A functional SNP upstream of the beta-2 adrenergic receptor gene (ADRB2) is associated with obesity in Oceanic populations.

OBJECTIVE: Obesity is a growing health concern in the Oceanic populations. To investigate the genetic factors associated with adult obesity in the Oceanic populations, the association of single nucleotide polymorphisms (SNPs) of the beta-2 adrenergic receptor (ADRB2) gene with obesity was examined in 694 adults living in Tonga and Solomon Islands. RESULTS: A screening for variation in 16 Oceanic subjects detected 17 SNPs in the entire region of ADRB2, of which nine SNPs including two non-synonymous ones, rs1042713 (Arg16Gly) and rs1042714 (Gln27Glu), were further genotyped for all subjects. The rs34623097-A allele, at a SNP located upstream of ADRB2, showed the strongest association with risk for obesity in a logistic regression analysis adjusted for age, sex, and population (P=5.6 × 10(-4), odds ratio [OR]=2.5, 95% confidence interval [CI]=1.5-4.2). The 27Glu was also significantly associated with obesity in the single-point association analysis (P=0.013, OR=2.0, 95%CI=1.2-3.4); however, this association was no longer significant after adjustment for rs34623097 since these SNPs were in linkage disequilibrium with each other. A copy of the obesity-risk allele, rs34623097-A, led to a 1.6 kg/m(2) increase in body mass index (BMI; defined as weight in kilograms divided by height in meters squared) (P=0.0019). A luciferase reporter assay indicated that rs34623097-A reduced the transcriptional activity of the luciferase reporter gene by approximately 10% compared with rs34623097-G. An electrophoretic mobility shift assay demonstrated that rs34623097 modulated the binding affinity with nuclear factors. An evolutionary analysis implies that a G>A mutation at rs34623097 occurred in the Neandertal genome and then the rs34623097-A allele flowed into the ancestors of present-day humans. CONCLUSION: The present results suggest that rs34623097-A, which would lead to lower expression of ADRB2, contributes to the onset of obesity in the Oceanic populations.

X-ray structure of T4 endonuclease V: an excision repair enzyme specific for a pyrimidine dimer.

The x-ray structure of T4 endonuclease V, an enzyme responsible for the first step of a pyrimidine-dimer-specific excision-repair pathway, was determined at a 1.6-angstrom resolution. The enzyme consists of a single compact domain classified into an all-alpha structure. This single domain has two distinct catalytic activities; it functions as a pyrimidine dimer glycosylase and as an apurinic-apyrimidinic endonuclease. The amino-terminal segment penetrates between two major helices and prevents their direct contact. The refined structure suggests the residues involved in the substrate binding and the catalysis of the glycosylation reaction.

The osteoclast-associated protease cathepsin K is expressed in human breast carcinoma.

Human cathepsin K is a novel cysteine protease previously reported to be restricted in its expression to osteoclasts. Immunolocalization of cathepsin K in breast tumor bone metastases revealed that the invading breast cancer cells expressed this protease, albeit at a lower intensity than in osteoclasts. In situ hybridization and immunolocalization studies were subsequently conducted to demonstrate cathepsin K mRNA and protein expression in samples of primary breast carcinoma. Expression of cathepsin K mRNA was confirmed by reverse transcription PCR and Southern analysis in a number of human breast cancer cell lines and in primary human breast tumors and their metastases. As this protease is known to degrade extracellular matrix, including bone matrix proteins, it is possible that cathepsin K may contribute to the invasive potential of breast cancer cells, including those that metastasize to bone. Thus, cathepsin K may be a potential target leading to the design of novel drugs for cancer therapy.

Preliminary crystallographic study of pyrimidine dimer-specific excision-repair enzyme from bacteriophage T4.

