Discontinuation of leisure time impact-loading exercise is related to reduction of a calcaneus quantitative ultrasound parameter in young adult Japanese females: a 3-year follow-up study.

UNLABELLED: A 3-year follow-up study on 334 young Japanese females enrolled in a university at the age of 18 years revealed that discontinuation of leisure time impact-loading exercises performed in junior high and/or high school was associated with increased risk of reduction in calcaneus osteo-sono assessment index (OSI). INTRODUCTION: Bone strength rapidly increases during puberty and reaches its peak by the end of adolescence. The aim of this study was to determine the lifestyle factors that influence the maintenance of calcaneus OSI in young adult females around the time when peak bone mass is attained. METHODS: Annual health checkups including OSI measurements, anthropometrics, lifestyle analysis, and blood examination were performed 4 times on 334 Japanese females enrolled in a university at the age of 18 years. According to the slope of OSI change during the 3-year follow-up, the subjects were grouped into two categories: OSI loss (the lowest tertile) and OSI gain/stable (the second and third tertiles). RESULTS: At the baseline assessment, the OSI loss group had higher OSI and height and an earlier menarche age than the OSI gain/stable group. Performing leisure time impact-loading exercise in junior high and/or high school but discontinuing it at university was associated with increased risk of OSI loss, independent of OSI, height and weight at the age of 18 years, weight change during follow-up, age of menarche, energy-adjusted nutrient intake, and alcohol drinking; the odds ratios were 4.1-4.9 compared with those performing impact-loading exercise at university. In particular, duration, frequency, and subjective intensity of impact-loading exercise during high school were positively associated with OSI loss. CONCLUSION: Discontinuation of leisure time impact-loading exercises performed during late adolescence is associated with an increased risk of OSI loss in young adult females during the 3-year follow-up period.

Development of Rat Caries-Induced Pulpitis Model for Vital Pulp Therapy.

Rodent animal models for vital pulp therapy are commonly used in dental research because their tooth anatomy and cellular processes are similar to the anatomy and processes in humans. However, most studies have been conducted using uninfected sound teeth, which makes it difficult to adequately assess the inflammatory shift after vital pulp therapy. In the present study, we aimed to establish a caries-induced pulpitis model based on the conventional rat caries model and then evaluate inflammatory changes during the wound-healing process after pulp capping in a model of reversible pulpitis induced by carious infection. To establish the caries-induced pulpitis model, the pulpal inflammatory status was investigated at different stages of caries progression by immunostaining targeted to specific inflammatory biomarkers. Immunohistochemical staining revealed that both Toll-like receptor 2 and proliferating cell nuclear antigen were expressed in moderate and severe caries-stimulated pulp, indicating that an immune reaction occurred at both stages of caries progression. M2 macrophages were predominant in moderate caries-stimulated pulp, whereas M1 macrophages were predominant in the severe caries-stimulated pulp. Pulp capping in teeth with moderate caries (i.e., teeth with reversible pulpitis) led to complete tertiary dentin formation within 28 d after treatment. Impaired wound healing was observed in teeth with severe caries (i.e., teeth with irreversible pulpitis). During the wound-healing process in reversible pulpitis after pulp capping, M2 macrophages were predominant at all time points; their proliferative capacity was upregulated in the early stage of wound healing compared with healthy pulp. In conclusion, we successfully established a caries-induced pulpitis model for studies of vital pulp therapy. M2 macrophages have an important role in the early stages of the wound-healing process in reversible pulpitis.

Novel Functional Peptide for Next-Generation Vital Pulp Therapy.

