Extremely low-frequency electromagnetic fields facilitate both osteoblast and osteoclast activity through Wnt/ß-catenin signaling in the zebrafish scale.

Electromagnetic fields (EMFs) have received widespread attention as effective, noninvasive, and safe therapies across a range of clinical applications for bone disorders. However, due to the various frequencies of devices, their effects on tissues/cells are vary, which has been a bottleneck in understanding the effects of EMFs on bone tissue. Here, we developed an in vivo model system using zebrafish scales to investigate the effects of extremely low-frequency EMFs (ELF-EMFs) on fracture healing. Exposure to 10 millitesla (mT) of ELF-EMFs at 60 Hz increased the number of both osteoblasts and osteoclasts in the fractured scale, whereas 3 or 30 mT did not. Gene expression analysis revealed that exposure to 10 mT ELF-EMFs upregulated wnt10b and Wnt target genes in the fractured scale. Moreover, ß-catenin expression was enhanced by ELF-EMFs predominantly at the fracture site of the zebrafish scale. Inhibition of Wnt/ß-catenin signaling by IWR-1-endo treatment reduced both osteoblasts and osteoclasts in the fractured scale exposed to ELF-EMFs. These results suggest that ELF-EMFs promote both osteoblast and osteoclast activity through activation of Wnt/ß-catenin signaling in fracture healing. Our data provide in vivo evidence that ELF-EMFs generated with a widely used commercial AC power supply have a facilitative effect on fracture healing.

Different effects of magnetic field on drug activity in human uterine sarcoma cell lines MES-SA and MES-SA/Dx5.

Previous studies reported that combined effect of magnetic field (MF) on cytotoxic drugs in human cancer cells. We focused on the effects of 60 Hz MF on drug activity in human uterine sarcoma MES-SA and drug-resistant variant MES-SA/Dx5 cells that overexpressed the membrane protein MDR1(P-glycoprotein), a drug efflux transporter for doxorubicin, daunorubicin, and etoposide, but not cisplatin. The cisplatin with MF caused 60% decrease in cell viability when compared with no MF treatment, cisplatin alone in MES-SA cells. Even in MES-SA/Dx5 cells, MF exposure equally enhanced cisplatin activity. Then, MF enhanced doxorubicin and daunorubicin activity in MES-SA cells and caused 60% decrease in the cell viability compared with these drugs only but had less effect on these drugs in MES-SA/Dx5 cells. Etoposide activity was unaffected by MF exposure in both cell lines, although etoposide is a MDR1 substrate as with doxorubicin and daunorubicin. Thus, MF had no direct impact on MDR1 in the cell membrane. However, the differences in doxorubicin and daunorubicin activity between MES-SA and MES-SA/Dx5 data revealed that the presence of MDR1 in abundance prevented the enhancing effects of MF on doxorubicin and daunorubicin activity. These results suggested that MF may act in the opposite direction of MDR1, affect the drug influx transporters for doxorubicin and daunorubicin, and facilitate anticancer drug uptake into the cells.

Exposure to 60 Hz magnetic field can affect membrane proteins and membrane potential in human cancer cells.

The experimental data support the hypothesis that extremely low frequency magnetic field (ELF-MF) can affect cell membranes. Since our previous studies suggested that MF changes the permeability of cell membrane, in this study we focused on the cell membrane and investigated the effect of 60 Hz, 50 mT MF on the membrane potential and membrane proteins. The membrane potentials of three cultured human cancer cell lines, A549, MES-SA, and MES-SA/Dx5, were increased by exposure to ELF-MF. When exposed to MF and an anticancer drug, changes in the membrane potentials were detected in A549 and MES-SA cells, but not in the multi drug-resistant cells, MES-SA/Dx5. We examined whether MF has an influence on the membrane proteins extracted from cultured A549 cells, using DiBAC4(3) dye enhanced fluorescence binding to a hydrophobic site. The increase in fluorescence observed following MF exposure for 10 min indicated that the structure of the hydrophobic site on the membrane proteins changed and became more likely to bind the probe dye. A decrease in fluorescence was detected following exposure to MF for 240 min. These results indicated that 60 Hz, 50 mT MF causes changes in the membrane potential of cultured cancer cells and the conformation of membrane proteins extracted from cultured cancer cells, and has different effects depending on the exposure time.

