Mechanical regulation of bone homeostasis through p130Cas-mediated alleviation of NF-κB activity.

Mechanical loading plays an important role in bone homeostasis. However, molecular mechanisms behind the mechanical regulation of bone homeostasis are poorly understood. We previously reported p130Cas (Cas) as a key molecule in cellular mechanosensing at focal adhesions. Here, we demonstrate that Cas is distributed in the nucleus and supports mechanical loading-mediated bone homeostasis by alleviating NF-κB activity, which would otherwise prompt inflammatory processes. Mechanical unloading modulates Cas distribution and NF-κB activity in osteocytes, the mechanosensory cells in bones. Cas deficiency in osteocytes increases osteoclastic bone resorption associated with NF-κB-mediated RANKL expression, leading to osteopenia. Upon shear stress application on cultured osteocytes, Cas translocates into the nucleus and down-regulates NF-κB activity. Collectively, fluid shear stress-dependent Cas-mediated alleviation of NF-κB activity supports bone homeostasis. Given the ubiquitous expression of Cas and NF-κB together with systemic distribution of interstitial fluid, the Cas-NF-κB interplay may also underpin regulatory mechanisms in other tissues and organs.

A novel and rapid diagnostic method for discriminating between feces of sika deer and Japanese serow by loop-mediated isothermal amplification.

Severe damages to natural vegetation, agriculture, and forestry caused by overpopulation of sika deer (Cervus nippon) have markedly increased in Japan in recent years. To devise a population management plan of sika deer, information on the distribution and population size of the animal in each region is indispensable. An easy and effective method to obtain this information is to count the fecal pellets in the field. However, the habitat of sika deer in Japan overlaps that of Japanese serow (Capricornis crispus). Additionally, it is difficult to discriminate between the feces of both animals. Here, we present a rapid and precise diagnostic method for discriminating between the feces of sika deer and Japanese serow using loop-mediated isothermal amplification (LAMP) targeting cytochrome b gene in the mitochondrial DNA. Our results showed that the LAMP can discriminate between the feces of sika deer and Japanese serow, and the method is simpler and more sensitive than the conventional molecular diagnostic method. Since LAMP method does not require special skills for molecular biology techniques, even the field researchers who have never done a molecular experiment can easily carry out the protocol. In addition, the entire protocol, from DNA extraction from fecal pellet to identification of species, takes only about 75 min and does not require expensive equipment. Hence, this diagnostic method is simple, fast, and accessible to anyone. As such, the method can be a useful tool to estimate distribution and population size of sika deer.

SH2-containing inositol 5'-phosphatase 2 selectively impairs hypothalamic insulin signalling and regulation of food intake in mice.

SH2-containing inositol 5'-phosphatase 2 (SHIP2) is a lipid phosphatase that negatively regulates the metabolic signalling of insulin in peripheral tissues; however, the expression of SHIP2 in the hypothalamus and its functional roles are largely unknown. In the present study, immunohistochemical analysis demonstrated that SHIP2 protein exists in neuronal cells expressing neuropeptide Y and pro-opiomelanocortin in the arcuate nucleus of the hypothalamus in C57BL/6J mice. Interestingly, the expression levels of SHIP2 in the hypothalamus were elevated in aged C57BL/6J mice and diabetic db/db mice. To clarify the significance of the increased expression of SHIP2 in the hypothalamus, we examined the central effects of insulin and leptin in transgenic mice overexpressing SHIP2 (SHIP2-Tg). Accumulation of phosphatidylinositol (3,4,5)-trisphosphate and phosphorylation of Akt in the hypothalamus, induced by i.c.v. injection of insulin, were attenuated in SHIP2-Tg compared to wild-type mice, whereas leptin-induced phosphorylation of signal transducer and activator of transcription 3 in the hypothalamus was comparable between them. The suppression of food intake after i.c.v. administration of insulin (but not leptin) was attenuated consistently in SHIP2-Tg. In addition, SHIP2-Tg showed increased food consumption after starvation and become heavier with visceral fat accumulation than wild-type mice, despite normal levels of oxygen consumption and spontaneous movement. These results suggest that SHIP2 contributes to the regulation of food intake mainly via the attenuation of insulin signalling in the hypothalamus of mice.

Leptographium pruni, sp. nov. from bark beetle-infested Prunus jamasakura in Japan.

