Intermittent pulsed electromagnetic field stimulation activates the mTOR pathway and stimulates the proliferation of osteoblast‐like cells

Pulsed electromagnetic fields (PEMFs) have been shown to be a noninvasive physical stimulant for bone fracture healing. However, PEMF stimulation requires a relatively long period of time and its mechanism of action has not yet been fully clarified. Recently, the mammalian target of rapamycin (mTOR) pathway has been shown to be involved in bone formation. This study aimed to investigate the effects of PEMFs on osteoblastic MC3T3‐E1 cells by examining various cellular responses including changes in the mTOR pathway. Continuous PEMF stimulation induced a transient phosphorylation of the mTOR pathway, whereas intermittent PEMF stimulation (1 cycle of 10 min stimulation followed by 20 min of stimulation pause) revitalized the reduced phosphorylation. Moreover, PEMF stimulation stimulated cell proliferation (bromodeoxyuridine incorporation) rather than differentiation (alkaline phosphatase activity), with a more notable effect in the intermittently stimulated cells. These results suggest that intermittent PEMF stimulation may be effective in promoting bone fracture healing by accelerating cell proliferation, and in shortening stimulation time. Bioelectromagnetics. 2019;40:412–421. © 2019 Bioelectromagnetics Society.     

Combined influences of gradual changes in room temperature and light around dusk and dawn on circadian rhythms of core temperature, urinary 6-hydroxymelatonin sulfate and waking sensation just after rising

The present experiment aimed at knowing how a gradual changes of room temperature (T(a)) and light in the evening and early morning could influence circadian rhythms of core temperature (T(core)), skin temperatures, urinary 6-hydroxymelatonin sulfate and waking sensation just after rising in humans. Two kinds of room environment were provided for each participant: 1) Constant room temperature (T(a)) of 27 degrees C over the 24 h and LD-rectangular light change with abrupt decreasing from 3,000 lx to 100 lx at 1800, abrupt increasing from 0 lx to 3,000 lx at 0700. 2) Cyclic changes of T(a) and with gradual decrease from 3,000 lx to 100 lx onset at 1700 (twilight period about 2 h), with gradual increasing from 0 lx to 3,000 lx onset at 0500 (about 2 h). Main results are summarized as follows: 1) Circadian rhythms of nadir in the core temperature (T(core)) significantly advanced earlier under the influence of gradual changes of T(a) and light than no gradual changes of T(a) and light. 2) Nocturnal fall of T(core) and morning rise of T(core) were greater and quicker, respectively, under the influence of gradual changes of T(a) and light than no gradual changes of T(a) and light. 3) Urinary 6-hydroxymelatonin sulfate during nocturnal sleep was significantly greater under the influence of gradual changes of T(a) and light. 4) Waking sensation just after rising was significantly better under the influence of gradual changes of T(a) and light. We discussed these findings in terms of circadian and thermoregulatory physiology.     

The role of nitric oxide synthase-derived reactive oxygen species in the altered relaxation of pulmonary arteries from lambs with increased pulmonary blood flow

Congenital cardiac defects associated with increased pulmonary blood flow (Q(p)) produce pulmonary hypertension. We have previously reported attenuated endothelium-dependent relaxations in pulmonary arteries (PA) isolated from lambs with increased Q(p) and pulmonary hypertension. To better characterize the vascular alterations in the nitric oxide-superoxide system, 12 fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt). Twin lambs served as controls. PA were isolated from these lambs at 4-6 wk of age. Electron paramagnetic resonance spectroscopy on fourth-generation PA showed significantly increased superoxide anion generation in shunt PA that were decreased to control levels following inhibition of nitric oxide synthase (NOS) with 2-ethyl-2-thiopseudourea. Preconstricted fifth-generation PA rings were relaxed with a NOS agonist (A-23187), a nitric oxide donor [S-nitrosyl amino penicillamine (SNAP)], polyethylene glycol-conjugated superoxide dismutase (PEG-SOD), or H(2)O(2). A-23187-, PEG-SOD-, and H(2)O(2)-mediated relaxations were impaired in shunt PA compared with controls. Pretreatment with PEG-SOD significantly enhanced the relaxation response to A-23187 and SNAP in shunt but not control PA. Inhibition of NOS with nitro-L-arginine or scavenging superoxide anions with tiron enhanced relaxation to SNAP and inhibited relaxation to PEG-SOD in shunt PA. Pretreatment with catalase inhibited relaxation of shunt PA to A-23187, SOD, and H(2)O(2). We conclude that NOS catalyzes the production of superoxide anions in shunt PA. PEG-SOD relaxes shunt PA by converting these anions to H(2)O(2), a pulmonary vasodilator. The redox environment, influenced by the balance between production and scavenging of ROS, may have important consequences on pulmonary vascular reactivity in the setting of increased Q(p).     