Cell concentration, viability and culture composition of airborne bacteria during a dust event in Beijing.

Airborne bacteria were measured when a dust storm passed Beijing in spring 2012 with a focus on cell concentration, viability and TSA- and R2A-cultured strain composition. The concentration varied at an order of 107cells/m3 with dust loading (demonstrated with PM10) and they had a very close correlation (RT2=0.91, p<0.01). At the time of highest PM10 of 652µg/m3, the bacterial concentration reached 1.4×108cells/m3, which was larger than that before and after the dust event by one order. Bacterial viability, the ratio of number concentration of viable cells to total cells, was 32%-64% and smaller in the dust plume than that before the dust arrival. Bacterial strains from the culture ranged between 2.5×104 and 4.6×105CFU/m3 and no correlation with PM10 was determined. Their composition was different before and after the dust arrival according to 16S rRNA gene sequences and strains belong to Actinomycetes and Firmicutes were the majority in the dust samples.

Bioprocess of Kosa bioaerosols: effect of ultraviolet radiation on airborne bacteria within Kosa (Asian dust).

Kosa (Asian dust) is a well-known weather phenomenon in which aerosols are carried by the westerly winds from inland China to East Asia. Recently, the frequency of this phenomenon and the extent of damage caused have been increasing. The airborne bacteria within Kosa are called Kosa bioaerosols. Kosa bioaerosols have affected ecosystems, human health and agricultural productivity in downwind areas. In order to develop a new and useful bacterial source and to identify the source region of Kosa bioaerosols, sampling, isolation, identification, measurement of ultraviolet (UV) radiation tolerance and experimental simulation of UV radiation conditions were performed during Kosa bioaerosol transportation. We sampled these bioaerosols using a Cessna 404 airplane and a bioaerosol sampler at an altitude of approximately 2900 m over the Noto Peninsula on March 27, 2010. The bioaerosol particles were isolated and identified as Bacillus sp. BASZHR 1001. The results of the UV irradiation experiment showed that the UV radiation tolerance of Kosa bioaerosol bacteria was very high compared with that of a soil bacterium. Moreover, the UV radiation tolerance of Kosa bioaerosol spores was higher than that of soil bacterial spores. This suggested that Kosa bioaerosols are transported across the atmosphere as living spores. Similarly, by the experimental simulation of UV radiation conditions, the limited source region of this Kosa bioaerosol was found to be southern Russia and there was a possibility of transport from the Kosa source area.

Cloning of two members of the calcitonin-family receptors from stingray, Dasyatis akajei: possible physiological roles of the calcitonin family in osmoregulation.

In cartilaginous fish, two cDNAs encoding calcitonin-family receptors were isolated for the first time from the stingray brain. The open reading frame of one receptor cDNA coded a 525-amino acid protein. The amino acid identity of this receptor to human calcitonin-receptor-like receptor (CRLR) is 64.5%, frog CRLR is 64.7%, and flounder CRLR is 61.2% and this was higher than to human calcitonin receptor (CTR) (46.1%), frog CTR (54.7%), and flounder CTR (48.9%). We strongly suggested that this receptor is a ray CRLR based on phylogenetic analysis. In case of the second receptor, amino acid identity among CRLRs (human 50.5%, frog 50.7%, flounder 48.0%) and CTRs (human 43.2%, frog 49.1%, flounder 41.8%) was similar. From phylogenetic analysis of both CRLRs and CTRs, we believe that this receptor is ray CTR. The expression of ray CRLR mRNA was predominantly detected in the nervous system (brain) and vascular system (atrium, ventricle, and gill), which reflects the similar localization of CGRP in the nervous and vascular systems as mammals. It was observed that the second receptor was expressed in several tissues, namely cartilage, brain, pituitary gland, gill, atrium, ventricle, pancreas, spleen, liver, gall bladder, intestine, rectal gland, kidney, testis and ovary. This localization pattern was very similar to flounder CTR. Both receptor mRNAs were strongly expressed in the gill. This suggests that the calcitonin-family members are involved in the osmoregulation of stingray as this fish is known to be euryhaline. When a stingray was transferred to diluted seawater (20% seawater), the expression of both receptors significantly decreased in the gill. Similar results were obtained in the kidney of the stingray. Thus, our cloning and isolation of both receptors in the stingray will be helpful for elucidation of their physiological role(s) such as osmoregulation including calcium metabolism of cartilaginous fish.