Leptographium species are anamorphs of Ophiostoma, commonly isolated from conifer. There are, however, a small number of these fungi that have been collected from angiosperm hosts. In this study, we describe Leptographium pruni, sp. nov. isolated from the bark of Prunus jamasakura infested by the bark beetle Polygraphus ssiori. This new species is unusual in having a distinct Sporothrix synanamorph with ramoconidia. No evidence of a teleomorph was found, but a high level of tolerance to the antibiotic cycloheximide and the presence of a Sporothrix synanamorph suggest that L. pruni is an Ophiostoma anamorph. Analysis of sequence data for the domain 1 region of the LSUrDNA operon also supports the phylogenetic relationship of L. pruni with Ophiostoma. In addition, sequence data suggest that L. pruni is related to other species of Leptographium rather than Pesotum species with distinct Sporothrix synanamorphs.

[Preliminary report: first identification of known mutation in the ryanodine receptor gene in a Japanese malignant hyperthermia pedigree].

In Japan, the rate of Ca-induced Ca release (CICR) using skinned fibers of skeletal muscle has been employed as a diagnostic test for malignant hyperthermia (MH) susceptibility, since most of the typical fulminant MH patients showed an enhancement of CICR rate. Recently the Ca releasing channel responsible for the CICR was reported to be mainly the ryanodine binding Ca protein which was identified as the causative site of MH muscle according to recent genetic studies. Among patients with enhanced CICR rate, one point mutation for Arg 2434 His was recognized in a family. Although this site is related to the central core disease (CCD), this pathological change could not be observed in this patient. This is the first report describing a gene mutation in a MH family identified in Japan.

Early Symptom Development and Histological Changes Associated with Migration of Bursaphelenchus xylophilus in Seedling Tissues of Pinus thunbergii.

In order to clarify the mechanism of pine wilt caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus, nematode migration in tissues and disease symptoms in Pinus thunbergii seedlings were investigated. One-year-old seedlings were inoculated with different pathogenic isolates of PWN under two different temperatures. At an early stage of symptom development, a virulent isolate of PWN multiplied in both bark and xylem and was distributed in cortical resin canals, cortical tissue, and xylem resin canals at 30°C. Cell death and disease symptoms developed in both bark and xylem. The virulent isolate of PWN at 25°C and the avirulent isolate of PWN at 30°C were distributed mainly in cortical resin canals, but rarely in xylem resin canals and cortical tissue. Disease symptoms and cell death occurred in cortical resin canals and rarely occurred in other tissues. These results demonstrated that the virulent isolate of PWN at low temperature and avirulent nematodes could not easily migrate to xylem resin canals and cortical tissue. It was shown that cell death and early symptom development coincided with PWN migration and, therefore, PWN invasion induces cell death and early symptom development.

Associations of medical status and physical fitness with periodontal disease.

To determine the possible associations of medical status and physical fitness with periodontal disease, a cross-sectional study was conducted. The subjects were 517 males and 113 females aged 23 to 83 years who participated in a multiphasic health test at the Aichi Prefectural Center of Health Care, Japan, from 1992 to 1997. Their periodontal status was assessed by means of the CPITN scoring system. To assess the strength of associations between the examined factors and the score, odds ratios were computed using ordinal logistic models. Conventional risk factors such as old age, smoking habits, and higher fasting plasma glucose and simplified debris index increased the risk of periodontal disease. Hypertension, hematuria, leucocytosis or thrombocytosis, positive C-reactive protein and higher serum alkaline phosphatase were positively associated with the score, whereas higher serum high-density lipoprotein cholesterol was related to a lower risk. Poor physical fitness affecting aerobic capacity, foot balance and reaction was associated with a higher CPITN score. These associations were independent of the conventional risk factors. Although these new potential risk factors should be further investigated for their causal relationship, our findings suggested a close relationship of oral health to medical status and physical fitness.

Favorable life-style modification and attenuation of cardiovascular risk factors.

