, monothioglycerol, into the cryoprotectant can significantly increase the in vitro fertilization (IVF) rate in many mouse strains, including the most widely used C57BL/6 strain. This method is quick, inexpensive, and requires only two breeding age male mice, but it still remains tricky and strain-dependent. A small change in experimental conditions can lead to significant variations in the outcome. In this chapter, we describe in detail our sperm cryopreservation, IVF, and oviduct transfer procedures for storing and reviving genetically modified mouse lines.

The Ohsaki Cohort 2006 Study: design of study and profile of participants at baseline.

BACKGROUND: Large-scale cohort studies conducted in Japan do not always include psychosocial factors as exposures. In addition, such studies sometimes fail to satisfactorily evaluate disability status as an outcome. METHODS: This prospective cohort study comprised 49 603 (22 438 men and 27 165 women) community-dwelling adults aged 40 years or older who were included in the Residential Registry for Ohsaki City, Miyagi Prefecture, in northeastern Japan. The baseline survey, which included psychosocial factors, was conducted in December 2006. Follow-up of death, immigration, cause of death, cancer incidence, and long-term care insurance certification was started on 1 January 2007. RESULTS: The response rate was 64.2%. In general, lifestyle-related conditions in the study population were similar to those of the general Japanese population; however, the proportion of male current smokers was higher in the cohort. The association between age and the proportion of those reporting psychological distress showed a clear U-shaped curve, with a nadir at age 60 to 69 years in both men and women, although more women were affected by such distress than men. The proportion of those who reported a lack of social support was highest among those aged 40 to 49 years. Most men and women surveyed did not participate in community activities. Among participants aged 65 years or older, 10.9% of participants were certified beneficiaries of the long-term care insurance system at baseline. CONCLUSIONS: The Ohsaki Cohort 2006 Study is a novel population-based prospective cohort study that focuses on psychosocial factors and long-term care insurance certification.

Green tea consumption is associated with depressive symptoms in the elderly.

BACKGROUND: Green tea is reported to have various beneficial effects (eg, anti-stress response and antiinflammatory effects) on human health. Although these functions might be associated with the development and progression of depressive symptoms, no studies have investigated the relation between green tea consumption and depressive symptoms in a community-dwelling population. OBJECTIVE: The aim of this study was to investigate the relations between green tea consumption and depressive symptoms in elderly Japanese subjects who widely consumed green tea. DESIGN: We conducted a cross-sectional study in 1058 community-dwelling elderly Japanese individuals aged >or=70 y. Green tea consumption was assessed by using a self-administered questionnaire, and depressive symptoms were evaluated by using the 30-item Geriatric Depression Scale with 2 cutoffs: 11 (mild and severe depressive symptoms) and 14 (severe depressive symptoms). If a participant was consuming antidepressants, he or she was considered to have depressive symptoms. RESULTS: The prevalence of mild and severe and severe depressive symptoms was 34.1% and 20.2%, respectively. After adjustment for confounding factors, the odds ratios (95% CI) for mild and severe depressive symptoms when higher green tea consumption was compared with green tea consumption of or=4 cups green tea/d (0.56; 95% CI: 0.39, 0.81) (P for trend: 0.001). Similar relations were also observed in the case of severe depressive symptoms. CONCLUSION: A more frequent consumption of green tea was associated with a lower prevalence of depressive symptoms in the community-dwelling older population.

Factors associated with psychological distress in a community-dwelling Japanese population: the Ohsaki Cohort 2006 Study.

BACKGROUND: In Asia, there has been no population-based epidemiological study using the K6, a 6-item instrument that assesses nonspecific psychological distress. METHODS: Using cross-sectional data from 2006, we studied 43,716 (20,168 men and 23,548 women) community-dwelling people aged 40 years or older living in Japan. We examined the association between psychological distress and demographic, medical, lifestyle, and social factors by using the K6, with psychological distress defined as 13 or more points out of a total of 24 points. RESULTS: The following variables were significantly associated with psychological distress among the population: female sex, young and old age, a history of serious disease (hypertension, diabetes mellitus, stroke, myocardial infarction, or cancer), current smoking, former alcohol drinking, low body mass index, shorter daily walking time, lack of social support (4 of 5 components), and lack of participation in community activities (4 of 5 components). Among men aged 40 to 64 years, only "lack of social support for consultation when in trouble" and a history of diabetes mellitus remained significant on multivariate analysis. Among men aged 65 years or older, age was not significantly associated with psychological distress, and the significant association with current smoking disappeared on multivariate analysis. Among women aged 40 to 64 years, a history of stroke was not associated with psychological distress. Among women aged 65 years or older, the significant association with current smoking disappeared on multivariate analysis. CONCLUSIONS: A number of factors were significantly associated with psychological distress, as assessed by the K6. These factors differed between men and women, and also between middle-aged and elderly people.

Serum C-reactive protein even at very low (<1.0 mg/l) concentration is associated with physical performance in a community-based elderly population aged 70 years and over.

