[A multicenter study of reference intervals for 15 laboratory parameters in Chinese children].

Objective: To establish comprehensive laboratory reference intervals for Chinese children. Methods: This was a cross-sectional multicenter study. From June 2013 to December 2014, eligible healthy children aged from 6-month to 17-year were enrolled from 20 medical centers with informed consent. They were assessed by physical examination, questionnaire survey and abdominal ultrasound for eligibility. Fasting blood samples were collected and delivered to central laboratory. Measurements of 15 clinical laboratory parameters were performed, including estradiol (E2), testosterone(T), luteinizing hormone(LH), follicle-stimulating hormone(FSH), alanine transaminase(ALT), serum creatinine(Scr), cystatin C, immunoglobulin A(IgA), immunoglobulin G(IgG), immunoglobulin M(IgM), complement (C3, C4), alkaline phosphatase(ALP), uric acid(UA) and creatine kinase(CK). Reference intervals were established according to central 95% confidence intervals for reference population, stratified by age and sex. Results: In total, 2 259 children were enrolled. Finally, 1 648 children were eligible for this study, including 830 boys and 818 girls, at a mean age of 7.4 years. Age- and sex- specific reference intervals have been established for the parameters. Reference intervals of sex hormones increased gradually with age. Concentrations of ALT, cystatin C, ALP and CK were higher in children under 2 years old. Serum levels of sex hormones, creatinine, immunoglobin, CK, ALP and urea increased rapidly in adolescence, with significant sex difference. In addition, reference intervals were variable depending on assay methods. Concentrations of ALT detected by reagents with pyridoxal 5'-phosphate(PLP) were higher than those detected by reagents without PLP. Compared with enzymatic method, Jaffe assay always got higher results of serum creatinine, especially in children younger than 9 years old. Conclusion: This study established age- and sex- specific reference intervals, for 15 clinical laboratory parameters based on defined healthy children.

Comparison of pregnancy outcomes between women with gestational diabetes and overt diabetes first diagnosed in pregnancy: a retrospective multi-institutional study in Japan.

AIMS: To determine differences in pregnancy outcomes including diabetic complications, maternal and perinatal complications between gestational diabetes mellitus and overt diabetes in pregnancy in Japan. METHODS: A multi-institutional retrospective study compared pregnancy outcomes between gestational diabetes mellitus and overt diabetes in pregnancy. We examined pregnant women who met the former criteria for gestational diabetes mellitus and received dietary intervention with self-monitoring of blood glucose with or without insulin. Overt diabetes in pregnancy was defined as ≥2 abnormal values on 75-g oral glucose tolerance test, fasting glucose ≥126 mg/dl (7.0 mmol/l) and 2-h postprandial glucose ≥200 mg/dl (11.1 mmol/l), or glycated hemoglobin levels ≥6.5% (48 mmol/mol). RESULTS: Data were collected on 1267 women with gestational diabetes and 348 with overt diabetes in pregnancy. Pregestational body mass index was higher (26.2 ± 6.1 vs. 24.9 ± 5.7 kg, P<0.05) and gestational age at delivery was earlier (37.8 ± 2.5 weeks vs. 38.1 ± 2.1 weeks, P<0.05) in overt diabetes than in gestational diabetes. Glycated hemoglobin (6.8 ± 1.1% [51 mmol/mol] vs. 5.8 ± 0.5% [40 mmol/mol], P<0.05) and glucose on 75-g oral glucose tolerance test and prevalence of retinopathy (1.2% vs. 0%, P<0.05) and pregnancy-induced hypertension (10.1% vs. 6.1%, P<0.05) were higher in overt diabetes than in gestational diabetes. Pregnancy-induced hypertension was associated with pregestational body mass index, gestational weight gain, chronic hypertension, and nulliparity but not with 75-g oral glucose tolerance test. CONCLUSIONS: Overt diabetes in pregnancy is significantly associated with maternal complications such as retinopathy and pregnancy-induced hypertension.

Simultaneous assay of activated platelet count and platelet-activating capacity by P-selectin detection using K2-EDTA-treated whole blood for antiplatelet agents.