In order to develop an effective counseling system for prevention of cardiovascular diseases, the association of a favorably changed life-style with improved risk factors was examined. Participants were 7,321 office workers aged 30-69 years from in and around Nagoya city. The age-adjusted odds ratio (OR) and its 95% confidence interval (CI) were calculated to assess the likelihood of risk factor improvement by favorable life-style modifications during a 3-year period. Those who began to eat breakfast and increased their vegetable intake normalized their previously abnormal diastolic blood pressure with more than twice the likelihood (adjusted OR [95% CI] 2.89 [1.29-6.46] and 2.60 [1.18-5.75], respectively). 'Began to eat breakfast' was also significantly associated with normalized total cholesterol (TC) (1.84, [1.05-3.21]). 'Stopped eating till full' significantly normalized the body mass index (2.03; [1.25-3.28]), uric acid (1.65; [1.07-2.52]) and TC (1.43; [1.04-1.97]). Those who started regular exercise significantly normalized their high-density lipoprotein-cholesterol (HDL-C) abnormality with 1.69-times the likelihood (1.69; [1.24-2.29]) and those who began to walk briskly also improved their TC abnormality (1.85; [1.19-2.89]). HDL-C was normalized with 2.55-times the likelihood in those who quit smoking (2.55; [1.68-3.86]). Because favorable life-style modifications can attenuate abnormal cardiovascular risk factors, then proper advice on specific risk factors should be routinely given at each health check-up in order to prevent the onset of cardiovascular diseases in subsequent years.

A study on how a 6-month aerobic exercise program can modify coronary risk factors depending on their severity in middle-aged sedentary women.

It is well known that physical exercise can reduce coronary risk factors. But how an aerobic exercise modifies coronary risk factors in relation to severity and physical fitness is still controversial.Fifty-four middle-aged women (mean age, 55 years) completed a 6-month on-site and home-based anaerobic threshold-level exercise program. The changes in coronary risk factor profiles were observed during the pre-intervention and intervention periods. Before the intervention (during control period), most coronary risk factors showed a rather unfavorable trend. After the program, their mean body weight decreased from 56.7 to 55.7 kg (p>0.05) and the proportion of body fat from 30.9 to 27.9% (p>0.05) without any reduction in lean body mass. Systolic blood pressure (SBP) decreased from 129.0 to 125.0 mm Hg (p>0.05) and diastolic blood pressure from 79.5 to 76.6 mm Hg (p>0.05). Fasting plasma glucose (FPG) declined from 109.6 to 103.4 mg/dl (p>0.05). Changes in SBP and FPG were most remarkable in their respective worst tertile. Serum lipids improved only modestly. Maximum oxygen uptake increased from 23.6 to 26.1 ml/kg/min (p>0.01). However, no significant correlations were found between changes in coronary risk factors and those in physical fitness. We conclude that the 6-month aerobic exercise program would modify women's coronary risk factors depending on their initial values, probably independently of the changes in physical fitness.

Staged reperfusion preserves the coronary flow reserve, especially in the regions not severely damaged by ischemic injury in the canine heart.

The objective of this study was to determine the effects of staged reperfusion on the progressive reduction in coronary blood flow (CBF) and coronary flow reserve during reperfusion and on the infarct size in the canine heart. Fifteen dogs underwent 90 min of left circumflex coronary artery occlusion and 3 hr of reperfusion. In the abrupt reperfusion group, the occluder was released completely at the initiation of reperfusion. In the staged reperfusion group, CBF was maintained at 20% of preocclusion values for 10 min after initiation of reperfusion, then gradually released, and completely released 20 min after initiation of reperfusion. There was no significant difference in CBF between the staged (n = seven) and abrupt (n = eight) groups after 3 hr of reperfusion. The repayment of flow debt in the staged reperfusion group was significantly greater than in the abrupt reperfusion group after 3 hr of reperfusion (260+/-120% vs 100+/-60%, staged vs abrupt at 3 hr, p < 0.03). The ratio of peak reactive hyperemic flow to resting flow in the staged reperfusion group was significantly greater than in the abrupt reperfusion group throughout the reperfusion phase (4.4+/-1.0 vs 2.6+/-0.6 at 3 hr, p < 0.001), and had returned to the preocclusion values after 3 hr of reperfusion. This preservation of the coronary flow reserve in the staged reperfusion group was observed in the epicardium (4.1+/-0.6 vs 2.8+/-0.7, staged vs abrupt at 3 hr, p < 0.01), but not in the endocardium or midmyocardium. Infarct size did not differ significantly between the two groups. Staged reperfusion in this study did not appear to attenuate the reduction of CBF, or to reduce infarct size, however preserved the coronary flow reserve, especially in the regions not severely damaged by ischemic injury.

Antineoplastic agents. 398. Isolation and structure elucidation of cephalostatins 18 and 19.

