Calreticulin mutant mice develop essential thrombocythemia that is ameliorated by the JAK inhibitor ruxolitinib.

Mutations of calreticulin (CALR) are detected in 25-30% of patients with essential thrombocythemia (ET) or primary myelofibrosis and cause frameshifts that result in proteins with a novel C-terminal. We demonstrate that CALR mutations activated signal transducer and activator of transcription 5 (STAT5) in 293T cells in the presence of thrombopoietin receptor (MPL). Human megakaryocytic CMK11-5 cells and erythroleukemic F-36P-MPL cells with knocked-in CALR mutations showed increased growth and acquisition of cytokine-independent growth, respectively, accompanied by STAT5 phosphorylation. Transgenic mice expressing a human CALR mutation with a 52 bp deletion (CALRdel52-transgenic mice (TG)) developed ET, with an increase in platelet count, but not hemoglobin level or white blood cell count, in association with an increase in bone marrow (BM) mature megakaryocytes. CALRdel52 BM cells did not drive away wild-type (WT) BM cells in in vivo competitive serial transplantation assays, suggesting that the self-renewal capacity of CALRdel52 hematopoietic stem cells (HSCs) was comparable to that of WT HSCs. Therapy with the Janus kinase (JAK) inhibitor ruxolitinib ameliorated the thrombocytosis in TG mice and attenuated the increase in number of BM megakaryocytes and HSCs. Taken together, our study provides a model showing that the C-terminal of mutant CALR activated JAK-STAT signaling specifically downstream of MPL and may have a central role in CALR-induced myeloproliferative neoplasms.

The Relation between Hamstring Strain Injury and Physical Characteristics of Japanese Collegiate Sepak Takraw Players.

The aim of this study was to investigate the injuries in Japanese collegiate sepak takraw players. We primarily focused on hamstring strain injury (HSI), and investigated the associated physical characteristics. The study included 77 Japanese collegiate sepak takraw players who were interviewed; data were collected regarding injuries sustained by them during the game within the past year. The hip range of motion (ROM) was measured. The total number of injuries was 48 in a year. The rate of HSI was the highest (31.3%) among all the injuries. All HSIs occurred in the dominant leg because of the sunback spike. Using the Mann-Whitney U test, significant differences in age and sport-related experience were observed between the injured group and uninjured group. Upon using logistic regression analysis, the presence of a HSI was found to be associated with the sport-related experience (adjusted odds ratio [OR], 0.30; 95% confidence interval [CI], 0.12-0.77) and the hip extension ROM (adjusted OR, 0.81; 95% CI, 0.66-0.99) after adjusting for sex, sport-related experience, and the hip ROM. HSI is the most common injury in Japanese collegiate sepak takraw players. Short sport-related experience and small hip extension ROM are related with the occurrence of HSI.

Effective CpG DNA delivery using amphiphilic cycloamylose nanogels.

Unmethylated CpG oligodeoxynucleotides induce inflammatory immune responses through cytokine production and have attracted increasing attention as an immunostimulator. However, there remains a challenging issue of the use of 'native CpG DNA'. In the present study, we prepared cationic nanometer-sized gels (nanogels) consisting of cycloamylose modified with cholesterol and diethylaminoethane to form hydrophobic cross-linking points and to add positively charged groups, respectively. The cationic nanogels and native CpG DNA formed nanometer-sized complexes. Complexes of native CpG DNA with cationic nanogels delivered native CpG DNA to macrophage-like cells and induced cytokine production. In addition, complexes of negative control oligonucleotides with cationic nanogels did not induce cytokine production, and the induction of cytokines using complexes of phosphorothioate-modified CpG with cationic nanogels was lower than that of native CpG DNA. These results suggest that the complex of native CpG DNA with cationic nanogels is a promising strategy for nucleic acid adjuvants.

Maternal restraint stress during pregnancy in mice induces 11ß-HSD1-associated metabolic changes in the livers of the offspring.