Bacteriophage T4 endonuclease V, which is an excision-repair enzyme specific to pyrimidine dimers within DNA, has been crystallized from polyethylene glycol 4000 solution by a vapour diffusion technique. The unit cell is monoclinic, space group P2(1), with unit cell parameters: a = 41.4 A, b = 40.1 A, c = 37.5 A, beta = 90.01 degrees. The unit cell contains two 16,000 Mr molecules. The crystals diffract X-rays beyond 2.3 A resolution and are suitable for structural analysis at high resolution.

Localization of cathepsin K in human osteoclasts by in situ hybridization and immunohistochemistry.

We have recently cloned cathepsin K from a human bone cDNA library. Since cathepsins are proposed to be involved in the degradation of mineralized bone matrix, we have investigated, by in situ hybridization and immunocytochemistry, the expression of the cathepsin K mRNA transcripts and protein in sections of bone and giant cell tumor to determine which cells express this enzyme. Within all tissues studied, cathepsin K was highly expressed in osteoclasts. Furthermore, the expression of cathepsin K mRNA in giant cell tumor tissue appeared to be confined to the periphery of the osteoclast indicating a compartmentalization of the mRNA. Immunohistochemistry confirmed the specific localization of cathepsin K to the osteoclast. In actively resorbing osteoclasts, the immunostaining was localized at the ruffled border, whereas in osteoclasts in sections of giant cell tumor, staining was observed in lysosomal vacuoles, which in some cases were seen to fuse with the cell membrane. Other cells within the bone, such as osteoblasts and osteocytes, did not express either the cathepsin K transcript or protein. However, there were very low levels of cathepsin K detected in a population of mononuclear cells, possibly representing osteoclast progenitor cells, within the marrow/stromal layer. The specific localization of cathepsin K within osteoclasts would therefore indicate the potential role of this enzyme in the bone resorptive process.

Males in rural Bangladeshi communities are more susceptible to chronic arsenic poisoning than females: analyses based on urinary arsenic.

Spot urine samples were collected from the inhabitants of two rural communities in northwestern Bangladesh. We compared arsenic levels in the urine samples ([As](u); n = 346) with those in water from tube wells ([As](tw); range < 1-535 microg/L; n = 86) on an individual basis. The small variation of [As](u) within subjects and highly positive correlation with [As](tw) indicate that [As](u) is a useful indicator of exposure. Analyses of [As](u) showed that creatinine correction was necessary, that [As](u) only reflected recent exposure, and that there were substantial interindividual differences for a given [As](tw) level. To evaluate the toxic effects of arsenic exposure, we constructed a system for rating skin manifestations, which revealed distinct sex-related differences. Comparison of males and females in the same households confirmed that skin manifestations were more severe in the males, and in the males of one community a dose-response relationship between [As](u) and the degree of skin manifestation was evident. The results of this study indicate that [As](u) in spot urine samples can be used as an exposure indicator for As. They suggest that there might be sex-related, and perhaps community-related, differences in the relationship between [As](u) and skin manifestations, although several confounding factors, including sunlight exposure and smoking habits, might contribute to the observed sex difference. The existence of such differences should be further confirmed and examined in other populations to identify the subpopulations sensitive to chronic arsenic toxicity.

Prolyl endopeptidase from Flavobacterium meningosepticum: cloning and sequencing of the enzyme gene.

The prolyl endopeptidase [EC 3.4.21.26] gene of Flavobacterium meningosepticum was cloned in Escherichia coli with the aid of an oligonucleotide probe which was prepared based on the amino acid sequence. The hybrid plasmid, pFPEP1, with a 3.5 kbp insert at the HincII site of pUC19 containing the enzyme gene, was subcloned into pUC19 to construct plasmid pFPEP3. The whole nucleotide sequence of an inserted HincII-BamHI fragment of plasmid pFPEP3 was determined by the dideoxy chain-terminating method. The purified prolyl endopeptidase was labeled with tritium DFP, and the sequence surrounding the reactive serine residue was found to be Ala (551)-Leu-Ser-Gly-Arg-*Ser-Asn(557). Ser-556 was identified as a reactive serine residue. The enzyme consists of 705 amino acid residues as deduced from the nucleotide sequence and has a molecular weight of 78,705, which coincides well with the value estimated by ultra centrifugal analysis. The amino acid sequence was 38.2% homologous to that of the porcine brain prolyl endopeptidase [Rennex et al. (1991) Biochemistry 30, 2195-2203] and 24.5% homologous to E. coli protease II, which has substrate specificity for basic amino acids [Kanatani et al. (1991) J. Biochem. 110, 315-320].