BACKGROUND: Although several studies have reported that C-reactive protein (CRP) is associated with physical performance, few studies have evaluated the relationships between CRP and physical performance among subjects who had a very low range of CRP. Therefore, it is still unclear whether a lower CRP is favorably associated with physical performance even within a very low range. OBJECTIVE: The aim of this study was to investigate the relationships between CRP and physical performance among a Japanese population with a low serum CRP concentration (CRP <1.0 mg/l). METHODS: We designed a cross-sectional survey for 775 persons aged 70 years and older living in Japan. High-sensitivity CRP was measured using a nephelometric method. The subjects whose serum CRP concentrations were higher than 10.0 mg/l were excluded. Physical performance was assessed using a 10-meter maximum walk test, leg extension power, and a timed 'up and go' test. RESULTS: The median value (interquartile range) of CRP was 0.55 (0.29-1.20) mg/l. After adjustment for potential confounding factors, an inverse relation of CRP with the 10-meter maximum walk test and leg power was observed in all subjects (p for trend = 0.10 and 0.04, respectively). For subjects who had a CRP <1.0 mg/l, these inverse relations were unchanged (p for trend = 0.03 and 0.02, respectively). CONCLUSIONS: Serum CRP concentration is favorably related to physical performance, even within a very low range in a community-based elderly population aged 70 years and over. The findings suggest that maintaining as low CRP levels as possible may potentially maintain better physical performance.

Time spent walking and risk of colorectal cancer in Japan: the Miyagi Cohort study.

Higher levels of physical activity have been consistently associated with a lower risk of colon cancer in earlier epidemiological studies. The specific benefits of walking, however, remain relatively unexplored. In 1990, 20 519 men and 21 469 women in Japan completed a self-administered questionnaire including a question on time spent walking per day. During 7 years of follow-up, 260 cases of colorectal cancer were documented in 305 790 person-years. We used the Cox proportional hazards regression model to estimate the relative risk of incident cancer (colorectal, colon, and rectal) according to three levels of walking. Time spent walking was inversely associated with risk of colorectal cancer incidence in men. Compared with men who walked 0.5 h or less per day, the multivariate relative risks were 1.06 (95% confidence interval 0.72-1.57) for men who walked between 0.5 and 1 h per day, and 0.57 (95% confidence interval 0.38-0.83) for men who walked 1 h or more per day (P for trend=0.003). Time spent walking per day was associated with a lower risk of colon cancer in Japanese men but not in women, and there was no association between time spent walking and the risk of rectal cancer.

Effects of transgenic expression of HIV-1 Vpr on lipid and energy metabolism in mice.

HIV infection is associated with abnormal lipid metabolism, body fat redistribution, and altered energy expenditure. The pathogenesis of these complex abnormalities is unclear. Viral protein R (Vpr), an HIV-1 accessory protein, can regulate gene transcription mediated by the glucocorticoid receptor and peroxisome proliferator-activated receptor-gamma and affect mitochondrial function in vitro. To test the hypothesis that expression of Vpr in liver and adipocytes can alter lipid metabolism in vivo, we engineered mice to express Vpr under control of the phosphoenolpyruvate carboxykinase promoter in a tissue-specific and inducible manner and investigated the effects of dietary fat, indinavir, and dexamethasone on energy metabolism and body composition. The transgenic mice expressed Vpr mRNA in white and brown adipose tissues and liver and immunoaffinity capillary electrophoresis revealed that they had free Vpr protein in the plasma. Compared with wild-type (WT) animals, Vpr mice had lower plasma triglyceride levels after 6 wk (P < 0.05) but not after 10 wk of a high-fat diet and lower plasma cholesterol levels after 10 wk of high-fat diet (P < 0.05). Treatment with dexamethasone obviated group differences, whereas indinavir had no significant independent effect on lipids. In the fasted state, Vpr mice had a higher respiratory quotient than WT mice (P < 0.05). These data provide the first in vivo evidence that HIV-1 Vpr expressed at low levels in adipose tissues and liver can 1) circulate in the blood, 2) regulate lipid and fatty acid metabolism, and 3) alter fuel selection for oxidation in the fasted state.

Detection and localization of proteinuria by dynamic contrast-enhanced magnetic resonance imaging using MS325.