In rats, maternal exposure to restraint stress during pregnancy can induce abnormalities in the cardiovascular and central nervous systems of the offspring. These effects are mediated by long-lasting hyperactivation of the hypothalamic-pituitary-adrenal axis. However, little is known about the potential effects of stress during pregnancy on metabolic systems. We examined the effect of restraint stress in pregnant mice on the liver function of their offspring. The offspring of stressed mothers showed significantly higher lipid accumulation in the liver after weaning than did the controls; this accumulation was associated with increased expression of lipid metabolism-related proteins such as alanine aminotransferase 2 diglyceride acyltransferase 1, peroxisome proliferator-activated receptor gamma and glucocorticoid receptor. Additionally, we observed increased levels of 11ß-hydroxysteroid dehydrogenase type 1, an intercellular mediator that converts glucocorticoid from the inactive to the active form, in the foetal and postnatal periods. These results indicate that restraint stress in pregnancy in mice induces metabolic abnormalities via 11ß-hydroxysteroid dehydrogenase type 1-related pathways in the foetal liver. It is therefore possible that exposure to stress in pregnant women may be a risk factor for metabolic syndromes (e.g. fatty liver) in children.

Chronobiology and nutrition.

Numerous long-term studies have investigated the circadian clock system in mammals, which organizes physiological functions, including metabolism, digestion, and absorption of food, and energy expenditure. Food or nutrition can be a synchronizer for the circadian clock systems, as potent as the external light-dark signal can be. Recent studies have investigated different kinds of food, frequency of consumption, and time of consumption for optimizing body clock and ensuring healthy habits. In this review, we discuss recent studies investigating chronobiology and nutrition, and then summarize available information as "Chrono-nutrition" for the development of a new standardized research strategy.

Biological clock dysfunction exacerbates contact hypersensitivity in mice.

BACKGROUND: Immediate-type skin allergic reactions, such as passive cutaneous anaphylactic reaction, are associated with circadian rhythm, but the role of circadian mechanisms on delayed-type skin allergic reactions, such as contact hypersensitivity (CHS), remains uncertain. In mice, CHS, a T-cell-mediated immune response, is a classic model of human allergic contact dermatitis. OBJECTIVES: We investigated whether biological clock dysfunction affects CHS pathogenesis in CLOCK mutant mice compared with wild-type (WT) mice. METHODS: Mice were treated with 2,4,6-trinitro-1-chlorobenzene (TNCB) on the abdominal skin on day 0 (sensitization) and then treated with TNCB on the ears on day 5 (challenge). RESULTS: We found that biological clock dysfunction resulted in severe inflammation. Ear swelling, serum immunoglobulin E level and mast cell number were significantly increased in CLOCK mutant mice compared with WT mice. These results provide evidence that CLOCK mutation promotes the T-helper type 2 immune response and exacerbates CHS. Corticosterone has a protective effect on CHS. The serum corticosterone level lost rhythmicity and showed a decreased daily level in CLOCK mutant mice compared with WT mice, supporting the exacerbating effect of CLOCK mutation on CHS. Adrenalectomy markedly worsened TNCB-induced CHS in WT mice but not in CLOCK mutant mice. In addition, dramatic dexamethasone-induced protection of CHS was observed in CLOCK mutant mice compared with WT mice. CONCLUSIONS: The present results suggest that circadian rhythm might be an important factor in the regulation of CHS via corticosterone rhythmicity and/or level.

Relationship between buccal mucosa ridging and viscoelastic behaviour of oral mucosa.

The aim of this study was to clarify the relationship between the buccal mucosa ridging (BMR), which has been mentioned to be a clinical sign of clenching, and the viscoelastic behaviour of oral mucosa. Twenty-three people with BMR and 21 people without BMR participated as volunteers in this study. Measurements of viscoelastic behaviour were performed using a suction viscoelastic meter on central part of lower labial mucosa. A suction pressure of 300 hPa was applied for 2 s and then released for 2 s, and the time-dependent changes in the deformation of the mucosa over this 4 s were recorded as a deformation curve. Distensibility, remaining deformation and elastic recovery, which describe viscoelastic behaviour, were calculated by the deformation curve. These parameters were compared between groups with and without BMR. No significant difference was found in distensibility between the two groups (P=0·349). There were significant differences for the remaining deformation (P=0·012) and the elastic recovery (P=0·032), and the group with BMR showed higher remaining deformation and lower elastic recovery than the group without BMR. Based on these results, it clarified that the BMR is related to the mucosal viscoelastic behaviour, in particular remaining deformation and elastic recovery.