Directed evolution to improve the thermostability of prolyl endopeptidase.

Prolyl endopeptidase is the only endopeptidase that specifically cleaves peptides at proline residues. Although this unique specificity is advantageous for application in protein chemistry, the stability of the enzyme is lower than those of commonly used peptidases such as subtilisin and trypsin. Therefore, we attempted to apply a directed evolution system to improve the thermostability of the enzyme. First, an efficient expression system for the enzyme in Escherichia coli was established using the prolyl endopeptidase gene from Flavobacterium meningosepticum. Then, a method for screening thermostable variants was developed by combining heat treatment with active staining on membrane filters. Random mutagenesis by error-prone PCR and screening was repeated three times, and as a result the thermostability of the enzyme was increased step by step as the amino acid substitutions accumulated. The most thermostable mutant obtained after the third cycle, PEP-407, showed a half-life of 42 min at 60 degrees C, which was 60 times longer than that of the wild-type enzyme. The thermostable mutant was also more stable with a high concentration of glycerol, which is a necessary condition for in vitro amidation.

An epidemiological study with risk analysis of liver diseases in the general population living in a methyl mercury polluted area.

STUDY OBJECTIVE: The aim was to determine the actual prevalence of liver disease and to investigate the contribution of various risk factors to liver disease among the population in a methyl mercury polluted area. DESIGN: The study was a population based cross sectional mass screening survey. A case-control study was designed to estimate the role of various risk factors for liver diseases. SETTING: The study was confined to a small rural town 10 km north of Minamata City. SUBJECTS: 1406 persons aged 50 to 69 years were examined (78.3% of the total population of this age in the locality). MEASUREMENTS AND MAIN RESULTS: Measurements of liver disease were made on the basis of haematological, physical, and ultrasonographic examinations. Data on liver risk factors were collected by questionnaire, and by measurement of body height, weight (obesity), and hepatitis B surface antigen (HBsAg). The prevalence rate of liver tumour was 0.5% in males, liver cirrhosis was found in 0.5% of males and 0.1% of females, and hepatitis was seen in 5.4% of males and 1.0% of females. Frequency rates of risk factors for liver disease among subjects with obesity were significantly higher in the female patient group, and the frequency rate among subjects with alcoholic drinking habits was significantly higher in the male patient group. The odds ratio of past history of blood transfusion showed the highest value among other related factors (7.73) and the attributable risk for this was very high (87.1%); HBsAg was next in rank (odds ratio 3.04; attributable risk 67.1%). CONCLUSIONS: The prevalence of liver disease in this methyl mercury polluted area was not increased, contrary to what was expected based on the standard mortality ratios. The main risk factors for liver disease in this area appear to be alcoholic drinking habits and a history of blood transfusion.

Microinjection of T4 endonuclease V produced by a synthetic denV gene stimulates unscheduled DNA synthesis in both xeroderma pigmentosum and normal cells.