INTRODUCTION: It is well recognized that examinations of activated platelets (aPLTs) and platelet-activating capacity are very important to observe and prevent embolic diseases (events) such as ischemic stroke and myocardial infarction. Previously, we reported an appropriate measurement technique of aPLT for clinical assay. In this paper, we investigated stable conditions for measurement of activating capacity of platelets. METHODS: Blood samples were taken from healthy volunteers using anticoagulants of 2K-EDTA, sodium citrate and heparin, and platelets were stimulated with adenosine diphosphate (ADP) or collagen. We demonstrated platelet-activating capacity by detection of scattering light, absorbance, microscopic observation, and P-selectin (CD62P) expression. We also performed basic experiments in seven healthy volunteers to test the clinical application of these assays with monitoring aspirin therapy. RESULTS: We judged that samples of whole blood with 2K-EDTA were suitable for CD62P expression assay as functional assessments of platelet activity, because platelets treated with anticoagulants such as sodium citrate and heparin were extremely damaged after stimulation, and it was difficult to measure the CD62P expression by flow cytometry. For optimal results, samples should be tested within 1 h after the drawing of blood and stimulated with ADP or collagen for 10 min. The CD62P-positive platelet value of blood from volunteers who had taken aspirin was decreased, and platelet activation was inhibited as well. CONCLUSION: The simultaneous assay of aPLT and platelet-activating capacity by CD62P detection using whole blood treated with the K2-EDTA anticoagulant was useful for the monitoring of antiplatelet drugs.

A new predictive indicator for development of pressure ulcers in bedridden patients based on common laboratory tests results.

BACKGROUND: Various scales have been devised to predict development of pressure ulcers on the basis of clinical and laboratory data, such as the Braden Scale (Braden score), which is used to monitor activity and skin conditions of bedridden patients. However, none of these scales facilitates clinically reliable prediction. AIMS: To develop a clinical laboratory data-based predictive equation for the development of pressure ulcers. METHODS: Subjects were 149 hospitalised patients with respiratory disorders who were monitored for the development of pressure ulcers over a 3-month period. The proportional hazards model (Cox regression) was used to analyse the results of 12 basic laboratory tests on the day of hospitalisation in comparison with Braden score. RESULTS: Pressure ulcers developed in 38 patients within the study period. A Cox regression model consisting solely of Braden scale items showed that none of these items contributed to significantly predicting pressure ulcers. Rather, a combination of haemoglobin (Hb), C-reactive protein (CRP), albumin (Alb), age, and gender produced the best model for prediction. Using the set of explanatory variables, we created a new indicator based on a multiple logistic regression equation. The new indicator showed high sensitivity (0.73) and specificity (0.70), and its diagnostic power was higher than that of Alb, Hb, CRP, or the Braden score alone. CONCLUSIONS: The new indicator may become a more useful clinical tool for predicting presser ulcers than Braden score. The new indicator warrants verification studies to facilitate its clinical implementation in the future.

Effect of aging on blood pressure in Leh, Ladakh, a high-altitude (3524 m) community, by comparison with a Japanese town.

The effect of aging on blood pressure (BP) and heart rate (HR) was investigated in a cross-sectional study in the high-altitude community of Leh, Ladakh (altitude: 3524 m) and a Japanese community in U town, Hokkaido (altitude: 25 m). BP and HR were obtained in a sitting position from 332 subjects 13-81 years of age in Ladakh, and from 216 Japanese citizens, 24-79 years of age. Measurements were taken after a 2-min rest, using a semi-automated BP device (UA-767 PC, A and D Co. LTD, Tokyo). High-altitude people showed higher diastolic BP and HR values than lowland people (83.2 vs. 76.9 mmHg and 78.6 vs. 69.2 bpm, P < 0.001), but no difference in systolic BP. Highland people also showed a steeper BP increase with age than the lowland people (systolic BP: 0.7476 vs. 0.3179 mmHg/year, P < 0.0005; diastolic BP: 0.3196 vs. 0.0750 mmHg/year, P < 0.001). This chronoecologic investigation in Ladakh examined the circulation as a physiological system at high-altitude. Our data indicate the need for a more comprehensive cardiovascular assessment for a better diagnosis and a more fruitful treatment. Longitudinal observations of effects of socio-ecologic factors on the cardiovascular system should help prevent strokes and other cardiovascular events, especially at high altitude.

Chronoecological health watch of arterial stiffness and neuro-cardio-pulmonary function in elderly community at high altitude (3524 m), compared with Japanese town.