Although vital pulp therapy should be performed by promoting the wound-healing capacity of dental pulp, existing pulp-capping materials were not developed with a focus on the pulpal repair process. In previous investigations of wound healing in dental pulp, we found that organic dentin matrix components (DMCs) were degraded by matrix metalloproteinase-20, and DMC degradation products containing protein S100A7 (S100A7) and protein S100A8 (S100A8) promoted the pulpal wound-healing process. However, the direct use of recombinant proteins as pulp-capping materials may cause clinical problems or lead to high medical costs. Thus, we hypothesized that functional peptides derived from recombinant proteins could solve the problems associated with direct use of such proteins. In this study, we identified functional peptides derived from the protein S100 family and investigated their effects on dental pulp tissue. We first performed amino acid sequence alignments of protein S100 family members from several mammalian sources, then identified candidate peptides. Next, we used a peptide array method that involved human dental pulp stem cells (hDPSCs) to evaluate the mineralization-inducing ability of each peptide. Our results supported the selection of 4 candidate functional peptides derived from proteins S100A8 and S100A9. Direct pulp-capping experiments in a rat model demonstrated that 1 S100A8-derived peptide induced greater tertiary dentin formation compared with the other peptides. To investigate the mechanism underlying this induction effect, we performed liquid chromatography-tandem mass spectrometry analysis using hDPSCs and the S100A8-derived peptide; the results suggested that this peptide promotes tertiary dentin formation by inhibiting inflammatory responses. In addition, this peptide was located in a hairpin region on the surface of S100A8 and could function by direct interaction with other molecules. In summary, this study demonstrated that a S100A8-derived functional peptide promoted wound healing in dental pulp; our findings provide insights for the development of next-generation biological vital pulp therapies.

Correlation between genotype and supernumerary tooth formation in cleidocranial dysplasia.

INTRODUCTION: Cleidocranial dysplasia (CCD, MIM#119600), for which the responsible gene is RUNX2, is a genetic disorder characterized by hypoplasia or aplasia of the clavicles, patent fontanelles, and a short stature. Supernumerary teeth and delayed eruption and impaction of permanent teeth are frequently associated with CCD. Our previous study reported wide intrafamilial variation in supernumerary tooth formation associated with a mutation in the RUNT-domain of RUNX2, suggesting a low correlation between the genotype and supernumerary tooth formation. To further clarify this point, a more precise evaluation was performed. DESIGN: Gene mutational analysis of nine Japanese individuals with CCD was performed. Dental and skeletal characteristics were examined based on patient examinations and radiographs. RESULTS: Four different gene mutations, including one novel mutation in RUNX2 gene (NM_001024630), were identified. Among them, four individuals had the R225Q mutation, three siblings had the P224S mutation, and the other two individuals had different frame-shift mutations. Wide variations in supernumerary tooth formation were observed in individuals with identical gene mutations, and discordance was seen between monozygotic twins. Asymmetric supernumerary tooth formation was noted in five out of the nine individuals. CONCLUSION: Individuals with identical gene mutations showed a wide variation in the supernumerary tooth formation. Not only the genotype but also environmental factors and a complex system including epigenetics and copy number variation might regulate supernumerary tooth formation in CCD.

A Mongolian patient with hypohidrotic ectodermal dysplasia with a novel P121S variant in EDARADD.

INTRODUCTION: Hypohidrotic ectodermal dysplasia is a genetic disorder characterized by diminished or a lack of sweating, congenital missing teeth, and sparse or absent hair. Three genes, EDA, EDAR, and EDARADD, all related to tumor necrosis factor signaling, have been reported as responsible genes for this disorder. Among them, the largest numbers of mutations have been identified in EDA, and only two mutations identified in EDARADD. MATERIALS AND METHODS: DNA analysis of EDA, EDAR, and EDARADD was performed on a Mongolian patient by polymerase chain reaction-direct sequencing. RESULTS: The 5-year-old Mongolian individual had no erupted deciduous or permanent teeth. A panoramic radiograph showed only one tooth in the right mandible. His hair and eyebrows were sparse, but he did not have a short stature. He showed diminished sweating. The nails of his fingers and toes were normal. Based on these conditions, he was diagnosed with hypohidrotic ectodermal dysplasia. There was no gene mutation of EDA or EDAR. A novel heterozygous variant (P121S; c.361C>T) was identified in the death domain of EDARADD (NM_080738). No other member of his family was affected, and this variant was not identified in his parents or maternal grandparents. CONCLUSION: This study reports an individual affected with hypohidrotic ectodermal dysplasia with a novel heterozygous P121S variant in the death domain of EDARADD.

Multistep control of pituitary organogenesis.