A structural gene for T4 endonuclease V was constructed by ligating synthetic oligonucleotides. The endonuclease V was overproduced in E. coli under control of the E. coli tryptophan promoter and purified to apparent homogeneity. The product had comparable DNA glycosylase and apurinic/apyrimidinic (AP) endonuclease activities to the natural enzyme in vitro. When this endonuclease V was microinjected into the cytoplasm of xeroderma pigmentosum (XP) cells of complementation group A, B, C, D, F, G or H, unscheduled DNA synthesis (UDS) above the residual level was detected in all the cells at a dose of about 10(3) molecules following UV irradiation. The gain numbers of UDS in these XP cells increased with increase in the dose of enzyme and reached a plateau at the normal cell level on introduction of about 10(4) molecules. Introduction of more enzyme into either XP cells or normal human cells did not increase the grain number under regular labelling conditions (2.5 h, 37 degrees C). In normal mouse cells, introduction of the enzyme increased the grain number more than 4-fold under the same conditions during at least 8.5 h following UV irradiation. Furthermore, with a labelling time of 30 min, the enzyme more than doubled the grain number even in normal human cells.

Postprandial resting metabolic rate and body composition in the moderately obese and normal-weight adult subjects at sitting posture.

A reduced metabolic rate in the etiology of obesity has been a subject of controversy. The prediction of the energy requirements for the obese using reference values may therefore be distorted. In order to examine this possibility, resting metabolic rate (RMR) while the subject was sitting comfortably in a chair was measured in a total of 134 moderately obese and normal-weight subjects (68 women aged 20 to 71 with a mean of 53.1 and 66 men aged 20 to 63 with a mean of 36.5). RMR per kg of body weight was significantly lower in the female obese subjects, but not in the male obese subjects. There was no evidence of difference in RMR between obese and normal-weight subjects in either sex when RMR was indexed with fat-free mass (FFM), indicating no substantial decrease in the metabolism due to obesity. Multiple regression analyses indicate that standardization of RMR by FFM eliminates the apparent difference in RMR between the sexes, and the diminution of RMR with age was not observed. While the best and logical prediction of RMR is to use FFM, regression analyses suggest an alternative way of predicting RMR by an incorporation of subscapular skinfold thickness to adjust the different body composition in lean and obese subjects. Prediction equations of postprandial RMR (kcal/24-h) while sitting are RMR = 24.5 x FFM(kg) + 303.7, and RMR = 22.7 x weight(kg) - 13.6 x SSF (subscapular skinfold: mm) + 350.6. Problems in predicting RMR are discussed.

Creatinine height index in a sample of Japanese adults under sedentary activities.

The value of creatinine height index (CHI), defined as milligrams of 24-h urinary excretion of creatinine divided by height in centimeters, was investigated on 21 healthy Japanese adults engaged in sedentary work. The average values were 10.58 in males and 6.68 in females. High partial correlation coefficients controlling sex were found among CHI, weight, fat-free mass, arm muscle area (AMA) and 24-h urinary excretion of creatinine. These indices had high loadings in the first factor of body size by principal component analysis. Regression of CHI on AMA differed in the intercept (i.e. the level of CHI at corresponding AMA) but not in the slope, from that studied under low-creatine diet in the U.S.A. Correcting AMA using arm bone area for Japanese and CHI with dietary factor, the relation between CHI and AMA in this study seemed to be in good agreement with that found in the U.S.A.

Scintigraphic evaluation of pamidronate and corticosteroid therapy in a patient with progressive diaphyseal dysplasia (Camurati-Engelmann disease).

A 27-year-old woman with progressive diaphyseal dysplasia (Camurati-Engelmann disease) received pamidronate and corticosteroid therapy for bone pain. During therapy, disease activity was assessed serially using bone scintigraphy with Tc-99m HMDP. With pamidronate administration, the bone pain became worse and diaphyseal uptake of Tc-99m HMDP increased, whereas corticosteroid administration improved the bone pain and reduced the diaphyseal uptake. In this case, pamidronate and corticosteroid produced different effects. Bone scintigraphy allowed an objective assessment of the response to these treatments, accurately reflecting clinical symptoms.

Whole-body vibration and health effects in the agricultural machinery drivers.