Effects of high altitude on arterial stiffness and neuro-cardio-pulmonary function were studied. Blood pressure (BP) and heart rate (HR) were measured in a sitting position on resting Ladakhis, living at an altitude of 3250-4647 m (Phey village, 3250 m: 17 men and 55 women; Chumathang village, 4193 m: 29 men and 47 women; Sumdo village, 4540 m: 38 men and 57 women; and Korzok village, 4647 m: 84 men and 70 women). The neuro-cardio-pulmonary function, including the Kohs block design test, the Up and Go, the Functional Reach and the Button tests, was examined in 40 elderly subjects (19 men and 21 women, mean age: 74.7 +/- 3.3 years) in Leh, Ladakh (altitude: 3524 m), for comparison with 324 elderly citizens (97 men and 227 women, mean age: 80.7 +/- 4.7 years) of Tosa, Japan (altitude: 250 m). Cardio-Ankle Vascular Index (CAVI) was measured as the heart-ankle pulse wave velocity (PWV) in these subjects using a VaSera CAVI instrument (Fukuda Denshi, Tokyo). SpO(2) decreased while Hb and diastolic BP increased with increasing altitude. At higher altitude, residents were younger and leaner. Women in Leh vs. Tosa had a poorer cognitive function, estimated by the Kohs block design test (3.7 +/- 3.6 vs. 16.4 +/- 9.6 points, P < 0.0001) and poorer ADL functions (Functional Reach: 13.7 +/- 7.0 cm vs. 25.3 +/- 8.7 cm, P < 0.0001; Button test: 22.5 +/- 4.8 vs. 14.8 +/- 5.7 s, P < 0.0001). Time estimation was shorter at high altitude (60-s estimation with counting: 41.1% shorter in men and 23.0% shorter in women). A higher voltage of the QRS complex was observed in the ECG of Leh residents, but two times measurement of CAVI showed no statistically significant differences between Leh and Tosa (two times of CAVI measures; 9.49 vs. 10.01 m/s and 9.41 vs. 10.05 m/s, respectively), suggesting that most residents succeed to adapt sufficiently to the high-altitude environment. However, correlation of CAVI with age shows several cases who show an extreme increase in CAVI. Thus, for the prevention of stroke and other adverse cardiovascular outcomes, including dementia, CAVI may be very useful, especially at high altitude. In conclusion, elderly people living at high altitude have a higher risk of cardiovascular disease than low-latitude peers. To determine how these indices are associated with maintained cognitive function deserves further study by the longitudinal follow-up of these communities in terms of longevity and aging in relation to their neuro-cardio-pulmonary function.

Depressive mood is independently related to stroke and cardiovascular events in a community.

