No effect of extremely low-frequency magnetic field observed on cell growth or initial response of cell proliferation in human cancer cell lines

An effect on the tumor promotion process, as represented by accelerated cell growth, has been indicated as one example of areas that demonstrate the possibility of biological effects of extremely-low frequency magnetic fields. We, therefore, exposed the five cell lines (HL-60, K-562, MCF-7, A-375, and H4) derived from human tumors to a magnetic field for 3 days to investigate the effects on cell growth. Prior to exposure or sham exposure, the cells were precultured for 2 days in low serum conditions. The number of growing cells was counted in a blind manner. To investigate the effect on the initial response of cell proliferation, two cell lines were synchronized in G1 phase by serum starvation and then exposed to a magnetic field for 18 h (H4 cells) or 24 h (MCF-7 cells), both with and without serum stimulation. The rate of DNA synthesis, taken as a measure of the cell proliferation, was determined by following the incorporation of [(3)H]-thymidine into the DNA. Three different magnetic field polarizations at both 50 and 60 Hz were used: linearly polarized (vertical); circularly polarized; and an elliptically polarized field. Magnetic field flux densities were set at 500, 100, 20 and 2 microT (rms) for the vertical field and at 500 microT (rms) for the rotating fields. No effect of magnetic field exposure was observed on either cell growth or the initial response of cell proliferation.     

Intracisternal administration of Angiotensin II AT1 receptor antisense oligodeoxynucleotides protects against cerebral ischemia in spontaneously hypertensive rats

Pharmacological blockade of peripheral and brain Angiotensin II (Ang II) AT(1) receptors protects against brain ischemia. To clarify the protective role of brain AT(1) receptors, we examined the effects of specific antisense oligodeoxynucleotides (AS-ODN) targeted to AT(1) receptor mRNA administered intracisternally to spontaneously hypertensive rats (SHRs), 4 and 7 days before middle cerebral artery (MCA) occlusion, and we determined the infarct size and tissue swelling 24 h after surgery. A single intracisternal injection of AT(1) mRNA receptor antisense oligodeoxynucleotides reduced systemic blood pressure for 5 days and AT(1) receptor binding for at least 4 days in the area postrema and the nucleus of the solitary tract. A similar injection of scrambled oligodeoxynucleotides (SC-ODN) was without effect. Both blood pressure and AT(1) receptor binding returned to normal 7 days after antisense receptor mRNA administration. Both the infarction size and the tissue swelling after middle cerebral artery occlusion were reduced when the antisense oligodeoxynucleotide was administered 7 days, but not 4 days, before the operation. We conclude that 4 to 5 days of decrease in brain AT(1) receptor binding by a single administration of an AT(1) receptor mRNA oligodeoxynucleotide are sufficient to significantly protect the brain against ischemia resulting from total occlusion of a major cerebral vessel.     

No effects of extremely low frequency magnetic fields found on cytotoxic activities and cytokine production of human peripheral blood mononuclear cells in vitro

Several epidemiologic studies have suggested an association between exposure to extremely low frequency (ELF) magnetic fields (MFs) and cancer in adults and children. A possible target of MFs is the immune system. The effects of the exposure to ELF MFs on the immunological functions of human peripheral blood mononuclear cells (PBMCs) obtained from healthy male volunteers were assessed by measuring the natural killer (NK) and lymphokine activated killer (LAK) activities and the production of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), and interleukin-10 (IL-10). The PBMCs were exposed to three different MF: linearly polarized (vertical), circularly polarized, and elliptically polarized, at 50 and 60 Hz. Magnetic flux densities were set at 500, 100, 20, and 2 microT (rms) for vertical field and at 500 microT (rms) for the rotating fields. Using cytotoxicity assay and enzyme-linked immunosorbent assay (ELISA) for cytokine production, we could not find any effects of ELF MFs on the cytotoxic activities and the cytokines production of human PBMCs.     

Mutations of the RNA-specific adenosine deaminase gene (DSRAD) are involved in dyschromatosis symmetrica hereditaria

Dyschromatosis symmetrica hereditaria (DSH) (also called "reticulate acropigmentation of Dohi") is a pigmentary genodermatosis of autosomal dominant inheritance characterized by a mixture of hyperpigmented and hypopigmented macules distributed on the dorsal aspects of the hands and feet. To determine the gene responsible for this disease, we performed a genomewide search in three families with DSH and mapped the DSH locus to chromosome 1q21.3. The mutations involved in causing DSH have been identified in the gene that encodes double-stranded RNA-specific adenosine deaminase (DSRAD) as the disease gene.     

Antibacterial activity and mechanism of action of tick defensin against Gram-positive bacteria

Defensins are a major group of antimicrobial peptides and are found widely in vertebrates, invertebrates and plants. Invertebrate defensins have been identified from insects, scorpions, mussels and ticks. In this study, chemically synthesized tick defensin was used to further investigate the activity spectrum and mode of action of natural tick defensin. Synthetic tick defensin showed antibacterial activity against many Gram-positive bacteria but not Gram-negative bacteria and low hemolytic activity, characteristic of invertebrate defensins. Furthermore, bactericidal activity against pathogenic Gram-positive bacteria including Bacillus cereus, Enterococcus faecalis and methicillin-resistant Staphylococcus aureus was observed. However, more than 30 min was necessary for tick defensin to completely kill bacteria. The interaction of tick defensin with the bacterial cytoplasmic membrane and its ability to disrupt the membrane potential was analyzed. Tick defensin was able to disrupt the membrane potential over a period of 30-60 min consistent with its relatively slow killing. Transmission electron microscopy of Micrococcus luteus treated with tick defensin showed lysis of the cytoplasmic membrane and leakage of cellular cytoplasmic contents. These findings suggest that the primary mechanism of action of tick defensin is bacterial cytoplasmic membrane lysis. In addition, incomplete cell division with multiple cross-wall formation was occasionally seen in tick defensin-treated bacteria showing pleiotropic secondary effects of tick defensin.     