Consecutive oral administration of Bifidobacterium longum MM‐2 improves the defense system against influenza virus infection by enhancing natural killer cell activity in a murine model

Bifidobacterium, one of the major components of intestinal microflora, shows anti‐influenza virus (IFV) potential as a probiotic, partly through enhancement of innate immunity by modulation of the intestinal immune system. Bifidobacterium longum MM‐2 (MM‐2), a very safe bacterium in humans, was isolated from healthy humans and its protective effect against IFV infection in a murine model shown. In mice that were intranasally inoculated with IFV, oral administration of MM‐2 for 17 consecutive days improved clinical symptoms, reduced mortality, suppressed inflammation in the lower respiratory tract, and decreased virus titers, cell death, and pro‐inflammatory cytokines such as IL‐6 and TNF‐α in bronchoalveolar lavage fluid. The anti‐IFV mechanism of MM‐2 involves innate immunity through significant increases in NK cell activities in the lungs and spleen and a significant increase in pulmonary gene expression of NK cell activators such as IFN‐γ, IL‐2, IL‐12 and IL‐18. Even in non‐infected mice, MM‐2 administration also induced significant enhancement of both IFN‐γ production by Peyer's patch cells (PPs) and splenetic NK cell activity. Oral administration of MM‐2 for 17 days activates systemic immunoreactivity in PPs, which contributes to innate immunity, including NK cell activation, resulting in an anti‐IFV effect. MM‐2 as a probiotic may function as a prophylactic agent in the management of an IFV epidemic.     

Isolation and characterization of mutants of a marine Vibrio strain that are defective in production of extracellular proteins

A marine Vibrio strain, Vibrio sp. strain 60, produces several extracellular proteins, including protease, amylase, DNase, and hemagglutinin. Mutants of Vibrio sp. strain 60 (epr mutants) pleiotropically defective in production of these extracellular proteins were isolated. They fell into two classes, A and B. In class A, no protease activity was detected in the cells either, whereas in class B, considerable protease activity was detected in the cells. Gel electrophoretic analysis revealed that the protease detected in class B mutant cells was similar to the protease excreted by the parent strain. In addition, the protease in class B mutant cells was found to be localized in the periplasmic space. These results suggest that the passage of the protease through the outer membrane is blocked in class B mutants. Comparison of membrane protein profiles by polyacrylamide gel electrophoresis revealed that all the epr mutants contained an increased amount of a 94,000-Mr protein that may be an outer membrane protein. Four epr mutations were mapped in two different regions of the Vibrio chromosome by transduction; two class A mutations and one class B mutation were located close to each other, whereas another class B mutation was located in a different region of the chromosome.     

Synthesis of selenocysteine and selenomethionine derivatives from sulfur-containing amino acids

Selenium-containing amino acids have attracted increasing interest from view points of the importance as active centers of several selenoenzymes, the biological synthesis, the metabolism, and the use for structure determination of proteins. In this article, our recent progresses in the transformation from sulfur-containing amino acids to selenocysteine (SeCys) and selenomethionine (SeMet) derivatives are reviewed along with the surveys of general organic methodologies for the synthesis of SeCys and SeMet derivatives in the literature. The S-->Se modification (i.e., the chemical atomic mutation) would be a useful approach to peptide synthesis involving selenoamino acid residues.     