By means of a multivariate Cox model, we investigated the predictive value of a depressive mood on vascular disease risk in middle-aged community-dwelling people. In 224 people (88 men and 136 women; mean age: 56.8 +/- 11.2 years) of U town, Hokkaido (latitude: 43.45 degrees N, longitude: 141.85 degrees E), a chronoecological health watch was started in April 2001. Consultations were repeated every 3 months. Results at the November 30, 2004 follow-up are presented herein. 7-day/24-h blood pressure (BP) and heart rate (HR) monitoring started on a Thursday, with readings taken at 30-min intervals between 07:00 h and 22:00 h and at 60-min intervals between 22:00 h and 07:00 h. Data stored in the memory of the monitor (TM-2430-15, A and D company, Japan) were retrieved and analyzed on a personal computer with a commercial software for this device. Subjects were asked to answer a self-administered questionnaire inquiring about 15 items of a depression scale, at the start of study and again after 1-2 years. Subjects with a score higher by at least two points at the second versus first screening were classified as having a depressive mood. The other subjects served as the control group. The mean follow-up time was 1064 days, during which four subjects suffered an adverse vascular outcome (myocardial infarction: one man and one woman; stroke: two men). Among the variables used in the Cox proportional hazard models, a depressive mood, assessed by the Geriatric Depression Scale (GDS), as well as the MESOR of diastolic (D) BP (DBP-MESOR) and the circadian amplitude of systolic (S) BP (SBP-Amplitude) showed a statistically significant association with the occurrence of adverse vascular outcomes. The GDS score during the second but not during the first session was statistically significantly associated with the adverse vascular outcome. In univariate analyses, the relative risk (RR) of developing outcomes was predicted by a three-point increase in the GDS scale (RR = 3.088, 95% CI: 1.375-6.935, P = 0.0063). Increases of 5 mmHg in DBP-MESOR and of 3 mmHg in SBP-Amplitude were associated with RRs of 2.143 (95% CI: 1.232-3.727, P = 0.0070) and 0.700 (95% CI: 0.495-0.989, P = 0.0430), respectively. In multivariate analyses, when both the second GDS score and the DBP-MESOR were used as continuous variables in the same model, GDS remained statistically significantly associated with the occurrence of cardiovascular death. After adjustment for DBP-MESOR, a three-point increase in GDS score was associated with a RR of 2.172 (95% CI: 1.123-4.200). Monday endpoints of the 7-day profile showed a statistically significant association with adverse vascular outcomes. A 5 mmHg increase in DBP on Monday was associated with a RR of 1.576 (95% CI: 1.011-2.457, P = 0.0446). The main result of the present study is that in middle-aged community-dwelling people, a depressive mood predicted the occurrence of vascular diseases beyond the prediction provided by age, gender, ABP, lifestyle and environmental conditions, as assessed by means of a multivariate Cox model. A depressive mood, especially enhanced for 1-2 years, was associated with adverse vascular outcomes. Results herein suggest the clinical importance of repetitive assessments of a depressive mood and the need to take sufficient care of depressed subjects. Another result herein is that circadian and circaseptan characteristics of BP variability measured 7-day/24-h predicted the occurrence of vascular disease beyond the prediction provided by age, gender, depressive mood and lifestyle, as assessed by means of a multivariate Cox model. Earlier, we showed that the morning surge in BP on Mondays was statistically significantly higher compared with other weekdays. Although a direct association between the Monday surge in BP and cardiovascular events could not be demonstrated herein, it is possible that the BP surge on Monday mornings may also trigger cardiovascular events. We have shown that depressive people exhibit a more prominent circaseptan variation in SBP, DBP and the double product (DP) compared to non-depressed subjects. In view of the strong relation between depression and adverse cardiac events, studies should be done to ascertain that depression is properly diagnosed and treated. Chronodiagnosis and chronotherapy can reduce an elevated blood pressure and improve the altered variability in BP and HR, thus reducing the incidence of adverse cardiac events. This recommendation stands at the basis of chronomics, focusing on prehabilitation in preference to rehabilitation, as a public service offered in several Japanese towns.

Arterial stiffness independently predicts cardiovascular events in an elderly community -- Longitudinal Investigation for the Longevity and Aging in Hokkaido County (LILAC) study.

We investigated the predictive value of arterial stiffness to assess cardiovascular risk in elderly community-dwelling people by means of a multivariate Cox model. In 298 people older than 75 years (120 men and 178 women, average age: 79.6 years), brachial-ankle pulse wave velocity (baPWV) was measured between the right arm and ankle in a supine position. The LILAC study started on July 25, 2000, consultation was repeated yearly, and the last follow-up ended on November 30, 2004. During this follow-up span of 1227 days, there were nine cardiovascular deaths, the cause of death being myocardial infarction for two men and three women or stroke for two men and two women. In Cox proportional hazard models, baPWV as well as age, Mini-Mental State Examination (MMSE), Hasegawa Dementia Scale Revised (HDSR) and the low-frequency/high-frequency (LF/HF) ratio showed a statistically significant association with the occurrence of cardiovascular death. A two-point increase in MMSE and HDSR score significantly protected against cardiovascular death, the relative risk (RR) being 0.776 (P = 0.0369) and 0.753 (P = 0.0029), respectively. The LF/HF ratio also was significant (P = 0.025), but the other indices of HRV were not. After adjustment for age and HDSR, a 200 cm/s increase in baPWV was associated with a 30.2% increase in risk (RR = 1.302, 95% CI: 1.110-1.525), and a 500 cm/s increase in baPWV with a 93.3% increase in risk (RR = 1.933, 95% CI: 1.300-2.874, P = 0.0011), whereas the LF/HF ratio was no longer associated with a statistically significant increase in cardiovascular mortality. In elderly community-dwelling people, arterial stiffness measured by means of baPWV predicted the occurrence of cardiovascular death beyond the prediction provided by age, gender, blood pressure and cognitive functions. baPWV should be added to the cardiovascular assessment in various clinical settings, including field medical surveys and preventive screening. The early detection of risk by chronomics allows the timely institution of prophylactic measures, thereby shifting the focus from rehabilitation to prehabilitation medicine, as a public service to several Japanese towns.

Fractal analysis of heart rate variability and mortality in elderly community-dwelling people -- Longitudinal Investigation for the Longevity and Aging in Hokkaido County (LILAC) study.