Lhx3 and Lhx4 (Gsh4), two closely related LIM homeobox genes, determine formation of the pituitary gland in mice. Rathke's pouch is formed in two steps-first as a rudiment and later as a definitive pouch. Lhx3 and Lhx4 have redundant control over formation of the definitive pouch. Lhx3 controls a subsequent step of pituitary fate commitment. Thereafter, Lhx3 and Lhx4 together regulate proliferation and differentiation of pituitary-specific cell lineages. Thus, Lhx3 and Lhx4 dictate pituitary organ identity by controlling developmental decisions at multiple stages of organogenesis.

Atelocollagen-mediated local and systemic applications of myostatin-targeting siRNA increase skeletal muscle mass.

RNA interference (RNAi) offers a novel therapeutic strategy based on the highly specific and efficient silencing of a target gene. Since it relies on small interfering RNAs (siRNAs), a major issue is the delivery of therapeutically active siRNAs into the target tissue/target cells in vivo. For safety reasons, strategies based on vector delivery may be of only limited clinical use. The more desirable approach is to directly apply active siRNAs in vivo. Here, we report the effectiveness of in vivo siRNA delivery into skeletal muscles of normal or diseased mice through nanoparticle formation of chemically unmodified siRNAs with atelocollagen (ATCOL). ATCOL-mediated local application of siRNA targeting myostatin, a negative regulator of skeletal muscle growth, in mouse skeletal muscles or intravenously, caused a marked increase in the muscle mass within a few weeks after application. These results imply that ATCOL-mediated application of siRNAs is a powerful tool for future therapeutic use for diseases including muscular atrophy.

Phosphorylation of cofilin by LIM-kinase is necessary for semaphorin 3A-induced growth cone collapse.

Semaphorin 3A is a chemorepulsive axonal guidance molecule that depolymerizes the actin cytoskeleton and collapses growth cones of dorsal root ganglia neurons. Here we investigate the role of LIM-kinase 1, which phosphorylates an actin-depolymerizing protein, cofilin, in semaphorin 3A-induced growth cone collapse. Semaphorin 3A induced phosphorylation and dephosphorylation of cofilin at growth cones sequentially. A synthetic cell-permeable peptide containing a cofilin phosphorylation site inhibited LIM-kinase in vitro and in vivo, and essentially suppressed semaphorin 3A-induced growth cone collapse. A dominant-negative LIM kinase, which could not be activated by PAK or ROCK, suppressed the collapsing activity of semaphorin 3A. Phosphorylation of cofilin by LIM-kinase may be a critical signaling event in growth cone collapse by semaphorin 3A.

The efficiency variation method for 4pibeta-gamma coincidence counting by ink-jet printing.

In order to vary the counting efficiencies in the 4pibeta-gamma coincidence extrapolation technique, a radioactive source was coated directly with varying amounts of an electrical conducting pigment using an ink-jet printer. This method can be used to efficiently prepare the multiple sources needed to generate efficiency extrapolation curves, and was successfully applied to the standardization of a (54)Mn source.

Effects of fermented soymilk with Lactobacillus casei Shirota on skin condition and the gut microbiota: a randomised clinical pilot trial.

Several clinical studies have shown that isoflavones and Lactobacillus casei Shirota (LcS) have beneficial effects on skin condition and the gut microbiota, respectively. Thus, we investigated the effects of consecutive intake of fermented soymilk (FSM) with LcS on skin condition and the gut microbiota, as well as isoflavone bioavailability, in a randomised, double-blind, placebo-controlled trial as a pilot study. Sixty healthy premenopausal Japanese women received FSM containing a moderate level of isoflavone aglycones and a probiotic LcS, or soymilk (SM) containing neither of them, twice a day for 8 weeks. Skin condition was assessed by a subjective questionnaire for face and morphological analysis of the stratum corneum on the inner forearm. Faecal microbiota and urinary isoflavone were analysed by 16S rRNA gene amplicon sequencing and high-performance liquid chromatography tandem mass spectrometry, respectively. Both the FSM and SM groups had improved skin condition as assessed from scores of overall satisfaction, dryness, moisture, elasticity, coarseness, pigmentation and/or stratum corneum morphology, as well as significantly increased levels of urinary isoflavones during the intake period compared with the pre-intake period, although there were no significant differences between the two groups. There was a significant positive correlation between urinary isoflavone levels and skin questionnaire scores. In contrast, the relative abundance levels of Lactobacillaceae significantly increased and those of Bifidobacteriaceae tended to increase during the intake period compared with the pre-intake period. For the after-intake period they only decreased significantly in the FSM group. The levels of Enterobacteriaceae and Porphyromonadaceae significantly decreased during the intake period in the FSM group. These findings suggest that daily intake of FSM, as well as SM, provides health benefits that improve skin condition via increased levels of isoflavone absorption in the body, and that only FSM beneficially modifies the gut microbiota in premenopausal healthy women.