Influence of concentration of fragrances on salivary alpha-amylase.

The objective is to reveal the influence of the concentration of fragrances on salivary biomarkers, which reflect the human stress system, in 15 female young healthy adults. Lavandula officinalis and Citrus aurantium were used as the test samples. Salivary biomarkers such as alpha-amylase activity (AMY), cortisol (CORT) and dehydroepiandrosterone (DHEA) were measured during baseline, inhalation and post-inhalation periods. Our results indicated that (i) a significant difference was not observed for the control and the 3 wt% test samples, however, the AMY was decreased by inhalation of the 1 wt% test samples (P < 0.05); (ii) AMY levels changed more significantly than did the hormone levels; (iii) a tendency of negative correlation was not observed between DHEA and CORT. It was considered that the time-course change of AMY might be a useful index of the inhalation of fragrances, which reflects the acute psychosomatic reactivity of humans.

Development of ET, primary myelofibrosis and PV in mice expressing JAK2 V617F.

An acquired JAK2 V617F mutation is found in most patients with polycythemia vera (PV), and about half of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF). Mice transplanted with bone marrow cells in which JAK2 V617F was retrovirally expressed developed PV-like features, but not ET or PMF. To address the contribution of this mutation to the pathogenesis of these three MPDs, we generated two lines of JAK2 V617F transgenic mice. One line showed granulocytosis after 4 months of age. Among 43 mice, 8 (19%) showed polycythemia and 15 (35%) showed thrombocythemia. The second line showed extreme leukocytosis and thromobocytosis. They showed anemia that means Hb value from 9 to 10 g per 100 ml when 1 month old. Myeloid cells and megakaryocytes were predominant in the bone marrow of these animals, and splenomegaly was observed. The expression of JAK2 V617F mRNA in bone marrow cells was 0.45 and 1.35 that of endogenous wild-type JAK2 in the two lines, respectively. In vitro analysis of bone marrow cells from both lines showed constitutive activation of ERK1/2, STAT5 and AKT, and augmentation of their phosphorylations by cytokine stimulation. We conclude that in vivo expression of JAK2 V617F results in ET-, PMF- and PV-like disease.

Benchmark experiments for cyclotron-based neutron source for BNCT.

In the previous study, we found the feasibility of a cyclotron-based BNCT using the Ta(p,n) neutrons at 90 degrees bombarded by 50 MeV protons, and the iron, AlF(3), Al and (6)LiF moderators by simulations using the MCNPX code. In order to validate the simulations to realize the cyclotron-based BNCT, we measured the epithermal neutron energy spectrum passing through the moderators with our new spectrometer consisting of a (3)He gas counter covered with a silicon rubber loaded with (nat)B and polyethylene moderator and the depth distribution of the reaction rates of (197)Au(n,gamma)(198)Au in an acrylic phantom set behind the rear surface of the moderators. The measured results were compared with the calculations using the MCNPX code. We obtained the good agreement between the calculations and measurements within approximately 10% for the neutron energy spectra and within approximately 20% for the depth distribution of the reaction rates of (197)Au(n,gamma)(198)Au in the phantom. The comparison clarified a good accuracy of the calculation of the neutron energy spectrum passing through the moderator and the thermalization in a phantom. These experimental results will be a good benchmark data to evaluate the accuracy of the calculation code.

Chart analysis of body composition change among pre- and postadolescent Japanese subjects assessed by underwater weighing method.