Peritoneal exudate cells treated with calcitonin gene-related peptide suppress murine experimental autoimmune uveoretinitis via IL-10

Immunization with retinal Ag induces experimental autoimmune uveoretinitis (EAU) in mice. We investigated the suppression of murine EAU by peritoneal exudate cells (PEC) cultured with calcitonin gene-related peptide (CGRP). PEC derived from mice were treated with CGRP and residues 1-20 of human interphotoreceptor retinoid-binding protein (hIRBP 1-20). The hIRBP 1-20-immunized mice were injected i.v. with PEC treated with CGRP and hIRBP 1-20. After immunization, Ag-specific delayed hypersensitivity (DH) was measured and EAU was assessed histopathologically. Both EAU- and Ag-specific DH were suppressed by injection of PEC treated with CGRP (100 ng/ml) and hIRBP 1-20. However, hIRBP 1-20-mediated EAU was not suppressed by injection of PEC treated with CGRP and BSA. Both EAU- and Ag-specific DH were not suppressed by injection of PEC treated with CGRP and hIRBP 1-20 into splenectomized mice. In mice adoptively transferred spleen cells from hIRBP 1-20-immunized mice, EAU was also suppressed by injection of CGRP-treated PEC. EAU was markedly inhibited in hIRBP 1-20-immunized mice adoptively transferred T cells obtained from mice injected with hIRBP 1-20-pulsed, CGRP-treated PEC. Furthermore, EAU- and Ag-specific DH were not suppressed by injection of PEC treated with CGRP and hIRBP 1-20 when the recipient mice were given anti-IL-10 Ab i.p., or when the PEC were derived from IL-10 knockout mice. The present results indicate that PEC treated with CGRP suppress murine EAU in an Ag-specific manner, even in the efferent phase, and IL-10 secreted from PEC might play an important role in the CGRP-mediated suppression of murine EAU.     

Immunolocalization of Protein 4.1B in the Rat Digestive System

Protein 4.1 family proteins are thought to interact with membrane proteins and also membrane skeletons. In this study, immunohistochemical studies by light and electron microscopy were performed with a specific antibody against protein 4.1B. Specific protein 4.1B immunolabeling was observed in simple columnar epithelium in the adult rat large intestine, small intestine and stomach. Protein 4.1B immunolabeling was localized along the membranes facing the adjacent cells (lateral portion) and also facing the extracellular matrix (basal portion). Moreover, a spatial protein 4.1B expression gradient was observed along the crypt-villus axis of the rat small and large intestinal epithelium: strong protein 4.1B expression was present within the villus, with the crypt showing barely any detectable protein 4.1B. The expression of protein 4.1B was not detected in the stratified squamous epithelium in the forestomach or the esophagus. By immunoelectron microscopy, the immunolabeling of the cells was observed to be restricted to the cytoplasmic side just beneath the plasma membrane, including the membranes adjacent to the next cells, except for the tight junctions. We conclude that the protein 4.1B expression pattern is related to the maturation of simple columnar epithelium in the rat digestive system, probably by the effect of adhesion.     

Immunolocalization of protein 4.1B/DAL-1 during neoplastic transformation of mouse and human intestinal epithelium

Recently, we have reported that the protein 4.1B immunolocalization occurred only in matured columnar epithelial cells of normal rat intestines. This finding suggested that protein 4.1B expression could be examined for a possible change during neoplastic transformation of the intestinal mucosa. In the present study, we first present the distribution of mouse protein 4.1B in normal intestinal epithelial cells and tumor cells using the adenomatous polyposis coli (Apc) mutant mouse model. A low level of protein 4.1B expression coincided with the phenotypic transition to carcinoma. To examine the protein 4.1B expression in human intestinal mucosa, we used another antibody against an isoform of the human protein 4.1B, DAL-1 (differentially expressed adenocarcinoma of the lung). Human DAL-1 was also expressed in matured epithelial cells in human colons, with a definite expression gradient along the crypt axis. In human colorectal cancer cells, however, DAL-1 expression was not detected. These results suggest that mouse protein 4.1B and human DAL-1 might have a striking analogy of functions, which may be integrally involved in epithelial proliferation. We propose that loss of protein 4.1B/DAL-1 expression might be a marker of intestinal tumors, indicative of a tumor suppressor function in the intestinal mucosa.     

SMXA-5 mouse as a diabetic model susceptible to feeding a high-fat diet

The SMXA-5 strain, a new mouse model for type 2 diabetes, is a recombinant inbred strain derived from non-diabetic SM/J and A/J strains. As dietary fat is a key component in the development of diabetes, we compared the glucose tolerance and diabetes-related traits among the SMXA-5, SM/J, and A/J strains while feeding a high-fat diet for 10 weeks. SMXA-5 fed on a high-fat diet showed an increased serum insulin concentration. Judging from the hyperinsulinemia in SMXA-5, this strain showed insulin resistance, an inability of peripheral tissues to respond to insulin, which was strengthened by feeding with a high-fat diet. When fed on a high-fat diet for 5 weeks, the SMXA-5 mice showed severely impaired glucose tolerance. On the other hand, SM/J showed mildly impaired glucose tolerance, even when fed on a high-fat diet for 10 weeks. These results indicate that SMXA-5 would be available for use as a diabetic model susceptible to a high-fat diet.