Studies of immune functions of patients with systemic lupus erythematosus: antibodies to desialized, rather than intact, T cells preferentially bind to and eliminate suppressor effector T cells.

Patients with systemic lupus erythematosus (SLE) were found to have in their plasma antibodies specific for desialized T cells. Adsorption studies with intact or desialized T cells indicated that SLE anti-T cell antibodies consisted of two populations with different target cell specificities, one capable of recognizing unique determinants on desialized T cells and another able to bind to both intact and desialized T cells. Normal T cells did not remove the antibodies specific for desialized T cells. moreover, the antibodies to desialized T cells were not removed by adsorption with either desialized non-T cells or desialized erythrocytes. Thus, the antibodies to desialized T cells recognize a determinant that is unique to a T cell subset and also includes a sugar. Inhibition studies with various sugars indicated that lactose was the most potent inhibitor of antibody binding. The anti-desialized T cell antibody appears to recognize a T cell determinant which includes lactose, probably in the form of a beta-galactosyl residue, but which also includes additional T cell determinants. The antibodies to desialized T cells were found to bind preferentially to concanavalin A-induced autorosetting T cells, which had been already demonstrated to contain suppressor effector cells. Indeed, such antibodies were effective in eliminating suppressor effector function without interfering with T cells necessary for such activation (such as precursor or inducer cells). Finally, studies of patients with SLE yielded a highly significant correlation (r = 0.92) between impaired suppressor effector function of their cells and the presence of antibodies to desialized T cells in their plasma.

Development of the fast neutron standard using a Be({alpha},n) reaction at the National Metrology Institute of Japan.

This paper describes the 8-MeV neutron field where the neutrons are generated in the (9)Be(alpha,n)(12)C reaction by bombardment of a beryllium target with a 2.4-MeV (4)He(+) beam from a Van de Graaff accelerator. The neutron field is being prepared for a new national standard on neutron fluence in Japan. Absolute measurement of the neutron fluence was taken using a proton recoil neutron detector, consisting of a silicon surface barrier detector with a polyethylene radiator. Neutron spectra were measured using a newly developed recoil proton spectrometer and a liquid organic scintillation detector. The gamma rays existing in the field were also characterised using a liquid organic scintillation detector. The ambient dose equivalents of the gamma rays were estimated to be <100 microSv at the neutron fluence of 10(7) neutrons cm(-2).

Neutralizing antibodies to human interleukin 6 reverse hypercalcemia associated with a human squamous carcinoma.

Bone resorbing cytokines may be associated with abnormalities in bone cell function and calcium homeostasis in a number of pathological conditions. One of these cytokines is interleukin 6 (IL-6), which is a multifunctional cytokine which has been shown to be associated with increased formation of bone resorbing osteoclasts in vitro. In this report, we demonstrate that neutralizing antibodies to human IL-6 lower the blood calcium in nude mice carrying a human tumor associated with increased IL-6 production. There was no effect on blood calcium in hypercalcemic nude mice carrying tumors not associated with increased IL-6 production and normocalcemic tumor-bearing nude mice. These results suggest that increased production of IL-6 may contribute to disturbances in calcium homeostasis in some malignancies, and suggest that neutralization of IL-6 effects can lower the serum calcium in these tumors.

Overexpression of laminin gamma2 chain monomer in invading gastric carcinoma cells.

