Genome scanning for detecting adaptive genes along environmental gradients in the Japanese conifer, Cryptomeria japonica.

Local adaptation is important in evolutionary processes and speciation. We used multiple tests to identify several candidate genes that may be involved in local adaptation from 1026 loci in 14 natural populations of Cryptomeria japonica, the most economically important forestry tree in Japan. We also studied the relationships between genotypes and environmental variables to obtain information on the selective pressures acting on individual populations. Outlier loci were mapped onto a linkage map, and the positions of loci associated with specific environmental variables are considered. The outlier loci were not randomly distributed on the linkage map; linkage group 11 was identified as a genomic island of divergence. Three loci in this region were also associated with environmental variables such as mean annual temperature, daily maximum temperature, maximum snow depth, and so on. Outlier loci identified with high significance levels will be essential for conservation purposes and for future work on molecular breeding.

Apolipoprotein E polymorphism in elderly Japanese-Brazilian immigrants does not explain the reduced cardiovascular risk factor incidence.

Study of immigrant populations may contribute to a better understanding of the epidemiology of diseases associated with the aging process. We examined the prevalence of cardiovascular risk factors, including apolipoprotein E (ApoE) polymorphism, in elderly subjects who were born in Japan, migrated to South Brazil and have lived in that region for over 40 years, versus a group of elderly, locally born Brazilians living in the same region. These Japanese subjects came to Brazil after World War II (1950-1960) from several Japanese cities, mainly Nagasaki, Kumamoto and Hokkaido. Among 1007 subjects genotyped for ApoE polymorphism, we selected 540 elderly subjects (>60 years old), consisting of 270 Japanese-Brazilians and 270 Brazilians of European ancestry from Rio Grande do Sul State (Gaucha population). The Japanese-Brazilian group had significantly lower prevalences of obesity, type 2 diabetes mellitus, dyslipidemia, and metabolic syndrome than did the Gaucho population group. ApoE polymorphism frequencies were similar in the two groups. The differences in cardiovascular risk factors observed in the two populations cannot be explained by ApoE polymorphism; they could be related to conservation of Japanese lifestyle habits, such as diet.

Continuous erythropoietin delivery by muscle-targeted gene transfer using in vivo electroporation.

It has been demonstrated that gene transfer by in vivo electroporation of mouse muscle increases the level of gene expression by more than 100-fold over simple plasmid DNA injection. We tested continuous rat erythropoietin (Epo) delivery by this method in normal rats, using plasmid DNA expressing rat Epo (pCAGGS-Epo) as the vector. A pair of electrodes was inserted into the thigh muscles of rat hind limbs and 100 microg of pCAGGS-Epo was injected between the electrodes. Eight 100-V, 50-msec electric pulses were delivered through the electrodes. Each rat was injected with a total of 400 microg of pCAGGS-Epo, which was delivered to the medial and lateral sides of each thigh. The presence of vector-derived Epo mRNA at the DNA injection site was confirmed by RT-PCR. The serum Epo levels peaked at 122.2 +/- 33.0 mU/ml on day 7 and gradually decreased to 35.9 +/- 18.2 mU/ml on day 32. The hematocrit levels increased continuously, from the preinjection level of 49.5 +/- 1.1 to 67.8 +/- 2.2% on day 32 (p < 0.001). In pCAGGS-Epo treated rats, endogenous Epo secretion was downregulated on day 32. In a control experiment, intramuscular injection of pCAGGS-Epo without subsequent electroporation did not significantly enhance the serum Epo levels. These results demonstrate that muscle-targeted pCAGGS-Epo transfer by in vivo electroporation is a useful procedure for the continuous delivery of Epo.

Role of calcium-sensitive tyrosine kinase Pyk2/CAKbeta/RAFTK in angiotensin II induced Ras/ERK signaling.