AIM: Fractal analysis of heart rate (HR) variability (HRV) has been used as a new approach to evaluate the risk of mortality in various patient groups. Aim of this study is to examine the prognostic power of detrended fluctuation analysis (DFA) and traditional time- and frequency-domain analyses of HR dynamics as predictors of mortality among elderly people in a community. METHODS: We examined 298 people older than 75 years (average age: 79.6 years) and 1-h ambulatory ECG was monitored. During the last 10 min, deep respiration (6-s expiration and 4-s inspiration) was repeated six times in a supine position. Time-domain and frequency-domain measures were determined by the maximum entropy method. Scaling exponents of short-term (<11 beats, alpha 1) and longer-term (>11 beats, alpha 2) were determined by the DFA method. Six estimates, obtained from 10-min segments, were averaged to derive mean values for the entire recording span. These average values were denoted Alpha 1 and Alpha 2, estimates obtained during the first 10-min segment Alpha 1 S and Alpha 2 S, and those during the last 10-min segment Alpha 1E and Alpha 2E, respectively. The LILAC study started on July 25, 2000 and ended on November 30, 2004. We used Cox regression analysis to calculate relative risk (RR) and 95% confidence interval (CI) for all-cause mortality. Significance was considered at a value of P < 0.05. RESULTS: Gender, age and Alpha 2E showed a statistically significant association with all-cause mortality. In univariate analyses, gender was significantly associated with all-cause mortality, being associated with a RR of 3.59 (P = 0.00136). Age also significantly predicted all-cause mortality and a 5-year increase in age was associated with a RR of 1.49 (P = 0.01809). The RR of developing all-cause mortality predicted by a 0.2-unit increase in Alpha 2E was 0.58 (P = 0.00390). Other indices of fractal analysis of HRV did not have predictive value. In multivariate analyses, when both Alpha 2E and gender were used as continuous variables in the same model, Alpha 2E remained significantly associated with the occurrence of all-cause mortality (P = 0.02999). After adjustment for both gender and age, a 0.2-unit increase in Alpha 2E was associated with a RR of 0.61 (95% CI: 0.42-0.90, p = 0.01151). CONCLUSION: An intermediate-term fractal-like scaling exponent of RR intervals was a better predictor of death than the traditional measures of HR variability in elderly community-dwelling people. It is noteworthy that the longer-term (alpha 2) rather than the short-term fractal component (alpha 1) showed predictive value for all-cause mortality, which suggests that an increase in the randomness of intermediate-term HR behavior may be a specific marker of neurohumoral and sympathetic activation and therefore may also be associated with an increased risk of mortality.

Common carotid intima-media thickness is predictive of all-cause and cardiovascular mortality in elderly community-dwelling people: Longitudinal Investigation for the Longevity and Aging in Hokkaido County (LILAC) study.

Several cohort studies have examined the association of carotid intima-media thickness (IMT) with the risk of stroke or myocardial infarction in apparently healthy persons. We investigated the predictive value of IMT of cardiovascular mortality in elderly community-dwelling people, beyond the prediction provided by age and MMSE, assessed by means of a multivariate Cox model. Carotid IMT and plaque were evaluated bilaterally with ultrasonography in 298 people older than 75 years (120 men and 178 women, average age: 79.6 years). The LILAC study started on July 25, 2000. Consultations were repeated every year. The follow-up ended on November 30, 2004. During the mean follow-up span of 1152 days, 30 subjects (21 men and nine women) died. Nine deaths were attributable to cardiovascular causes (myocardial infarction: two men and three women; stroke: two men and two women). The age- and MMSE-adjusted relative risk (RR) and 95% confidence interval (95% CI) of developing all-cause mortality was assessed. A 0.3 mm increase in left IMT was associated with a RR of predicted 1.647 (1.075-2.524), and a similar increase in right IMT with a RR of 3.327 (1.429-7.746). For cardiovascular mortality, the corresponding RR values were 2.351 (1.029-5.372) and 2.890 (1.059-7.891), respectively. Carotid IMT assessed by ultrasonography is positively associated with an increased risk of all-cause and cardiovascular death in elderly community-dwelling people.

[Standardization of statistical procedures and evaluation scheme in external quality-control survey].