Laminin (LN)-5, a heterotrimer of alpha3, beta3, and gamma2 chains, has been suggested to be involved in tumor cell invasion. The present immunohistochemical study investigated the distribution of the LN gamma2 chain in 48 different human gastric adenocarcinomas. The immunohistochemical analysis showed two distinct patterns of LN gamma2 chain expression: (a) extracellular deposition; and (b) cytoplasmic accumulation. The extracellular deposition of the LN gamma2 chain was typically observed at neoplastic basement membranes of well-differentiated adenocarcinomas. The immunoreactivity was continuous along tumor basement membranes in these tumors but was irregular and diffuse in poorly differentiated carcinomas. These tumor cells coexpressed the LN alpha3 and beta3 chains, suggesting that the LN gamma2 chain was deposited as the LN-5 complex. In contrast, tumor cells at the invading fronts showed strong cytoplasmic staining for the LN gamma2 chain without any detectable signal for the LN alpha3 or beta3 chain in both well- and poorly differentiated carcinomas. On the other hand, in vitro analysis by two-dimensional SDS-PAGE demonstrated that human gastric carcinoma cells secrete a high level of LN gamma2 chain monomer in addition to the LN-5 complex into culture medium. These results indicate that the LN gamma2 chain can be secreted as a single subunit and might be involved in tumor cell invasion.

Detection of a point mutation in cholesteryl ester transfer protein gene by polymerase chain reaction-mediated site-directed mutagenesis.

We describe a method for the rapid and non-radioactive examination of DNA samples for a mutation of cholesteryl ester transfer protein using a polymerase chain reaction-mediated site-directed mutagenesis. CETP deficiencies were studied in 554 Japanese subjects (370 men, 184 women) aged between 18 and 91 (mean 48.3 years). By this method, we detected one homozygote and 3 heterozygotes of the CETP deficiency.

Characterization of two new human apolipoprotein A-I variants: apolipoprotein A-I Tsushima (Trp-108-->Arg) and A-I Hita (Ala-95-->Asp).

Two unrelated subjects with new variants of apolipoprotein (apo) A-I were found during screening with isoelectric focusing (IEF) gel analysis. In the first case, apo A-I Tsushima, sequencing following amplification by the polymerase chain reaction (PCR) revealed a residue 108 missense mutation (TGG-->CGG, Trp-->Arg) in exon 4. The proband of apo A-I Tsushima was heterozygous for this mutation. The second case, apo A-I Hita, revealed a residue 95 missense mutation (GCC-->GAC, Ala-->Asp) in exon 4. The proband of apo A-I Hita was compound heterozygous with apo A-I (Ala-37-->Thr). These two subjects exhibited normal plasma concentrations of apo A-I and HDL cholesterol. In screening normal high school students (n = 198), we used a PCR-mediated site-directed mutagenesis to rapidly detect the substitution of G to A at codon 37 because the apo A-I (GC-->ACC, Ala-37-->Thr) mutation is unrelated to the charge difference on IEF. The frequency of the A allele was 0.04: the substitution G to A at codon 37 did not affect the plasma concentrations of lipids and lipoproteins.

Apolipoprotein E1 Lys-146----Glu with type III hyperlipoproteinemia.

During the screening of samples obtained from 5 individuals with type III hyperlipidemia, we identified a variant of apolipoprotein (apo) E which exhibited a discrepancy in apo E phenotype showing the E3/E1 isoform on isoelectric focusing (IEF) analysis and E3/E3 on gene analysis. Sequence analysis of the DNA of the proband that was amplified by PCR and subcloned, revealed a single substitution of one lysine (AAG) for one glutamic acid (GAG) at position 146, thereby adding two negatively charged units to apo E3. This defect had been described only for apo E1 to date (Mann et al. (1989) Clin. Res. 37, 520A (abstract)). In this case, PCR-mediated site-directed mutagenesis was used to identify the structural alterations forming the abnormal E1 genotype in the proband's family. Purified apo E1 Lys-146----Glu showed less than 10% of binding activity to apo B, E receptor on human skin fibroblasts compared with apo E3. This substitution demonstrates that Lys-146 is essential for the binding of apo E to the receptor.

Characterization of a novel variant of apolipoprotein E, E2 Fukuoka (Arg-224 --> Gln) in a hyperlipidemic patient with xanthomatosis.

