Effect of sinusoidal 50 Hz magnetic field on the testosterone production of mouse primary Leydig cell culture

This study evaluated the effect of sinusoidal 50 Hz magnetic field on the basal and human chorionic gonadotropin (hCG)-stimulated testosterone (T) production of 48-h mouse Leydig cell culture. The luteinizing hormone (LH) analog hCG was used to check the T response of the controls and to evaluate the possible effect of the applied magnetic field on the steroidogenic capacity of the exposed cells. Leydig cells were obtained from the testes of 35- to 45-g CFLP mice and isolated by mechanical dissociation without enzyme treatment. The cell cultures were exposed to sinusoidal 50 Hz 100 μT (root mean square) AC magnetic field during the entire time of a 48-h incubation. Testosterone content of the culture media was measured by radioimmunoassay. In cultures exposed to the magnetic field, a marked increase of basal T production was found (P < .05), compared with the unexposed controls, whereas no significant difference was seen between the exposed or unexposed cultures in the presence of maximally stimulating concentration of hCG. These findings demonstrate that sinusoidal 50 Hz 100 μT magnetic fields are able to stimulate the basal T production of primary mouse Leydig cell culture, leaving the steroidogenic responsiveness to hCG unaltered. Bioelectromagnetics 19:429–431, 1998. © 1998 Wiley-Liss, Inc.     

Nifedipine is an antagonist to cyclotron resonance enhancement of Ca incorporation in human lymphocytes

The incorporation of 45Ca in mixed human lymphocytes was measured following one-hour exposures of the cells to combined steady and periodic magnetic fields designed to probe for cyclotron resonance response in calcium incorporation. Measurements were made as a function of magnetic field frequency, up to 30 Hz, and as a function of magnetic field amplitude, up to 1.5 x 10(-4) Trms. The amplitude measurements demonstrated that the relative 45Ca uptake at resonance follows different mechanisms of interaction above and below 0.2 x 10(-4) Trms. After adjusting the magnetic field configuration for maximum incorporation, we then determined the effects of the calcium influx blocker nifedipine on 45Ca incorporation, with and without simultaneous exposure to this specific magnetic field combination. The presence of nifedipine in both unexposed and exposed cell suspensions resulted in decreased 45Ca uptake, presumably through the slow inward calcium channels. Evidence was found suggesting that nifedipine acts antagonistically to the 45Ca cyclotron resonance tuning signal.     

Tissue and species distribution of mRNA encoding two ADP-ribosylation factors, 20-kDa guanine nucleotide binding proteins

Cholera toxin catalyzed ADP-ribosylation of Gs alpha, the stimulatory guanine nucleotide binding protein of the adenylyl cyclase system, is enhanced by approximately 20-kDa guanine nucleotide binding proteins, termed ADP-ribosylation factors or ARFs. ARF is an allosteric activator of the A1 catalytic protein of the toxin. Bovine ARF cDNA clones, ARF-1 isolated from adrenal (Sewell & Kahn, 1988) and ARF-2B from retina (Price et al., 1988), exhibit nucleotide and deduced amino acid sequences that are 80% and 96% identical, respectively, in the coding region. To determine tissue and species distribution of ARF-like mRNAs, bovine ARF-2B and human ARF-1 cDNAs and 30- or 48-base oligonucleotide probes that distinguish between ARF-1 and ARF-2B cDNAs in coding and 3'-untranslated regions were used for Northern analysis of poly(A+) RNA from different tissues and species. On the basis of hybridization with specific oligonucleotide probes, all bovine tissues contained mRNAs of 1.7 and 2.1 kb that were related to ARF-1 and ARF-2B, respectively. Northern analysis of brain poly(A+) RNA from different species with ARF-2B and ARF-1 cDNAs at low stringency demonstrated several bands varying in size from 0.9 to 3.7 kb. A 1.7-kb band consistently hybridized with an ARF-1 30-base coding-region probe but not with a probe for the 3'-untranslated region. Similar ARF-2B oligonucleotide probes did not hybridize with rat, mouse, rabbit, or human brain mRNA. Cleavage of ARF-2B cDNA with PvuII generated two fragments, one containing coding and the other 3'-noncoding region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Absence of DNA damage after 60-Hz electromagnetic field exposure combined with ionizing radiation,hydrogen peroxide,or c-Myc overexpression

The principal objective of this study was to assess the DNA damage in a normal cell line system after exposure to 60 Hz of extremely low frequency magnetic field (ELF-MF) and particularly in combination with various external factors, via comet assays. NIH3T3 mouse fibroblast cells, WI-38 human lung fibroblast cells, L132 human lung epithelial cells, and MCF10A human mammary gland epithelial cells were exposed for 4 or 16 h to a 60-Hz, 1 mT uniform magnetic field in the presence or absence of ionizing radiation (IR, 1 Gy), H₂O₂ (50 μM), or c-Myc oncogenic activation. The results obtained showed no significant differences between the cells exposed to ELF-MF alone and the unexposed cells. Moreover, no synergistic or additive effects were observed after 4 or 16 h of pre-exposure to 1 mT ELF-MF or simultaneous exposure to ELF-MF combined with IR, H₂O₂, or c-Myc activation.     