Numerous external quality assessment surveys are being conducted by variety of organizations throughout Japan, but the statistical processing and evaluation scheme are not compatible. Standardization of the procedures is essential to make comparison of results among surveys possible. A coding system is available only for names of analytes and analytical equipments. Systematic coding for analytical principles, manufacturers and standard materials is necessary. Regarding computation of peer-group statistics, the mean and SD are often biased when there are many, or wildly, outlying values. Therefore it is recommended to use an iterative method. The methodology removes a relatively large proportion of the population in the tails of the distribution and re-inflates the SD to compensate for the trimming, thus reaching an unbiased mean and SD by iteration. It is also useful to compute between-method CV and within method-CV by one-way analysis of variance. They represent overall levels of standardization and reproducibility of the analyte, respectively. The evaluation of results is usually based on the peer-group mean and SD. The scheme is unfair for those belonging to a peer-group with a narrow SD. It is recommended to use so-called "common CV evaluation scheme", which is based on a within-method CV computed from overall test results after excluding those peer-groups with large CVs. The common CV is applied to the unbiased peer-group mean to get the evaluation SD. For standardized data processing and statistical analysis, it is crucial to develop a unified, generalized soft ware. We developed its prototype named SurveyMaster I & II and herein introduce their potentials.

Simvastatin and atorvastatin enhance hypotensive effect of diltiazem in rats.

Effects of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, simvastatin and atorvastatin, on diltiazem-induced hypotension were examined in anaesthetized rats and compared to that of pravastatin. Vehicle, 2 mg/kg/day simvastatin, 2 mg/kg/day atorvastatin, or 4 mg/kg/day pravastatin was administered orally for 4 days. Diltiazem at 3 mg/kg was given orally 2 hours after the final administration of the inhibitors. Arterial blood pressure was measured via a cannula introduced into the left carotid artery, and heart rate was counted from the pulse pressure. In all groups, diltiazem significantly decreased the mean arterial blood pressure without any changes in heart rate. Pretreatment with simvastatin and atorvastatin significantly enhanced the hypotensive effect of diltiazem, while that with pravastatin did not. Heart rate was not modified by pretreatment with the inhibitors. The results indicate that concomitant use of diltiazem with simvastatin or atorvastatin enhances diltiazem-induced hypotension, probably by competitive inhibition of diltiazem metabolism with simvastatin and atorvastatin metabolisms.

Visualization of the stop of microtubule depolymerization that occurs at the high-density region of microtubule-associated protein 2 (MAP2).

Individual microtubules (MTs) repeat alternating phases of polymerization and depolymerization, a process known as dynamic instability. Microtubule-associated proteins (MAPs) regulate the dynamic instability by increasing the rescue frequency. To explore the influence of MAP2 on in vitro MT dynamics, we correlated the distribution of MAP2 on individual MTs with the dynamic phase changes of the same MTs. MAP2 was modified selectively on its projection region by X-rhodamine iodoacetamide without altering the MT-binding activity. When the labeled MAP2 was added to MTs, the fluorescence was distributed along almost the entire length of individual MTs. However, the inhomogeneity of the distribution gradually became obvious due to the fluorescence bleaching, and the MTs appeared to consist of rapidly bleached portions (RBPs) and slowly bleached portions (SBPs), which were distributed randomly along the MT. By measuring the duration of fluorescence bleaching, the density of MAP2 in SBP was estimated to be approximately 2.5 times higher than the RBP. The average tubulin:MAP2 ratio in SBP was calculated to be 16. When the MT dynamics were observed by dark-field microscopy after determining the MAP2 distribution, rescues were always found to occur only at the SBPs. MTs also displayed intermittent shortening by repeated depolymerization phases separated by pause phases. In these cases, depolymerization phases stopped only at the SBPs. Not every SBP stopped depolymerization, but depolymerization always stopped at an SBP. Taken together, we suggest that there is a minimum density of MAP2 that is necessary to stop depolymerization.

Site-directed mutagenesis of the putative distal helix of peroxygenase cytochrome P450.