Phylogenetic analysis of atmospheric halotolerant bacterial communities at high altitude in an Asian dust (KOSA) arrival region, Suzu City.

The microbial communities transported by Asian desert dust (KOSA) events have attracted much attention as bioaerosols because the transported microorganisms are thought to influence the downwind ecosystems in Korea and Japan. However, the atmospheric microbial community has not been investigated at high altitude in the KOSA arrival area. In this study, to estimate the viability and diversity of atmospheric halotolerant bacteria, which are expected to resist to various environmental stresses as well as high salinities, bioaerosol samples were collected at 10 and 600 m above the ground within the KOSA arrival area, Suzu City, Japan, during KOSA events. During the sampling period, the particle numbers at 600 m were higher than those at 10 m, suggesting that large particles of aerosol fall from the high altitude of 600 m to the ground surface. The microorganisms in bioaerosol samples grew in media containing up to 15% NaCl concentrations demonstrating the viability of the halotolerant bacteria in bioaerosol samples. The PCR-DGGE analysis using 16S rDNA revealed that the bacterial species in NaCl-amended cultures were similar to the bacteria detected from the genomic DNA directly extracted from the bioaerosol samples. The 16S rDNA sequences of bacterial communities in bioaerosol samples were classified into 4 phylotypes belonging to the Bacilluscereus or Bacillussubtilis group. The bioaerosol samples collected at 600 m included 2 phylotypes belonging to B. subtilis, and one phylotype among all 4 phylotypes was identical between the samples at 10 and 600 m. In the atmosphere at 600 m, the halotolerant bacterial community was expected to remain viable, and the species composition was expected to include a few species of the genus Bacillus. During this investigation period, these atmospheric bacteria may have been vertically transported to the ground surface, where the long-range KOSA particle transport from China is frequently observed.

Molecular analysis of the lysis protein Lys encoded by Lactobacillus plantarum phage phig1e.

The putative cell-lysis gene lys of Lactobacillus plantarum G1e phage phig1e encodes for a 442 amino-acids protein Lys. The N-terminal region (about 80 amino acids) of Lys consists of two discrete regions (the signal-peptide-like domain and the DE domain containing putative active sites of endolysin). To elucidate functions of the regions of Lys, mutational (random, site-directed, and/or fusion) analysis was performed. The plasmid pNdEHL, expressing the wild type Lys protein under promoter of lacZ' gene in Escherichia coli, was constructed. Two molecular species (44 kDa; referred to as pre-Lys, and 42 kDa; mature-Lys) from the protein extract of XL1-Blue/pNdEHL were detected on a sodium dodecyl sulfate gel and zymogram with L. plantarum G1e cells. Based on the N-terminal amino acid sequences, the two molecules were determined as; pre-Lys (the amino acid position deduced from lys gene, 1-7) MKLKNKL, mature-Lys (27-33) QTLSSQS. The mature Lys was hardly detected in the cells treated with sodium azide. These results suggested that the N-terminal 26 amino acids region of Lys precursor form is possibly processed posttranslationally, by a SecA-dependent manner at least in E. coli. Analysis of the point mutants (pLD36A, pLE39A, pLE55A, pLE67A and pLD71A), indicated that the acidic residues (aspartic acids at position 36, 71 and glutamic acids at position 39, 55) of N-terminal region and the serine at the position 48 of phig1e Lys are essential for the lytic activity.

Characterization of the major tail protein gpP encoded by Lactobacillus plantarum phage phi gle.

Lactobacillus plantarum temperate phage phi g1e encodes a major virion protein gpP. In the present study, the gpP protein was overproduced in Escherichia coli under plac, and purified. Like the native-gpP protein from phi gle particles (Kakikawa et al., 1996), the purified-gpP protein had an apparent molecular mass of 26.0 kDa on SDS polyacrylamide gel electrophoresis (PAGE), larger than that (18.8 kDa) predicted from the DNA sequence, and was deficient in the first methionine as revealed by the N-terminal protein sequencing. In addition, analysis by immunoelectron microscopy demonstrated that immunogold particles (associated with antigpP-sera) specifically bound to the tails of phi gle particles, indicating that gpP is a main tail component (putatively a tube protein).