OBJECTIVE: To examine the effect of age on the relationship between fat-free mass (FFM) and fat mass (FM), and fat-free mass index (FFMI) and fat mass index (FMI) by applying body composition chart analysis on pre- and postadolescent Japanese subjects. SUBJECTS AND METHOD: A sample of 516 children (244 boys and 272 girls) ranging in age from 11 to 17 y and 840 adults (288 male and 552 female subjects) ranging in age from 18 to 59 y were studied to determine a body composition by an underwater weighing method. FMI and FM were put on an x- and y-axis in body composition chart 1, and FFMI (FFM/ height(2)) and FMI (FM/height(2)) were taken on an x- and y-axis in body composition chart 2. RESULTS: In body composition chart 1, the plots for male subjects stayed flat from 11 to 14 y and after that a steady growth of FFM concurring with the FM growth was observed. During the adult stage, steady increments of FM and gradual decreases of FFM were observed. In body composition chart 2, steady increases of FFMI and gradual decreases of FMI were indicated in the male preadolescent period. In the adult stage, FFMI decreased year by year, although the FMI continued to increase. In female subjects, a conspicuous increase of FMI was observed throughout all periods of the present subjects. After middle age, the decline of FFMI was characteristically demonstrated in the chart. CONCLUSION: The relationships between FFM and FM are characteristically delineated on the body composition charts demonstrating clear gender differences. The change of body mass index was not reflecting the change of adiposity level in male subjects, although it was occurring along with the changes of the adiposity level in female subjects.

Over-expression system for secretory phospholipase D by Streptomyces lividans.

The structural gene for phospholipase D (PLD) of an actinomycete, Streptoverticillium cinnamoneum, together with its promoter region was introduced into Streptomyces lividans using a shuttle vector-pUC702-for Escherichia coli and S. lividans. The transformant was found to secrete a large amount of PLD (about 2.0x10(4) U/l, 42 mg/l) when cultured in a jar fermentor. Both an initial glucose concentration of 17.5 g/l and the feeding of carbon and nitrogen sources are effective for efficient secretion of PLD; under these culture conditions, the amount of PLD secreted reached a maximum level (about 5.5x10(4) U/l, 118 mg/l) after about 60 h. In contrast to the original producer, Stv. cinnamoneum, which secretes only a small amount of PLD (about 1.1x10(3) U/l, 2 mg/l) along with other extracellular proteins, this heterologous expression system is markedly more efficient in production of secretory PLD.

Inactivation of atmospheric bacteria using lytic enzyme mixture.

The mass of air breathed by a human per day is equivalent to 10-times the mass of food consumed in that time. However, fundamental safety measures for atmospheric bacterial control have not yet been implemented. The purpose of our research is to develop a cell wall Iytic filter using a cell wall Iytic enzyme, which can inactivate the bacteria in air that cause infectious diseases by decomposing their cell envelope. In this study, the use of Iytic enzyme mixture was suggested, including glycosidase, protease and lipase. The performance of the Iytic enzyme mixture was evaluated using lysozyme, a typical Iytic enzyme, as a control. The substrate that we used was Micrococcus luteus, a gram-positive bacteria. The experimental results showed that the use of the Iytic enzyme mixture exhibited a Iytic rate per hour that was 13 - 39% greater than the control. Furthermore, although there are some different phases during bacterium multiplication, the Iytic rate per hour improved for all of the phases when the Iytic enzyme mixture was used.

Age-related pattern of body density and body composition of Japanese men and women 18-59 years of age.