In cardiac fibroblasts, angiotensin II (Ang II) induced a rapid increase in extracellular signal regulated kinase (ERK) activity in a pertussis toxin insensitive manner. This ERK activation was abolished by the Gq-associated phospholipase C inhibitor U73122 but was insensitive to protein kinase C (PKC) inhibitors or PKC downregulation by phorbol ester. Intracellular Ca2+ chelation by BAPTA-AM or TMB-8 abolished Ang II induced ERK activation, whereas treatment with EGTA or nifedipine did not affect it. Ca2+ ionophore A23187 also induced a rapid increase in ERK activity to an extent similar to that of Ang II stimulation. Calmodulin inhibitors (W7 and calmidazolium) and tyrosine kinase inhibitors (genistein and ST638) completely blocked ERK activation by Ang II and A23187. Both Ang II and A23187 caused a rapid increase in the binding of GTP to p21(Ras), which was nearly abolished by genistein and calmidazolium. Transfection with the dominant negative mutant of Ras and the Ras inhibitor manumycin completely inhibited Ang II induced ERK activation. It was also found for the first time that cardiac fibroblasts abundantly expressed Ca2+-sensitive tyrosine kinase Pyk2/CAKbeta/RAFTK and that Ang II markedly induced its activation in a Ca2+/calmodulin-sensitive manner. Overexpression of the dominant negative mutant of Pyk2 significantly attenuated Ang II or A23187-induced ERK activities (36% and 38% inhibition compared with that in mock-transfected cells, respectively) and ERK tyrosine phosphorylation levels, as well as an increase in the binding of GTP to p21(Ras). These findings demonstrate that in cardiac fibroblasts, Ang II induced Ras/ERK activation is dominantly regulated by Gq-coupled Ca2+/calmodulin signaling and that Pyk2 plays an important role in the signal transmission for efficient activation of the Ang II induced Ras/ERK pathway.

Differential kinetics of circulating angiotensin IV and II after treatment with angiotensin II type 1 receptor antagonist and their plasma levels in patients with chronic renal failure.

BACKGROUND: Angiotensin II (Ang II) C-terminal hexapeptide (referred to as Ang IV) possesses the characteristics of a real hormone with specific receptors and biological effects. Clinical application of Ang II type 1 receptor (AT1-R) antagonists cause an increase in plasma Ang II level, which may result in enhanced production of Ang IV. PATIENTS AND METHODS: In this study, we measured plasma Ang IV and Ang II levels in patients with chronic renal failure (CRF), and also examined the changes in Ang IV and Ang II levels after administration of an ATI-R antagonist. RESULTS: Ang II and Ang IV levels in CRF patients untreated with hemodialysis (n = 16) were 15.8+/-3.6 and 6.0+/-1.1 pg/ml, respectively, which did not differ significantly from Ang II (20.6+/-2.4) and Ang IV levels (8.6+/-1.1) in normal controls (n = 23). The ratio of Ang IV to Ang II was 38%, similar to that in the controls (41%). Ang II or Ang IV levels in CRF patients treated with hemodialysis (n = 12) were also similar to the control values. Ang IV levels had a significant correlation with Ang II levels (r = 0.59). When hypertensive patients were treated with an AT1-R antagonist candesartan for 7 days, Ang II and Ang IV levels were increased 5.5- and 4.1-fold relative to the control levels, respectively. Ang II levels 28 and 56 days after treatment were significantly lower than those 7 days after treatment, whereas Ang IV levels did not differ significantly from those 7 days after treatment. Similar differential kinetics in Ang II and Ang IV levels after long-term (90 days) treatment with an AT1-R antagonist was also confirmed in experiments using rats. Significant decrease in blood pressure continued during long-term treatment with an AT1-R antagonist. CONCLUSION: These findings demonstrated that plasma Ang IV levels in patients with CRF did not differ significantly from those in normal subjects, and that treatment with an AT1-R antagonist caused marked increases in both Ang II and Ang IV levels. In contrast, during long-term treatment plasma Ang II levels were more rapidly decreased than Ang IV levels, suggesting longer-lasting enhancement of the action of Ang IV rather than that of Ang II after treatment with an AT1-R antagonist.