Recently farm mechanization has been widespread and developing rapidly, in particular riding farm machines are increasingly used in paddy fields in Japan. We have no information available on the actual situation regarding whole-body vibration on the seats of these farm machines from the standpoint of labour protection. Measurement and evaluation of whole-body vibration was performed on the seats of popular riding agricultural machineries. Whole-body vibration on the seats of combine harvesters and wheel tractors exceeded exposure limits and the fatigue-decreased proficiency boundary limit of 8 hr and also shortened the reduced comfort boundary limits of ISO 2631 (1985). Some combines, tractors and carieers had only less than one hour exposure duration as compared with the ISO 2631-1 standard (1997). On the other hand a questionnaire was also performed on the subject of agricultural machine operators. Any specific injury or other effects, i.e. low back injuries were not found among the group of operators as compared with those in non-operator farmers. It seems to be difficult to find out the health effects of whole-body vibration itself, because there may be a lot of causes, i.e. working posture, operating heavy materials, in farm working conditions.

Hand-arm vibration in tropical rain forestry workers.

Working conditions and health hazards including vibration syndrome related to forestry work using chain-saws were studied in Papua New Guinea and Indonesia. The subjects comprised 291 workers including 97 chain-saw operators. The health examination consisted of peripheral circulatory and sensory tests in the upper extremities. The vibration spectrum measured at the handle of the chain-saw indicated that these acceleration levels would lead to a moderately high risk of hand-arm vibration syndrome (HAVS). The peripheral circulatory function tests revealed dysfunction after more than five years vibration exposure. However, in general, the results of the function tests and subjective complaints showed fewer health problems compared to those of Japanese forestry workers. The reason of such differences of vibration effects seem to be the following: (1) warmer climate (more than 25 degrees C throughout the year), (2) young workers and short work experience. (3) short time vibration exposures on working days in the natural forests, (4) seasonal changes in logging work (5) healthy workers effects. Thus, we found no clear evidence that the workers of our study suffered from HAVS. A principal component analysis was applied. The factor score of the components of the reactive dynamics of peripheral circulation differed significantly after more than five years' exposure. On the other hand, we cannot deny the possibility that subclinical dysfunction of peripheral circulation may be caused by chain-saw operation in the tropics in future. Further investigations on the HAVS among forestry workers in the tropic environment are needed.

[Reproductive histories of coastal and inland women with and without exposure to methylmercury in Kumamoto].

To examine the difference in abnormal conception by region and generation and its possible causes in the town of Tsunagi, which experienced intensive methylmercury-pollution during the 1950s and 1960s, reproductive histories of 109 women in 3 coastal villages and 64 controls in 5 inland villages were obtained by visit- and interview-study in 1991. There were about 290 married women in respective regions. The subjects were divided into 4 age groups to form 10-year-birth cohorts from 1915 to 1954. The rate of sterility and those who had never become pregnant after marriage, did not differ between coastal (7.3%) and inland women (6.3%). The younger the birth cohort, the smaller the total number of conceptions. The rate of abnormal conception, i.e., abnormal conception (abortion+stillbirth) per normal conceptions (total number of conceptions--number of artificial abortions), in the coastal women tended to increase after the 1960s with its peak (19%) in 1965-70. The rate in the inland women was already as high as 15% in 1945-50, and was constantly above 10% afterwards. However, it did not differ between the two groups during 1950-1970, when severe methylmercury-pollution occurred in the area. Breakdown of the abnormal conceptions in the inland women showed 4 stillbirths and 19 abortions, among the latter of which 16 were related to climbing up and down the hills to fetch water, hard agricultural work and agitation. The abnormal conceptions in the coastal women showed 3 stillbirths and 19 abortions. Sixteen of the latter were not related to any specific physical causes.

[Work history, health conditions and hearing loss of Ishigaki fishermen].

