Effects of Arm-cranking Training with Electrical Muscle Stimulation on Vessel Function.

This study aimed to determine whether arm-cranking training with electrical muscle stimulation (EMS) results in a greater improvement in vessel function than performing the same exercise without EMS. First, nine healthy young men performed two 20-min arm-cranking trials at 50% V˙O2max with and without EMS applied to the lower limbs. The flow-mediated vasodilation (FMD) of the right brachial artery was measured using a high-resolution ultrasound device. Both FMD and normalized FMD were increased significantly after the arm-cranking with EMS trial, and significant differences were observed between the two trials. Second, 16 healthy adult men were randomly assigned to either the arm-cranking exercise training (A) group or arm-cranking training with EMS (A+EMS) group. The subjects were engaged in 20 min of arm-cranking at 50% V˙O2max twice a week for 8 weeks with/without EMS applied to the lower limbs. The FMD increased significantly after A+EMS training session and the FMD in A+EMS group was significantly higher than that in the A group. These results indicate that acute/chronic endurance arm-cranking with EMS applied to the lower limbs improves the brachial artery endothelial function more markedly than the same exercise without EMS.

Prevalence of infant and maternal anemia during the lactation period in Japan.

BACKGROUND: The perinatal period is associated with a high risk of infant anemia. The aim of the present study was to determine the prevalence of infant and maternal anemia during the late lactation period and the risk factors for anemia in Japan. METHODS: This retrospective cohort study was based on data from health checkups of healthy infants at 6-7 and 9-10 months of age and their mothers who visited Akitsu Children's Clinic between September 2013 and August 2015. Complete blood count data from infant blood samples obtained at 6-7 months and 9-10 months and from maternal blood samples obtained at 6-7 months, information on feeding methods, and other related parameters were analyzed. RESULTS: Data from 388 mother-infant pairs were analyzed. The prevalence of infant anemia was 21.1% at 6-7 months and 29.1% at 9-10 months. The prevalence of anemia in exclusively breast-fed infants was 28.4% at 6-7 months and 40.0% at 9-10 months. The risk factors for infant anemia at 9-10 months were exclusive breast-feeding, lower gestational age at birth, male sex, and high weight gain. The prevalence of maternal anemia was 10.5%. There was no correlation between infant and maternal hemoglobin in exclusively breast-fed infants. CONCLUSIONS: Japanese infants who were breast-fed exclusively had a high prevalence of anemia. A nationwide strategy to prevent anemia is required to prevent infant anemia, even in a nutrition-rich country such as Japan.

Phospholipase C-related, but catalytically inactive protein (PRIP) up-regulates osteoclast differentiation via calcium-calcineurin-NFATc1 signaling.

Phospholipase C-related, but catalytically inactive protein (PRIP) was previously identified as a novel inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-δ but lacking phospholipase activity. We recently showed that PRIP gene knock-out (KO) in mice increases bone formation and concomitantly decreases bone resorption, resulting in increased bone mineral density and trabecular bone volume. However, the role of PRIP in osteoclastogenesis has not yet been fully elucidated. Here, we investigated the effects of PRIP on bone remodeling by investigating dynamic tooth movement in mice fitted with orthodontic devices. Morphological analysis indicated that the extent of tooth movement was smaller in the PRIP-KO mice than in wild-type mice. Histological analysis revealed fewer osteoclasts on the bone-resorption side in maxillary bones of PRIP-KO mice, and osteoclast formation assays and flow cytometry indicated lower osteoclast differentiation in bone marrow cells isolated from these mice. The expression of genes implicated in bone resorption was lower in differentiated PRIP-KO cells, and genes involved in osteoclast differentiation, such as the transcription factor NFATc1, exhibited lower expression in immature PRIP-KO cells initiated by M-CSF. Moreover, calcineurin expression and activity were also lower in the PRIP-KO cells. The PRIP-KO cells also displayed fewer M-CSF-induced changes in intracellular Ca2+ and exhibited reduced nuclear localization of NFATc1. Up-regulation of intracellular Ca2+ restored osteoclastogenesis of the PRIP-KO cells. These results indicate that PRIP deficiency impairs osteoclast differentiation, particularly at the early stages, and that PRIP stimulates osteoclast differentiation through calcium-calcineurin-NFATc1 signaling via regulating intracellular Ca2.

Involvement of PRIP (Phospholipase C-Related But Catalytically Inactive Protein) in BMP-Induced Smad Signaling in Osteoblast Differentiation.