Transient fluctuation of serum melatonin rhythm is suppressed centrally by vitamin B12.

Vitamin B12 has been reported to improve sleep-wake rhythm disorders. Although the mechanism is still unclear, a change in the sensitivity of the circadian clock system to photic input is thought to be a possible mechanism of the effect. In this study, the effect of the vitamin B12 on the circadian aspect of the electroretinogram (ERG) and serum melatonin level was analyzed in rats. Vitamin B12, alpha-(5,6-dimethylbenzimidazolyl)-co-methyl-cobamide was daily administrated subcutaneously for 8 weeks to adult male Wister rats in the experimental group, and saline was given to the control group. The ERGs were recorded under dark adaptation during the night and day, and under light adaptation (0.1 lux) during the night. Blood was drawn before and after ERG recording. The amplitudes of the a-wave, b-wave, and trough-to-peak of both waves and latencies of ERG were analyzed following various exposures to stimuli of light intensity. These parameters in the group treated with vitamin B12 showed similar characteristics to the control group, and no significant difference was observed between the two groups. The melatonin levels of both groups before the measurement of ERG were similar under each measurement condition. The elevated serum melatonin concentration in the control group under dark adaptation at night was suppressed after the series of 10-msec light stimuli used for measurement of ERG. However, this suppressing effect of light pulses on melatonin level was significantly inhibited in the group treated with vitamin B12. Under light adaptation during the night and under dark adaptation during the day, melatonin levels after the measurement of ERG were not different between the groups. From these results, it is suggested that vitamin B12 if effective in suppressing melatonin rhythm disturbances introduced by transient light stimulation, and it affects the site more central than the retinal level.

Measurement of estrogenic activity of chemicals for the development of new dental polymers.

The estrogenic activities of 13 Bisphenol-A (BPA)-related chemicals for development of new polymers by three in vitro bioassay have been examined in the presence and absence of a post-mitochondrial metabolizing system (S9 mix). BPA, Bisphenol-B (BPB), Bisphenol-F (BPF), Bisphenol-S (BPS), 4,4-ethylidenebisphenol (BP1), 4,4-dihydroxybenzophenone (BP2), 2,2-bis (4-hydroxyphenyl)-hexafluoropropane (BP3), 4,4-(1,4-phenylenediisopropylidene) bisphenol (BP4), 4,4-cyclohexylidenebisphenol (BP5), 4,4-dihydroxydiphenyl ether (BP6), 4-hydroxydiphenylmethane (BP7), 4-cumylphenol (BP8) and 4,4-dihydroxydiphenyl sulfide (BP9) were each diluted with dimethyl sulfoxide to final concentrations ranging from 10(-7) to 10(-3) M in both the yeast two-hybrid system and in a fluorescence polarization system. Dilutions of 10(-9) to 10(-4) M were assayed in the E-screen, respectively. Except for BPS and BP4, the chemicals tested showed estrogenic activity in the absence of cut S9 mix preparation and the activity was enhanced with S9 mix. BPS, which was initially negative, was active with S9 mix in the yeast two-hybrid system. BP2 was weakly estrogenic with or without S9 mix. Chemicals other than BP2 were positive in the competition binding assay. All chemicals tested showed estrogenic activity in the E-screen, the concentration level of which was 10(4) times lower than those of the other two assays.

[Augmentation of therapeutic effect of adoptive immunotherapy through a synergy between transferred killer cells and host's fresh lymphocytes].