Based on our previous health survey among Ishigaki fishermen in 1979, this study aimed at clarifying the relations of their work histories to physical characteristics, physiological functions, blood conditions and hearing levels. Medical examination was conducted in 1987 on 118 fishermen inclusive of 33 followed-up fishermen, and in 1989 noise-level and noise-induced temporary threshold shift of hearing was measured for different fishing methods. The results were as follows; 1) Fishing history was not specifically associated with any physical characteristics. 2) High HDL cholesterol, which was observed among divers using diving apparatus, was considered to be a survival effect for divers, since HDL cholesterol is amplified by intensive muscle work. 3) Abnormalities of ECG except for a high R wave were notably observed among the divers, who once changed from unassisted diving to diving with apparatus and then to fishing lines. The main reason for switching jobs was claimed to be caisson disease, and it was suggested that diving with apparatus was related to a high risk of health hazards in the central nervous system and/or respiro-circulatory system. 4) Acoustic acuity greatly declined with aging, and which was commonly observed in groups with different work histories. By way of explanation, exposure to ship engine sounds during daily travelling was the most likely cause. It was also revealed that several hours' exposure to the engine sounds was needed to detect a temporary threshold shift of hearing before and after work.

Health status comparison by urinalysis (dipstick test) among four populations in Papua New Guinea.

The health status of four populations depending on traditional subsistence in Papua New Guinea was compared by the dipstick test urinalysis. Conspicuous inter-population difference in the distribution of urinary pH was attributed to the levels of protein intake and the balances of sodium and potassium intake. The percentage of positive findings on protein differed by population along with the percentages of urobilinogen and bilirubin; the higher percentage of protein positives (12-16%) found in less urbanized populations suggests a high risk of hepatic and/or renal disorders in traditional societies. The very low percentage, 0.3 percent, of positive findings on glucose among 1,132 urine samples tested indicated that diabetes mellitus was not yet the major problem. Simultaneously, however, the fact that glucose positives were found only in the most urbanized villages indicates increasing risk of diabetes even in the traditional populations during future urbanization.

Variation in malaria endemicity in relation to microenvironmental conditions in the Admiralty Islands, Papua New Guinea.

For 197 adults and adolescents in four villages of three small islands in the Admiralty Islands, Papua New Guinea, antimalarial antibody titers were examined using the indirect fluorescent antibody test (IFAT) and malaria parasites were detected by the microtiter plate hybridisation (MPH) method using polymerase chain reaction (PCR) technique. The parasite rate (either Plasmodium falciparum or P. vivax, or both) averaged 39.2%, varying from 31.1% to 44.8% among the four villages due to natural and artificial microenvironmental conditions related to breeding sites of mosquitoes (Anopheles farauti). The lack of flat zones owing to geomorphological formation contributed to the lowest parasite rate in the extremely small island. However, human-modified environments such as a wet-land (naturally formed but artificially reformed) and an open well played significant roles in other inter-village differences. The present findings imply significant roles of microenvironment in diversified malaria prevalence and suggest some ways of mitigation of malarial hazards.

[Susceptibility of the Chinese hamster (Cricetulus griseus) to parasitic infection. (2) Experimental infection with Schistosoma japonicum].

Chinese hamsters (Cricetulus griseus Milne-Edwards: CHA colony) were experimentally infected with Schistosoma japonicum cercariae by the percutaneous route and intraperitoneal inoculation to confirm their susceptibility to the infection with this parasite in comparison with that of mice (DD). Chinese hamsters proved to be highly susceptible to percutaneous and intraperitoneal infection with S. japonicum. The eggs of S. japonicum were recognized in feces of all Chinese hamsters and mice from about 6 weeks after infection. The mortality within 50 weeks post-infection was lower in Chinese hamsters than in mice. Eight out of 18 Chinese hamsters survived more than 50 weeks post-infection, seven of these showed eggs in their feces beyond this period, whereas no fecal eggs was detected in the same period in the five mice which survived more than 50 weeks post-infection. Chinese hamsters seem to be equally susceptible as mice to S. japonicum infection and more tolerant than mice to the worm load.