CYP152A1 is an unusual, peroxygenase enzyme that catalyzes the beta- or alpha-hydroxylation of fatty acids by efficiently introducing an oxygen atom from H2O2 to the fatty acid. To clarify the mechanistic roles of amino acid residues in this enzyme, we have used site-directed mutagenesis of residues in the putative distal helix and measured the spectroscopic and enzymatic properties of the mutant proteins. Initially, we carried out Lys-scanning mutagenesis of amino acids in this region to determine residues of CYP152A1 that might have a mechanistic role. Among the Lys mutants, only P243K gave an absorption spectrum characteristic of a nitrogenous ligand-bound form of a ferric P450. Further investigation of the Pro243 site revealed that a P243H mutant also exhibited a nitrogen-bound form, but that the mutants P243A or P243S did not. On the hydroxylation of myristic acid by the Lys mutants, we observed a large decrease in activity for R242K and A246K. We therefore examined other mutants at amino acid positions 242 and 246. At position 246, an A246K mutant showed a roughly 19-fold lower affinity for myristic acid than the wild type. Replacing Ala246 with Ser decreased the catalytic activity, but did not affect affinity for the substrate. An A246V mutant showed slightly reduced activity and moderately reduced affinity. At position 242, an R242A showed about a fivefold lower affinity than the wild type for myristic acid. The Km values for H2O2 increased and Vmax values decreased in the order of wild type, R242K, and R242A when H2O2 was used; furthermore, Vmax/Km was greatly reduced in R242A compared with the wild type. If cumene hydroperoxide was used instead of H2O2, however, the Km values were not affected much by these substitutions. Together, our results suggest that in CYP152A1 the side chain of Pro243 is located close to the iron at the distal side of a heme molecule; the fatty acid substrate may be positioned near to Ala246 in the catalytic pocket, although Ala246 does not participate in hydrophobic interactions with the substrate; and that Arg242 is a critical residue for substrate binding and H2O2-specific catalysis.

Limitation of heart growth in neonatal piglets by simvastatin and atorvastatin: comparison with pravastatin.

The pig heart grows rapidly in the first few days after birth. We examined the effects of simvastatin, atorvastatin, and pravastatin on heart growth in piglets. After vehicle, 2 mg x kg(-1) x day(-1) simvastatin, 2 mg x kg(-1) x day(-1) atorvastatin, or 4 mg x kg(-1) x day(-1) pravastatin were administered orally for 6 days, the thoracic cavity was opened, and the heart was removed under pentobarbital sodium (30 mg/kg ip) anesthesia. The heart was perfused to remove residual blood. After the heart was blotted dry, the right and left ventricular free walls were dissected. Each free wall was weighed and used for determination of DNA, RNA, and protein concentrations and mitogen-activated protein (MAP) kinase activity. Simvastatin and atorvastatin resulted in smaller increases with age in the weight, concentrations of RNA and protein, and activity of MAP kinase in the left ventricular free wall, whereas pravastatin did not. The parameters of heart growth in the right ventricular free wall were not appreciably affected by either drug. The blood pressure and heart rate were not changed by the treatments. These results suggest that simvastatin and atorvastatin interfere with heart growth in neonatal piglets after birth, especially in the left ventricular free wall.

Gray matter of the bovine cervical spinal cord is mechanically more rigid and fragile than the white matter.

The gray matter of the cervical spinal cord has been thought to be equally or less rigid than the white matter. Based on this assumption, various studies have been conducted on the changes of stress distributions within the spinal cord under mechanical compression, although the mechanical properties of the white and gray matters had not been fully elucidated. The present study measured the mechanical properties of the white and gray matter of bovine spinal cords. For both the white and gray matter, the stress-strain curves had a nonlinear region, followed by a linear region, and then a region where the stresses plateaued before failure. In the nonlinear region, stress was not significantly different between the gray and white matter samples (strain approximately 0-10%), while stress and Young's modulus in the gray matter was significantly higher than the white matter in the linear part of the curve. The gray matter ruptured at lower strains than the white matter. These findings demonstrated the gray matter is more rigid and fragile than the white matter, and the conventional assumption (i.e., the white matter is more rigid than the gray matter) is not correct. We then applied our data to computer simulations using the finite element method, and confirmed that simulations agreed with actual magnetic resonance imaging findings of the spinal cord under compression. In future computer simulations, including finite element method using our data, changes in stress and strain within the cervical spinal cord under compression would be clarified in more detail, and our findings would also help to elucidate the area which can easily receive histologic damage or which could have hemodynamic disorders under mechanical compression, as well as severity and location of biochemical and molecular biological changes.

[Establishment of a reference interval for highly sensitive CRP by exclusion of individuals with abnormal values in related laboratory tests].