Age-related patterns of body size and composition were studied in a cross-sectional sample of Japanese adults 18-59 years of age. Height, weight, the body mass index (BMI), body density (BD), percentage body fat (%Fat), fat mass (FM), fat-free mass (FFM), and the sum of seven skinfold thicknesses (SF) of 288 men and 552 women were considered. Body density was measured by underwater weighing densitometry. Mean values of height, weight, BMI, BD, %Fat, FM, FFM, and SF of males were 169.0 cm, 65.3 kg, 22.8 kg/m(2), 1.0600 g/ml, 17.0%, 11.4 kg, 53.9 kg, and 95.0 mm, respectively, while corresponding values for females were 157.4 cm, 52.9 kg, 21.4 kg/m(2), 1.0420 g/ml, 24.4%, 13.1 kg, 39.7 kg, and 128.2 mm, respectively. Height, BD, and FFM correlated negatively with age in both sexes, while weight, BMI, %Fat, FM, and SF correlated positively with age. The highest BD and the lowest %Fat were observed in males ages 20 to <25 years (G20) and in females 25 to <30 years (G25). The lowest BD and highest %Fat were observed in G50 in both sexes. Correlations among parameters of body size and composition were stable with age in each sex. Height correlated negatively with BMI and %Fat in females, but not in males. In males, FM started to increase between G20 and G25 and continued to increase until G50, while SF and BMI remained stable during this period. In females, FM accumulation started around 30 years of age and continued until G50 and was accompanied by increases in SF and BMI.

Age-related pattern of body density and body composition in Japanese males and females, 11 and 18 years of age.

The age-related pattern of body density and body composition in Japanese males (n = 266) and females (n = 318), 11.00 to 18.99 years of age was studied. Body density (BD) as well as height, body weight, and seven skinfold thicknesses were measured. Percentage fat (%Fat) was calculated using the age- and sex-specific equation of Lohman. Fat mass (FM), fat-free mass (FFM), and the body mass index (BMI) were calculated. The trend for BD in males was lowest at 11 years (1.0530 g/ml) and increased to 1.0695 g/ml at 14 years, and then decreased slightly at 15 to 17 years. In female, BD decreased from 1.0530 g/ml at 13 years to 1.0424 g/ml at 17 years. Mean %Fat was highest in males at 11 years (15.8%), and lowest at 14 years (10.1%). The highest mean %Fat in females occurred at 16 years (22.8%), and the lowest at age 11 years (15.2%). Overall, only 6.8% of males and 3.1% of females were classified as obese. Between 11 and 18 years, FFM of males differed by 20.7 kg or 67.9%, whereas females showed a difference of only 10.8 kg or 34.7%. Consequently, age effects explained approximately 60% of the male variance of FFM but only 26% in females. Body density of each sex and age group in this study did not differ significantly from previous Japanese studies, and the pooled BD data for 1,457 Japanese including the present study are reported as a reference.

Prevalence of age related maculopathy in a representative Japanese population: the Hisayama study.

AIM: To determine the prevalence of age related maculopathy (ARM) in a representative older Japanese population. METHODS: 1486 residents of Hisayama town, Fukuoka, Japan, aged 50 years or older were examined and the presence of ARM was determined by grading from fundus examination by indirect ophthalmoscope, slit lamp, and colour fundus photographs. RESULTS: The prevalence rate of drusen, which occurred with comparable frequency in men and women, was 9.6%. The frequency of drusen increased with age (p <0.01). Hyperpigmentation and/or hypopigmentation of the retina was present in 3.2%, geographic atrophy in 0.2%, and neovascular age related macular degeneration in 0.67%. The frequency of neovascular age related macular degeneration was significantly higher in the men (1.2% v. 0.34%, p <0.01). CONCLUSIONS: Early and late stage ARM is less common among Japanese people than among white people in Western countries, while late stage ARM is more common among Japanese than among black people.

[Validity of various indices of obesity calculated from height and weight data for adult males use of the underwater-weighing method as a reference].