Phospholipase C-related but catalytically inactive protein (PRIP) was first isolated as an inositol 1,4,5-trisphosphate binding protein. We generated PRIP gene-deficient mice which exhibited the increased bone mineral density and trabecular bone volume, indicating that PRIP is implicated in the regulation of bone properties. In this study, we investigated the possible mechanisms by which PRIP plays a role in bone morphogenetic protein (BMP) signaling, by analyzing the culture of primary cells isolated from calvaria of two genotypes, the wild type and a mutant. In the mutant culture, enhanced osteoblast differentiation was observed by measuring alkaline phosphatase staining and activity. The promoter activity of Id1 gene, responding immediately to BMP, was also more increased. Smad1/5 phosphorylation in response to BMP showed an enhanced peak and was more persistent in mutant cells, but the dephosphorylation process was not different between the two genotypes. The luciferase assay using calvaria cells transfected with the Smad1 mutated as a constitutive active form showed increased transcriptional activity at similar levels between the genotypes. The expression of BMP receptors was not different between the genotypes. BMP-induced phosphorylation of Smad1/5 was robustly decreased in wild type cells, but not in mutant cells, by pretreatment with DB867, an inhibitor of methyltransferase of inhibitory Smad6. Furthermore, BMP-induced translocation of Smad6 from nucleus to cytosol was not much observed in PRIP-deficient cells. These results indicate that PRIP is implicated in BMP-induced osteoblast differentiation by the negative regulation of Smad phosphorylation, through the methylation of inhibitory Smad6.

Use of non-invasive total hemoglobin measurement as a screening tool for anemia in children.

Universal screening for anemia is important in children, but invasive blood sampling is required. A new device (Radical-7® Pulse CO-Oximeter™, Masimo, Irvine, CA, USA) now enables non-invasive hemoglobin concentration (SpHb) measurement to be done, but the usefulness of this device for anemia screening in children is unclear. The objective of this study was to compare SpHb with complete blood count (CBC) using a hematology analyzer (Microsemi® LC-667CRP; Fukuda Denshi, Tokyo, Japan). SpHb measurement with Radical-7® was done as part of a medical check-up in 3-year-old children (n = 110). Another 43 pediatric patients were checked for CBC using Microsemi® and monitored with Radical-7®. The mean SpHb level of the 3-year-old children was 12.1 ± 0.64 g/dL (range, 10.8-13.7 g/dL). The correlation of Radical-7® and Microsemi® was 0.602 (P < 0.0001). On Bland-Altman comparison, bias was -0.6 ± 1.1 g/dL. Even though further improvement is required, Radical-7® offers many possibilities in the context of primary screening.

Involvement of PRIP, phospholipase C-related, but catalytically inactive protein, in bone formation.

PRIP (phospholipase C-related, but catalytically inactive protein) is a novel protein isolated in this laboratory. PRIP-deficient mice showed increased serum gonadotropins, but decreased gonadal steroid hormones. This imbalance was similar to that for the cause of bone disease, such as osteoporosis. In the present study, therefore, we analyzed mutant mice with special reference to the bone property. We first performed three-dimensional analysis of the femur of female mice. The bone mineral density and trabecular bone volume were higher in mutant mice. We further performed histomorphometrical assay of bone formation parameters: bone formation rate, mineral apposition rate, osteoid thickness, and osteoblast number were up-regulated in the mutant, indicating that increased bone mass is caused by the enhancement of bone formation ability. We then cultured primary cells isolated from calvaria prepared from both genotypes. In mutant mice, osteoblast differentiation, as assessed by alkaline phosphatase activity and the expression of osteoblast differentiation marker genes, was enhanced. Moreover, we analyzed the phosphorylation of Smad1/5/8 in response to bone morphogenetic protein, with longer phosphorylation in the mutant. These results indicate that PRIP is implicated in the negative regulation of bone formation.

Effect of Electrical Muscle Stimulation on Vascular Endothelial Function during Prolonged Sitting.

OBJECTIVE: While prolonged sedentary behaviors (SBs) increase cardiovascular disease (CVD) risk, interrupting prolonged sitting (PS) with frequent light exercise reduces arterial functional decline. Skeletal muscle electrical stimulation (EMS) enhances peripheral circulation through passive muscle contraction, suggesting that EMS reduces CVD risk by providing an alternative to active exercise for prolonged SBs. This study aimed to investigate the effects of EMS to skeletal muscles during PS on the endothelial function of the brachial artery (BA). METHODS: Study participants included 12 healthy adult men who were subjected to 15 min of supine rest, followed by 1 h of PS only (control [CON] trial), or 20 min of EMS to the lower extremities at 50% of the maximum tolerance intensity during PS (EMS trial). Flow-mediated dilation (FMD) of the BA was measured before and 30 min after PS, and normalized FMD (nFMD) was calculated. RESULTS: The nFMD of the CON trial significantly decreased 30 min after PS completion (6.21% ± 1.13%) compared with that before PS (7.26% ± 0.73%), and there was no significant change in the EMS trial before and after PS. The EMS trial showed a significant increase in the nFMD 30 min after PS completion (1.14 ± 0.77) compared with that before PS (0.84 ± 0.43). However, no significant difference was observed in the CON trials. CONCLUSION: Passive contraction of the lower extremity muscles by EMS increases BA nFMD, suggesting that prolonged sedentary lower extremity EMS use may reduce the risk of vascular endothelial dysfunction.