Among several approaches to augment the therapeutic effect of adoptive immunotherapy, we focused the antitumor synergy between transferred killer cells and host's fresh lymphocytes. Immunotherapy models using murine tumors or clinical experiments revealed that preadministration of immunostimulator such as OK-432, followed by chemotherapeutic agents such as cyclophosphamide, can induce host's non-cytotoxic fresh lymphocytes that act synergistically with cultured killer cells against autologous tumor cells. Immuno-chemo-lymphocytotherapy (a sequential treatment with OK-432, chemotherapy and adoptive immunotherapy) is useful to treat the patients with advanced cancer even if the number of transferred lymphocytes is limited.

Impact of diet on the cardiovascular risk profile of Japanese immigrants living in Brazil: contributions of World Health Organization CARDIAC and MONALISA studies.

1. Japanese immigrants from Okinawa living in Brazil have a higher mortality from cardiovascular diseases and have their mean life expectancy shortened compared with their counterparts living in Japan. 2. A cross-sectional study comparing Okinawans living in Okinawa (OO) and Okinawan immigrants living in Brazil (OB) was designed to characterize the dietary factors that could interfere with the profile of cardiovascular risk factors and with this reduction on the life expectancy when Okinawans emigrate to Brazil. 3. In total, 234 OO and 160 OB (aged 45-59 years) were recruited to the present study to undergo medical and dietary history, blood pressure measurement, electrocardiograph (ECG), blood tests and 24 h food/urine collection. 4. In the present study, OO subjects presented with 37% less obesity and 50% less systemic hypertension than OB. The OB subjects used threefold more antihypertensive medication than OO. Meat intake was 34% higher in OB than OO, whereas fish intake was sevenfold higher in OO than OB. Serum potassium levels were 10% higher in OO than OB. Urinary taurine (an index of seafood intake) was 43% higher in OO than OB. Urinary isoflavones (an index of the intake of soy products) were significantly lower in OB than in OO. Of acid (20:5) and docosahexaenoic acid (22:6) were two- and threefold higher in OO than OB, respectively. 5. The rate of ischaemic ECG changes in OO subjects was only 50% of that of OB subjects. 6. There were no differences in the smoking rate between OO and OB subjects. 7. The results of the present study suggest that coronary risk factors and cardiovascular health are not only regulated by genetic factors, but that the impact of lifestyle (mainly diet) can be large enough to modulate the expression of genes.

[Transarterial immuno-chemotherapy including adoptive transfer of autologous cultured lymphocytes for stage IV breast cancer patients with locally-advanced tumor].

Immuno-chemotherapy via a catheter in the subclavian artery using sequential treatment with OK-432, chemotherapeutic agents (ADM, 5-FU), and cultured autologous lymphocytes, was performed for 9 Stage IV breast cancer patients with locally-advanced primary tumor. Tumor reduction of more than 50% was observed in 8 patients including 4 whose breast tumors had disappeared. Among 11 evaluable distant metastatic lesions, 7 (1 pleural effusion, 2 lung, 2 liver, 2 bone metastases) regressed after local immunotherapy of breast or additional regional immunotherapy (1 lung, 1 liver, 1 pleural effusion). Median survival time to date is 56 months. Five patients are currently alive, although 3 of them did not undergo mastectomy. Local immuno-chemotherapy may be useful because (a) toxicity is limited, (b) low doses of anti-cancer agents during the therapy (median dose of ADM, 60 mg) do not limit subsequent systemic chemotherapy, and (c) distant metastases often regress concomitantly with the primary lesions.

Effect of the brain-derived neurotrophic factor and the apolipoprotein E polymorphisms on disease progression in preclinical Alzheimer's disease.