After renal transplantation, persistent glomerular disease affecting the native kidneys typically causes albuminuria, at least for a period of time, making it difficult to determine in a noninvasive fashion whether proteinuria originates in the native kidneys or the renal allograft. To address this problem, dynamic contrast-enhanced magnetic resonance imaging (MRI) using gadolinium (Gd)-based albumin-bound blood pool contrast agent (MS325) to localize proteinuria was investigated. Glomerular proteinuria was induced in Sprague-Dawley rats by intravenous injection of puromycin aminonucleoside (PAN), whereas control rats received physiologic saline vehicle. Both groups of animals underwent a 40-min dynamic contrast-enhanced MRI using radio frequency spoiled gradient echo imaging sequence after injection of Gd-labeled MS325. Contrast uptake and clearance curves for cortex and medulla were determined from acquired MR images. Compared with controls, proteinuric rats exhibited significantly lower elimination rate constants. The use of gadopentetate dimeglumine (Gd-DTPA) as a contrast agent showed smaller and less specific differences between proteinuric and control groups. In rats with one proteinuric kidney (PAN-treated) and one normal kidney (transplanted from a normal rat), MRI using MS325 was able to differentiate between the two kidneys. The results suggest that MRI with an albumin-bound blood pool contrast agent may be a useful noninvasive way to localize proteinuria. If this technique can be successfully applied in human patients, it may allow for the localization of proteinuria after kidney transplant and thereby provide a noninvasive way to detect disease affecting the renal allograft.

Walking and mortality in Japan: the Miyagi Cohort Study.

BACKGROUND: Although many studies in western populations demonstrated that time spent walking was associated with a reduced risk of all-cause mortality, data on Japanese has been sparse. METHODS: In 1990, 20,004 men and 21,159 women in Miyagi Prefecture in rural northern Japan (40-64 year of age) completed a self-administered questionnaire including a question on time spent walking. Cox regression was used to estimate relative risk (RR) of mortality according to three levels of walking (30 minutes or less, between 30 minutes and one hour, and one hour or more), with adjustment for age, education, marital status, past history of diseases, smoking, drinking, body mass index, and dietary variables. During 11 years of follow-up, 1,879 subjects had died. RESULTS: Time spent walking was inversely associated with risk of all-cause mortality: compared with men and women who walked one hour or more per day, multivariate RR (95% confidence intervals) was 1.06 (0.95-1.19) for subjects who walked between 30 minutes and one hour per day, and 1.16 (1.04-1.29) for subjects who walked 30 minutes or less per day (P for trend=0.007). Shorter duration of walking was associated with increased mortality among men who were never smokers (P for trend=0.081) and past smokers (P for trend=0.026), but not among currently smoking men (P for trend=0.751). We observed similar effect modification for women. CONCLUSIONS: Time spent walking was associated with a reduced risk for all-cause mortality, especially among nonsmoking men and women.

Health practices and mortality in Japan: combined effects of smoking, drinking, walking and body mass index in the Miyagi Cohort Study.

BACKGROUND: Evidence is limited regarding the association between the combinations of multiple health practices and mortality. METHODS: In 1990, 28,333 men and women in Miyagi Prefecture in rural northern Japan (40-64 year of age) completed a self-administered questionnaire. A lifestyle score was calculated by adding the number of high-risk practices (smoking, consuming > or = 22.8 g alcohol/d, walking < 1 hr/d, body mass index < 18.5 or > or = 30.0). Cox regression was used to estimate relative risk (RR) of mortality according to the lifestyle score, with adjustment for age, education, marital status, past history of diseases, and dietary variables. During 11 years of follow-up, 1,200 subjects had died. RESULTS: We observed linear increase in risk of death associated with increasing number of high-risk practices: compared with men who had no high-risk practices, multivariate RRs for men who had 1 to 4 practices were 1.20, 1.66, 1.94, and 3.96, respectively (P for trend<0.001), and corresponding RRs for women were 1.31, 2.14, 3.98, 5.56, respectively (P for trend<0.001). A unit increase in the number of high-risk practices corresponded to being 2.8 and 4.8 years older for men and women, respectively. CONCLUSIONS: In this prospective cohort study of middle-aged men and women in rural Japan, a larger number of high-risk practices was associated with linear increase in risk of all-cause mortality.

Inducible podocyte-specific gene expression in transgenic mice.

The podocyte plays a key role in glomerular function and glomerular disease. To facilitate studies of podocyte function, we have developed a transgenic mouse model with inducible expression in the podocyte. The tetracycline-inducible transgenic system facilitates gene expression with restricted cellular distribution and tight temporal control. Recently, Bujard and colleagues have developed a functionally improved reverse tetracycline-controlled transcriptional activator (rtTA) with substantially lower background in the off state (the absence of tetracycline) and greater inducibility in the on state (the presence of tetracycline). We used the human podocin (NPHS2) gene promoter to control expression of the rtTA cassette and bred these mice with a reporter mouse line that contains the cytomegalovirus minimal promoter and tetO promoter elements together with LacZ, encoding beta-galactosidase. Dual transgenic mice, bearing both podocin-rtTA and tetO-LacZ transgenes, had no detectable expression in kidney or other organs in the absence of tetracycline. Administration of tetracycline in the drinking water was associated with podocyte expression of beta-galactosidase, in a fashion that was time dependent (maximal at 1 wk) and dose-dependent (maximal at 2 mg/ml). Podocyte expression was confirmed in two ways: histochemical staining for beta-galactosidase and double-immunostaining using the podocyte marker WT-1 and beta-galactosidase. This transgenic system should aid future investigations of podocyte function.