Continued investigation of murine leukemia (P-388) active fractions from the African marine worm Cephalodiscus gilchristi has resulted in the discovery of cephalostatins 18 (1b) and 19 (1c). The structures were determined by interpretation of their highfield (500 MHz) 1H, 13C, and 2D NMR and HRMS. Both of these new methoxy steroidal alkaloids exhibited strong activity against the murine P-388 lymphocytic leukemia cell line (ED50 ca. 10(-3) microg/mL), a mini panel of human cancer cell lines (GI50 <10(-3) microg/mL), and the U.S. National Cancer Institute's 60 human cancer cell line panel (mean panel GI50 ca. 10(-9) M).

[Effect of temperature on Ca induced Ca release (CICR) rate].

The aim of this study is to assess the effect of temperature on CICR rate. The extensor digitorum longus muscles of male Hartley guinea pigs of about 450 g, were prepared for this study. According to Endo's method, CICR rates were measured using chemically skinned fibers. Two plates were prepared: one at 20 degrees C and the other at 30, 35, 37, 40 degrees C controlled by circulating water at an appropriate temperature placed underneath each plate. The whole procedure was carried out at 20 degrees C except the step of CICR using a plate at a given temperature. An increase in temperature (20 to 40 degrees C) accelerated CICR rate in a logarithmic manner. These experimental findings suggest that cooling of the patient's body is important not only to control metabolism but also to reduce an accelerated CICR rate.

Relation of electrocardiographic left ventricular hypertrophy with and without T-wave changes to systemic blood pressure, body mass, and serum lipids and blood glucose levels in Japanese men.

Left ventricular (LV) hypertrophy, especially combined with an abnormal ST-T, is considered 1 of many coronary risk factors. Seven hundred forty-nine Japanese men were selected according to their electrocardiographic findings, i.e., normal electrocardiogram, LV hypertrophy without an abnormal ST-T segment, LV hypertrophy with a flat T wave, and LV hypertrophy with a negative T wave. Coronary risk factors were compared among these 4 age-matched groups. Groups with LV hypertrophy with negative or flat T waves had larger body mass index (24.9 vs 22.9 kg/m2), higher mean systemic blood pressure (111 vs 95 mm Hg), larger LV mass (265 vs 157 g), higher blood glucose (110 vs 100 mg/dl), higher serum triglyceride (148 vs 122 mg/dl), higher total cholesterol (206 vs 198 mg/dl), and lower high-density lipoprotein cholesterol (47 vs 54 mg/dl) than the normal group or the group with LV hypertrophy without T-wave change. Among these risk factors, blood pressure and glucose remained higher even after the adjustment by body mass index or by body mass index and blood pressure. Electrocardiographic LV hypertrophy with a changed T wave signified higher risk of coronary artery disease in Japanese men.

Synthesis of a new potent anti-angiogenic agent, 17 alpha-acetoxy-9 alpha-fluoro-6 alpha-methylprogesterone (9 alpha-fluoromedroxyprogesterone acetate [FMPA]).

A new anti-angiogenic agent, 17 alpha-acetoxy-9 alpha-fluoro-6 alpha-methylprogesterone (9 alpha-fluoromedoroxyprogesterone acetate [FMPA, 9] was synthetized in a 10-step sequence. FMPA (9) had about two orders of magnitude stronger anti-angiogenic activity than medroxyprogesterone acetate (MPA), as estimated in a bioassay involving chorioallantoic membranes of growing chick embryos.

Oxygen uptake and its relation to physical activity and other coronary risk factors in asymptomatic middle-aged Japanese.

PURPOSE: Low physical activity is considered to be an important risk factor for atherosclerotic coronary artery disease. However, few data are reported on the Japanese general population. The authors have studied whether oxygen uptake in physical fitness evaluation is a quantitative index for physical activity and whether or not it has a relation to coronary risk factors. METHODS: Five hundred thirteen asymptomatic Japanese (40-64 years of age, 282 males and 231 females) were tested on a cycle ergometer for measurement of peak oxygen uptake (peak VO2) and oxygen uptake at anaerobic threshold (VO2AT). Physical activity was estimated by pedometer score. Data for oxygen uptake were adjusted by age or by age and body mass index (BMI), then its relationship to the following risk factors was investigated: physical activity, BMI, blood pressure, total cholesterol, HDL and LDL cholesterol, fasting blood glucose, and triglycerides. RESULTS: Subjects in the highest peak VO2 quartile walked significantly more than those in the lowest quartile in both males and females. Those in the highest quartile showed lower BMI, lower blood pressure, lower triglyceride, and higher HDL cholesterol. The same relationship was observed for VO2AT. CONCLUSIONS: Higher fitness level determined by peak VO2 or VO2AT is related to higher physical activity and lower coronary risk factors in the asymptomatic middle-aged Japanese. These data provide support for exercise prescription in the primary prevention of coronary heart disease in Japan.