Cell cycle-dependent regulation of the phosphorylation and metabolism of the Alzheimer amyloid precursor protein.

Accumulation of the amyloid A beta peptide, which is derived from a larger precursor protein (APP), and the formation of plaques, are major events believed to be involved in the etiology of Alzheimer's disease. Abnormal regulation of the metabolism of APP may contribute to the deposition of plaques. APP is an integral membrane protein containing several putative phosphorylation sites within its cytoplasmic domain. We report here that APP is phosphorylated at Thr668 by p34cdc2 protein kinase (cdc2 kinase) in vitro, and in a cell cycle-dependent manner in vivo. At the G2/M phase of the cell cycle, when APP phosphorylation is maximal, the levels of mature APP (mAPP) and immature APP (imAPP) do not change significantly. However, imAPP is altered qualitatively. Furthermore, the level of the secreted extracellular N-terminal domain (APPS) is decreased and that of the truncated intracellular C-terminal fragment (APPCOOH) is increased. These findings suggest the possibility that phosphorylation-dependent events occurring during the cell cycle affect the metabolism of APP. Alterations in these events might play a role in the pathogenesis of Alzheimer's disease.     

Bayesian Parameter Estimation and Segmentation in the Multi-Atlas Random Orbit Model

This paper examines the multiple atlas random diffeomorphic orbit model in Computational Anatomy (CA) for parameter estimation and segmentation of subcortical and ventricular neuroanatomy in magnetic resonance imagery. We assume that there exist multiple magnetic resonance image (MRI) atlases, each atlas containing a collection of locally-defined charts in the brain generated via manual delineation of the structures of interest. We focus on maximum a posteriori estimation of high dimensional segmentations of MR within the class of generative models representing the observed MRI as a conditionally Gaussian random field, conditioned on the atlas charts and the diffeomorphic change of coordinates of each chart that generates it. The charts and their diffeomorphic correspondences are unknown and viewed as latent or hidden variables. We demonstrate that the expectation-maximization (EM) algorithm arises naturally, yielding the likelihood-fusion equation which the a posteriori estimator of the segmentation labels maximizes. The likelihoods being fused are modeled as conditionally Gaussian random fields with mean fields a function of each atlas chart under its diffeomorphic change of coordinates onto the target. The conditional-mean in the EM algorithm specifies the convex weights with which the chart-specific likelihoods are fused. The multiple atlases with the associated convex weights imply that the posterior distribution is a multi-modal representation of the measured MRI. Segmentation results for subcortical and ventricular structures of subjects, within populations of demented subjects, are demonstrated, including the use of multiple atlases across multiple diseased groups.     

Roles of integral protein in membrane permeabilization by amphidinols

Amphidinols (AMs) are a group of dinoflagellate metabolites with potent antifungal activity. As is the case with polyene macrolide antibiotics, the mode of action of AMs is accounted for by direct interaction with lipid bilayers, which leads to formation of pores or lesions in biomembranes. However, it was revealed that AMs induce hemolysis with significantly lower concentrations than those necessary to permeabilize artificial liposomes, suggesting that a certain factor(s) in erythrocyte membrane potentiates AM activity. Glycophorin A (GpA), a major erythrocyte protein, was chosen as a model protein to investigate interaction between peptides and AMs such as AM2, AM3 and AM6 by using SDS-PAGE, surface plasmon resonance, and fluorescent-dye leakages from GpA-reconstituted liposomes. The results unambiguously demonstrated that AMs have an affinity to the transmembrane domain of GpA, and their membrane-permeabilizing activity is significantly potentiated by GpA. Surface plasmon resonance experiments revealed that their interaction has a dissociation constant of the order of 10 μM, which is significantly larger than efficacious concentrations of hemolysis by AMs. These results imply that the potentiation action by GpA or membrane integral peptides may be due to a higher affinity of AMs to protein-containing membranes than that to pure lipid bilayers.