In November, 1999, U.S. Food and Drug Administration approved a highly sensitive CRP(hs-CRP) assay that could assist medical doctors to predict the risk of cardiovascular accidents. Many doctors are now interested in the assay and trying to elucidate the relationship between serum CRP levels and cardiovascular diseases. In the past, it was difficult to establish a valid reference interval of serum CRP because of the poor analytical sensitivity and difficulty in sampling reference individuals. We have established a reference interval of serum CRP for the hs-CRP assay(Dade Behring). The study population consisted of 7,224 individuals(21-81 years old) who received a regular medical check-up. Potentially abnormal samples were excluded, depending on the results of other laboratory tests related to serum CRP variation. The upper limit of the reference interval was 0.15 mg/dl. The serum CRP was higher in smokers than in non-smokers, especially in men.

[Identification of microbial subtypes from antibiotic susceptibility data in clinical laboratory for nosocomial infection surveillance].

We developed an algorism to identify microbial subtypes automatically from daily antibiotic susceptibility data in clinical microbiology laboratory. The susceptibility pattern was expressed as a string of digits, each consisting of 0(resistant), 1(intermediate) or 2(susceptible) to respective antibiotics. Any two patterns were regarded identical and combined if the difference at each digit never exceeds 1. The combined pattern was expressed as an array of digit-by-digit weighted averages of the two. The second combination was based on a degree of similarity among the numerical patterns using a formula, which was designed to emphasize differences in highly variable elements. This subgrouping procedure was done every three months. Identity of the detected subtypes between the intervals was determined using the same algorism as for the second combination. The algorism was applied to data of clinical isolates of MRSA, Pseudomonas aeruginosa (PA), Klebsiella pneumoniae (KP), Enterococcus faecalis (EF), Streptococcus pneumoniae (SP), Escherichia coli (EC) that were obtained over a period of 4 years. Three major subtypes of MRSA, KP and EF were consistently detected with shifting mutual frequencies. Most of EC isolates belonged to two consistent subtypes. Although PA and SP had one or two consistent subtypes, there were multiple minor subtypes of varying frequencies. This analysis is regarded as an "infotyping", in contrast to serotype or genotype, of clinical microbial isolates, which is useful for nosocomial infection surveillance.

[A case of central nervous system anomalies (agenesis of corpus callosum, colpocephaly, hydrocephalus, congenital dermal sinus) associated with congenital heart disease(double-outlet right ventricle, complete endocardial cushion defect, atrial septal defect, pulmonary arterial stenosis, patent ductus arteriosus)].

A case of central nervous system anomalies(agenesis of corpus callosum, colpocephaly, hydrocephalus, congenital dermal sinus) associated with congenital heart disease(double-outlet right ventricle, complete endocardial cushion defect, atrial septal defect, pulmonary arterial stenosis, patent ductus arteriosus) is reported. Female patient had been already diagnosed as hydrocephalus during pregnancy and ventricular drainage was performed soon after the delivery. Prostaglandin E 1 was also applied for heart disease, but saturation of O2 decreased to 80% on arterial blood gas analysis. Blalock-Taussig operation and ligation of ductus arteriosus was done 41 days after the delivery and ventricle-peritoneal shunt was also made for the progressive hydrocephalus on the same day. Chromosome analysis showed no abnormality. The genesis of this complicated brain and heart anomaly is discussed from the viewpoint of neural crest cell abnormality.

Antioxidative effect of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on peroxidation of phospholipid liposomes.

The antioxidative effect of fluvastatin sodium (fluvastatin), a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on lipid peroxidation of phosphatidylcholine (PC) liposomes was investigated in various peroxidizing systems. Fluvastatin markedly inhibited the formation of thiobarbituric acid reactive substances in iron (II)-supported peroxidation of liposomes (IC50 = 1.2 x 10(-5) M). The order of magnitude of inhibition of each drug on the peroxidation was: butylated hydroxytoluene > fluvastatin > or = probucol >> pravastatin. Moreover, concentrations of fluvastatin ranging from 1 x 10(-6) to 1 x 10(-4) M inhibited peroxyl radical-mediated peroxidation of liposomes induced by water-soluble and lipid-soluble radical generators, 2,2'-azobis (2-amidinopropane) dihydro-chloride and 2,2'-azobis (2,4-dimethylvaleronitrile), respectively. However, pravastatin showed no effect against peroxyl radical-mediated peroxidation. These results indicate that fluvastatin acted non-enzymatically as an effective inhibitor against lipid peroxidation of PC liposomes and that the antioxidative effects of fluvastatin may be due to the scavenging action of fluvastatin on liposomal lipid peroxidation induced by peroxyl radicals generated in the aqueous and lipid phases.