Newly developed ST-T abnormalities on the electrocardiogram and chronologic changes in cardiovascular risk factors.

An ST-T abnormality on an electrocardiogram (ECG) is known to independently predict subsequent morbidity and mortality from cardiovascular diseases. But how ST-T abnormality develops in relation to chronologic changes in cardiovascular risk factors has not been fully discussed. Sixty-eight men whose ECG had been initially normal but who exhibited ST-T abnormality later (ST-T subjects) were identified among 21,579 apparently healthy adults who had undergone comprehensive health examinations for > 10 years. Echocardiography proved that 26 of 29 examinees among ST-T subjects had left ventricular hypertrophy. Antihypertensive drugs were given to 26 of the ST-T subjects. Their cardiovascular risk factors were chronologically reviewed from 10 years before the onset of definite ST-T abnormality, and were compared with those of 68 men whose ECG had remained consistently normal for 10 years (controls). Mean values of systolic and diastolic blood pressure gradually increased over 10 years (from 127/78 to 144/84 mm Hg) among ST-T subjects, but showed little change (from 122/76 to 124/77 mm Hg) during the same period in controls. The time course of blood pressure over 10 years was similar in ST-T subjects, irrespective of final blood pressure level. Mean serum cholesterol and glucose increased over 10 years in both ST-T and control subjects. Uric acid decreased over 10 years (from 6.1 to 5.6 mg/dl) only in ST-T subjects. Multivariate analysis revealed that blood pressure and uric acid before onset of ST-T abnormality were chronologically changed independent of other risk factors. The time course of risk factors may be of great importance in the development of cardiovascular disorders.

Body surface distribution of significant changes in QRST time-integral values after radiofrequency catheter ablation in patients with Wolff-Parkinson-White syndrome.

We analyzed 87-lead body surface QRST time-integral values (QRST values) in 29 patients with Wolff-Parkinson-White syndrome (group A, 17 patients with manifest left-sided accessory pathway; group B, 6 patients with manifest right-sided accessory pathway; and group C, 6 patients with concealed left-sided accessory pathway), before, 1 day after, and 1 week after radiofrequency catheter ablation (RCA). The number of leads with abnormal QRST values was significantly lower 1 week after RCA compared with those before RCA and 1 day after RCA in groups A and B (p < 0.05); there was no significant difference in QRST values before and 1 day after RCA in groups A and B. The QRST values over areas with preexisting repolarization abnormalities were significantly altered 1 week after RCA compared with before and 1 day after RCA in groups A and B (p < 0.01). However, there was no significant difference in the QRST values over areas without preexisting abnormalities before RCA. In group C, there were no significant differences in the QRST values or the number of leads with abnormal QRST values before, 1 day and 1 week after RCA. In conclusion, RCA did not significantly influence repolarization properties over areas without preexisting abnormalities, but gradually reduced preexisting repolarization abnormalities, which were closely related to the location of the accessory pathway in patients with manifest Wolff-Parkinson-White syndrome. Our results suggest that body surface QRST values are useful for assessment of repolarization abnormalities during the periablation period.

Spatial ventricular gradient in patients with Wolff-Parkinson-White syndrome in comparison with normal subjects: vectorcardiographic evidence for significant repolarization changes due to preexcitation.

We investigated the use of the spatial ventricular gradient (VG) from vectorcardiogram (VCG) to determine whether significant repolarization differences were present in patients with WPW syndrome compared with normal subjects and also examined which VG parameter (i.e., elevation, azimuth, and magnitude) reflected the differences in repolarization properties during preexcitation. VG was calculated in 49 patients of Wolff-Parkinson-White (WPW) syndrome (group A: left-sided accessory pathway, n = 29; group B: right-sided, n = 20). Group N consisted of 607 normal subjects. In group A, the azimuth of VG was significantly (p < 0.01) greater than in groups B and N. In group B, the elevation of VG was significantly (p < 0.01) greater than in groups A and N. There were no significant differences in the magnitude of VG among groups. QRS duration was significantly (p < 0.01) related with the elevation of VG in group B. These findings suggested that VG is useful for spatial evaluation of repolarization abnormalities during preexcitation, which are related to the site of the accessory pathway.