A new variant of apolipoprotein (apo) E, designated apo E2 Fukuoka, was identified in a 54-year-old Japanese woman who suffered from hyperlipoproteinemia (total cholesterol 29.7 mmol/l, triglyceride 12.0 mmol/l, when she was 48-year-old) with the presence of xanthoma in the palms, bones, and ocular fundi, and other sites. Foam-cell macrophages were observed in bone marrow specimens. Analysis of apo E phenotype showed the E3/E2 isoform on isoelectric focusing performed on plasma, but the E3/E3 isoform on restriction-fragment-length polymorphism of the apo E gene. This discrepancy indicated that the apo E had an amino-acid substitution outside of amino-acid residues at 112 and 158. Sequence analysis of the patient's DNA, which was amplified by PCR and subcloned, revealed a single substitution from arginine (CGG) to glutamine (CAG) at residue 224, thereby adding one negatively charged unit to apo E3. Recombinant apo E2 Fukuoka produced in COS-1 cells showed almost the same binding activity to the LDL receptor on human skin fibroblasts as compared with recombinant apo E3. Recombinant apo E2 Fukuoka showed the same heparin binding ability than recombinant apo E3. Findings indicated that apo E2 Fukuoka was not the primary cause of the hyperlipoproteinemia observed in this case.

Novel mutations in ABCA1 gene in Japanese patients with Tangier disease and familial high density lipoprotein deficiency with coronary heart disease.

Mutations in the ATP-binding cassette transporter 1 (ABCA1) gene have been recently identified as the molecular defect in Tangier disease (TD) and familial high density lipoprotein deficiency (FHA). We here report novel mutations in the ABCA1 gene in two sisters from a Japanese family with TD who have been described previously (S. Ohtaki, H. Nakagawa, N. Kida, H. Nakamura, K. Tsuda, S. Yokoyama, T. Yamamura, S. Tajima, A. Yamamoto, Atherosclerosis 49 (1983)) and a family with FHA. Both probands of TD and FHA developed coronary heart disease. Sequence analysis of the ABCA1 gene from the patients with TD revealed a homozygous G to A transition at nucleotide 3805 of the cDNA resulting in the substitution of Asp 1229 with Asn in exon 27, and a C to T at nucleotide 6181 resulting in the substitution of Arg 2021 with Trp in exon 47. Sequence analysis of the ABCA1 gene from the FHA patient revealed a homozygous 4 bp CGCC deletion from nucleotide 3787 to 3790 resulting in premature termination by frameshift at codon 1224. These mutations were confirmed by restriction digestion analysis, and were not found in 141 control subjects. Our findings indicate that mutations in the ABCA1 gene are associated with TD as well as FHA.

Effects of supine and lateral recumbent positions on pulmonary venous flow in healthy subjects evaluated by transesophageal Doppler echocardiography.

OBJECTIVES: This study attempted to evaluate the effects of supine and lateral recumbent positions on pulmonary venous flow by transesophageal Doppler echocardiography in healthy subjects. BACKGROUND: Although transesophageal echocardiographic examination is usually performed with the patient lying in the left lateral decubitus or supine position, little attention has been paid to the effects of these positions on pulmonary venous flow. METHODS: We performed pulsed Doppler transesophageal echocardiography of the left and right pulmonary veins in 16 normal subjects as they lay in the left and right lateral decubitus and supine positions. RESULTS: Data are reported as mean value +/- SD. Adequate recordings were obtained in 12 subjects (75%). In the left pulmonary vein, peak systolic velocity and time-velocity integral of systolic flow increased significantly in the left compared with the right lateral decubitus position (56 +/- 12 vs. 44 +/- 13 cm/s, p < 0.05, and 15 +/- 4 vs. 9 +/- 4 cm, p < 0.05, respectively). In the right pulmonary vein, peak systolic velocity and time-velocity integral of systolic flow decreased significantly in the left compared with the right lateral decubitus position (38 +/- 10 vs. 48 +/- 9 cm/s, p < 0.05, and 9 +/- 2 vs. 12 +/- 2 cm, p < 0.05, respectively). There were no significant differences between positions in peak diastolic flow velocity, time-velocity integral of diastolic flow or peak velocity of flow reversal at atrial contraction. CONCLUSIONS: Pulmonary venous systolic peak velocities and time-velocity integrals of systolic flow increase when the pulmonary venous recording is from the recumbent subject's lower side. Therefore, the effects of position should be considered in evaluating left ventricular diastolic function by transesophageal Doppler echocardiography.