Genetic factors, such as apolipoprotein E (ApoE) polymorphisms, are thought to play an important role in the etiology of Alzheimer's disease (AD). Recent association studies have suggested that the Val66Met polymorphism in the brain-derived neurotrophic factor (BDNF) gene could play a role in the development of AD. To identify genotypic effects of the BDNF and the ApoE genes on disease progression in preclinical AD, we assessed morphological changes using serial magnetic resonance imaging during the preclinical period of AD in 35 individuals. When all subjects were analyzed as one group, progressive atrophy was noted in the limbic, paralimbic and neocortical areas. Individuals of the BDNF Val/Val genotype showed progressive atrophy in the left medial temporal areas, whereas the BDNF Met allele carriers showed additional changes in the anterior cingulate cortex (ACC), posterior cingulate cortex (PCC) and the precuneus. An interaction between the BDNF genotype and progressive morphological changes was found in the PCC. The noncarriers for the ApoE epsilon4 allele showed progressive atrophy in the bilateral medial temporal areas. In addition to changes in the medial temporal areas, epsilon4 carriers showed progressive atrophy in the PCC, ACC and precuneus. An interaction between the ApoE genotype and progressive morphological change was noted in the right medial temporal area. The present preliminary study indicates that polymorphisms of the ApoE and the BDNF genes could affect disease progression in preclinical AD and implies that the Met-BDNF polymorphism could be an additional risk factor for rapid disease progression in preclinical AD.

Waste benefits of CO2 policies in Japan.

A linear programming model REAP (Regional Environmental strategy Analysis Program) has been developed for the analysis of the consequences of a CO2 tax for petrochemical products such as plastics (CO2, carbon dioxide, is the most important greenhouse gas). Special attention has been paid to the impacts on waste management. The results suggest that a 10,000 Y t(-1) CO2 tax would result in a significant reduction of CO2 emissions in the petrochemical life cycle, ranging from 40 Mt in 2015 to 70 Mt CO2 in later decades (more than 50% emission reduction). Waste quantities will be reduced simultaneously. The CO2 tax results in an 18% reduction of plastic waste weight in 2015 (3 Mt waste). Lower tax levels that may be politically more acceptable would result in proportionally lower environmental benefits. CO2 benefits and waste benefits of a CO2 tax are of equal importance in policy terms. Apart from changes in waste volume, CO2 taxes would affect the cost-effectiveness of waste handling technologies. Energy recovery in industrial kilns may replace conventional waste incineration. Recycling constitutes half of the total waste treatment. Given these results, current investments in new incineration capacity may suffer from insufficient waste availability during the next two decades in case CO2 taxes are introduced. A third effect of a CO2 tax is a significant increase of waste transportation. The results show that this increase is concentrated in the central part of Honshu (Kinki, Chubu & Kanto). Such transportation can result in new local environmental impacts that should be analysed in more detail. Given the strong impact of CO2 taxes on waste quantities and waste treatment it is recommended to co-ordinate CO2 policies and solid waste policies.

Diet and hypertension in Tanzania.

A survey was conducted on an urban population in the city of Dar es Salaam and on a rural community in both Handeni and the pastoral Masai in Monduli to investigate the relationship between diet and hypertension in Tanzania. Blood pressure (BP) was measured using an automatic BP-measuring machine. Biological markers of dietary intake were measured in 24-h urine and in blood. Hypertension was noted to be a bigger problem in the capital city, where the rate of obesity and salt intake were higher whereas potassium, protein, and polyunsaturated fatty acid intake were lower. Therefore, attention to dietary habits may reduce the growing problem of hypertension in Tanzania.

Systemic arterial hypertension: results of the change from pharmacological to nonpharmacological treatment.

The effect of nonpharmacological measures--diet (restriction to salt), weight reduction, stress avoidance, stopping smoking, and exercise--are analyzed after being applied to 145 hypertensive individuals (average age 60.3 years) using antihypertensive drugs for 6 months without nonpharmacological therapy. The initial systolic arterial pressure was 177 mm Hg; at the end of 4 weeks of treatment, it was 151.3 mm Hg. The initial diastolic arterial pressure was 98.5 mm Hg; at the end of 4 weeks it was 89.8 mm Hg (p less than 0.001). The proposal for treating hypertension with nonpharmacological measures represents a challenge and opens a new horizon to scientific research.