PURPOSE: We evaluated associations between excess body fat (%Fat) and various indices of obesity calculated from height and weight data. METHODS: In 147 adult males, %Fat was measured by the underwater-weighing method, and obesity indices were generated by the following 5 approaches: the Broca-Katsura (Katsura method), the Kato-Wataya (Kato method), Japan Society for the Study of Obesity (BMI method; based on the body weight at which the BMI is 22), and the Meiji Life Insurance Co. methods, and the Tables and Figures for Assessment of Obesity and Leanness published by the Ministry of Health and Welfare (MHW method). RESULTS: %Fat was 20% or more (obese) in 67 males (45.6%), 15-20% in 39 (26.5%), 10-15% in 35 (23.8%), and less than 10% in 6 (4.1%). The correlation coefficients between the obesity indices and %Fat were 0.612 for the Katsura method, 0.590 for the Kato-method, 0.611 for the BMI method, 0.612 for the Meiji Life Insurance Co. method, and 0.550 for the MHW method, being significant in each case (P < 0.01). When the cut-off point was set as 110% for each obesity index, sensitivity was highest with the Kato-method (82.1%), and specificity was highest with the Meiji Life Insurance Co. method (93.8%). With the MHW method, the receiver operating characteristic (ROC) curve was slightly farther from the point of sensitivity of 100% and 1-specificity of 0% than the others. CONCLUSION: Excess fat accumulation can not be accurately assessed by obesity indices calculated from body height and weight data. Validity was similar among obesity indices examined.

Transduction of antisense cyclin D1 using two-step gene transfer inhibits the growth of rat hepatoma cells.

Cyclin D1, one of the G(1) cyclins, is frequently overexpressed in several types of carcinomas and is thought to play an important role in tumorigenesis and tumor progression including hepatocellular carcinoma. We constructed a retrovirus vector-carrying rat cyclin D1 cDNA in the reverse orientation, resulting in expression of antisense (AS) cyclin D1 mRNA. For efficient transduction of this recombinant retrovirus, two-step gene transfer was performed. The rat hepatoma cell line (dRLh84) was infected with this recombinant retrovirus after preinfection with adenovirus expressing the retrovirus receptor. In the rat hepatoma cells, AS cyclin D1 mRNA was expressed, inducing a decrease in the expression of endogenous cyclin D1 mRNA and an inhibition of cell growth. Moreover, two-step gene transfer of AS cyclin D1 into s.c. hepatoma xenografts resulted in inhibition of tumor growth and prolonged animal survival. In the virus-infected tumor xenografts, expression of cyclin D1 was immunohistochemically inhibited, and apoptosis of hepatoma cells was detected. These findings suggest that transduction of AS cyclin D1 is useful as an adjunct to standard treatments for hepatocellular carcinoma.

Effects of lifetime volleyball exercise on bone mineral densities in lumbar spine, calcaneus and tibia for pre-, peri- and postmenopausal women.

To clarify the effects of habitual volleyball exercise on bone in women during the menopausal periods, we measured bone mineral densities (BMDs) of the lumbar spine, calcaneus and tibia every 12 months for 2 years and estimated factors related to the baseline values and annual loss rates. Forty Japanese female volleyball players 42-62 years of age, who had belonged to the district non-professional club for more than 10 years on average, were recruited. Twenty women had regular menstruation at the start, but 7 underwent menopause during the study. Fifty-nine healthy women who did not participate in habitual exercise, but were otherwise comparable with the players, were recruited as the controls. The lumbar and calcaneus BMDs were measured by dual-energy X-ray absorptiometry (DXA), and both the volumetric BMDs and cross-sectional geometry at the diaphysis of the tibia were measured by peripheral quantitative computed tomography. The baseline BMDs of all measured sites and the values of the cortical area and moment of inertia of the tibia in the players were significantly higher than the values in the control women. In the peri- and postmenopausal players, the baseline values of the lumbar and calcaneus BMDs related to total years of participating in regular exercises during adulthood including volleyball (TYE), body mass index (BMI) and years since menopause (YSM). Tibia cortical area and moment of inertia values related to TYE. Annual bone loss rates in the tibia and calcaneus of players were significantly smaller than those values in the controls. However, the bone loss rates in the lumbar spine did not differ significantly between the groups. The bone loss rate in the calcaneus was significantly related to the current number of training hours per week and YSM. The rate of bone loss in the tibia was related to BMI. These data indicated that the total number of years participating in exercise activity during adulthood have positive effects on lumbar and calcaneus BMDs and the cortical structure of the tibia. Habitual volleyball exercise apparently did not alleviate the menopause-related bone loss in the lumbar spine.