Characterization of the human PRIP-1 gene structure and transcriptional regulation.

The PRIP [phospholipase C related, but catalytically inactive protein] family has been isolated as a novel inositol 1,4,5-trisphosphate binding protein with a domain organization similar to phospholipase C-delta but lacking the enzyme activity, comprising PRIP-1 and PRIP-2. The PRIP-1 gene is expressed predominantly in the brain, while PRIP-2 exhibits a relatively ubiquitous expression in rats and mice. We also found that PRIP-1 plays an important role in type A receptor signaling for gamma-aminobutyric acid in the brain. In this study, we investigated PRIP-1 gene structure and the possible mechanisms involved in the expression. The tissue distribution pattern of PRIP gene expression in humans was similar to that in rodents. 5'RACE (rapid amplification of cDNA ends) analysis using PRIP-1 gene specific primers with human brain mRNA revealed the presence of three new exons, indicating that the PRIP-1 gene is organized into 8 exons intervened by 7 introns. Although three transcripts resulting from the alternative splicing of exon 2 and/or 3 were detected, a transcript lacking exons 2 and 3 was predominantly expressed in humans, suggesting that the translation start codon of human PRIP-1 exists in exon 1. To characterize the human PRIP-1 promoter, transient luciferase assay was carried out with luciferase constructs including various lengths of the 5' flanking region of the PRIP-1 gene. The results indicated that the positive regulatory region is located -237 to -108 bp upstream from the transcription start site. Gel shift assay revealed the specific binding of some nuclear proteins to this region, suggesting that the existence of transcription factors contributes to the positive regulation of PRIP-1 gene expression. Mutation analyses revealed that the binding of a transcription factor, MAZ to the regulatory site leads to the promoter activity, indicating that MAZ is involved in the expression regulation of the human PRIP-1 gene.

[The Xanthine Oxidase Inhibitory Activity and Hypouricemic Effects of Crude Drugs Obtained from the Silkworm in Mice].

This study evaluated the effects of crude drugs obtained from the silkworm in mice with oxonic acid-induced hyperuricemia using xanthine oxidase inhibitory activity and plasma uric acid levels. The plasma uric acid level was analyzed using an improved HPLC with UV detection (HPLC-UV) method, which enabled high-sensitivity analysis of a microliter of plasma. Using this method, we evaluated natural products administered orally to the hypouricemic mice. The plasma uric acid level of mice administered a water-soluble extract from silkworm larvae with botrytis (used in traditional Chinese medicine to reduce wind, lower blood pressure, and change platelet coagulation) was significantly lower than in the control group 1, 2, and 3 h after treatment. In addition, water soluble extracts from a fungus (NBRC 31161) metabolite and silkworm pupae and larvae reduced the plasma uric acid levels in mice compared with the control group.

The asymmetry of avian egg-shape: an adaptation for reproduction on dry land.

The present study describes the biological meaning of the asymmetrical shape in avian reproduction using quail. During the incubation of eggs, water was gradually lost and the air chamber which appeared in between the inner and outer shell membranes at the blunt end expanded, so that the angle made by the long egg-axis and the horizontal line increased, presumably because the centre of gravity of the egg contents moved toward the sharp end. The increase in angle occurred in both fertile and infertile eggs, suggesting that this phenomenon occurs irrespective of fertility and is due to the asymmetrical shape. The increase in the volume of the air chamber resulted in an increase in the area of the inner shell membrane at the chamber to satisfy the amount of gas exchange needed by the developing embryo for better hatching. We isolated a 300-kDa protein from the inner shell membrane. It was produced by cells in the luminal epithelium of the oviductal isthmus and was found in the cortex of the fibres of shell membranes and a lining surrounding the air chamber. The lining comprised a medial layer between the inner and outer shell membranes in uterine eggs. The asymmetrical ellipsoid produces the air chamber at the blunt end of the avian egg during its sojourn in the oviductal isthmus, to maintain the blunt end up after oviposition and to raise that end during incubation in a dry environment, leading to high hatchability.