Agonist regulation of beta-adrenergic receptor mRNA. Analysis in S49 mouse lymphoma mutants

Agonist-promoted down-regulation of beta-adrenergic receptor mRNA was investigated in S49 mouse lymphoma variants with mutations in elements of hormone-sensitive adenylate cyclase. In wild-type cells steady-state levels of beta-adrenergic receptor mRNA were established by DNA-excess solution hybridization to be 1.72 +/- 0.08 (n = 8) amol/microgram total cellular RNA. Receptor mRNA levels declined 35-45% in response to stimulation by the beta-adrenergic agonist (-)isoproterenol or forskolin as described previously in DDT1 MF-2 cells (Hadcock, J. R., and Malbon, C. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5021-5025). Agonist-promoted cAMP accumulation and down-regulation of receptor mRNA were analyzed in three variants with mutations in Gs alpha (H21a, unc, cyc-) and a single variant lacking cAMP-dependent protein kinase activity (kin-). H21a (Gs alpha coupled to receptor, but not to adenylate cyclase), unc (Gs alpha uncoupled from receptor), and cyc- (lacking Gs alpha) variants accumulated cAMP and down-regulated beta AR mRNA in response to forskolin. In unc and cyc- cells isoproterenol failed to stimulate cAMP; accumulation and down-regulation of receptor mRNA was not observed. H21a cells, in contrast, displayed agonist-promoted regulation of beta-adrenergic receptor mRNA but only basal levels of cAMP accumulation in response to isoproterenol. The kin- cells displayed cAMP accumulation in response to forskolin as well as to isoproterenol but no down-regulation of receptor mRNA or receptor expression. Taken together these data demonstrate several features of agonist-promoted down-regulation of mRNA: (i) cAMP-dependent protein kinase activity is required for down-regulation of mRNA (kin-), although elevated cAMP accumulation is not (H21a); (ii) functional receptor-Gs coupling is required (H21a), and clones lacking Gs alpha (cyc-) or receptor Gs coupling (unc) lack the capacity to down-regulate mRNA in response to agonist; and (iii) in the presence of basal levels of cAMP and cAMP-dependent protein kinase activity, functional receptor-Gs coupling (H21a) to some other effector other than adenylate cyclase may be propagating the signal.     

Extended exposure of adult and fetal mice to 50 Hz magnetic field does not increase the incidence of micronuclei in erythrocytes.

The flow cytometer-based micronucleus assay was used to study the effects on chromosomes in erythroid cells of CBA/Ca mice after extended exposure to 50 Hz magnetic field (MF), 14 microT, peak-to-peak (p-p). The study included two different experiments: (a) mice exposed in utero during 18 days of their prenatal stage, and (b) adult mice exposed for 18 days. In experiment (a) 35 days after exposure was terminated, peripheral blood was drawn from the mice exposed in utero to determine whether the exposure had a genotoxic effect on the pluripotent erythroid stem cells. About 200000 polychromatic erythrocytes (PCE) and 200000 normochromatic erythrocytes (NCE) were analysed from each of 20 exposed mice. The EMF exposure did not significantly change the frequency of micronucleated PCE or NCE in comparison with 20 sham-irradiated mice. There was no difference in the proportion of PCE between exposed and unexposed animals. Similarly, in experiment (b) no differences were seen between EMF exposed and unexposed adult mice when samples of peripheral blood were taken at the end of exposure and analyzed for micronuclei in PCE and NCE. The proportion of PCE was the same in both groups. The results indicate that exposure to EMF does not induce direct or indirect effects on chromosomes in erythroid cells expressed as increased levels of micronucleated erythrocytes of mice. No indications of delayed genetic effects were found.     

Sinusoidal 60 Hz electromagnetic fields failed to induce changes in protein synthesis in Escherichia coli

Escherichia coli JM83 {F^? ara Δ(lac-proAB) rpsL [?80dΔ(lacZ)M15]} in midlog growth phase at 30 °C were exposed to 60 Hz sinusoidal magnetic field of 3 mT of nonuniform diverging flux, inducing a nonuniform electric field with a maximum intensity of 32 μV/cm using an inductor coil. Exposed and unexposed control cells were maintained at 30.8 ± 0.1 °C and 30.5 ± 0.1 °C, respectively. Quadruplicate samples of exposed and unexposed E. coli cells were simultaneously radiolabeled with ³⁵S-L-methionine at 10 min intervals over 2 hr. Radiochemical incorporation into proteins was analyzed via liquid scintillation counting and by denaturing 12.5% polyacrylamide gel electrophoresis. The results showed that E. coli exposed to a 60 Hz magnetic field of 3 mT exhibited no qualitative or quantitative changes in protein synthesis compared to unexposed cells. Thus small prokaryotic cells (less than 2 μm × 0.5 μm) under constant-temperature conditions do not alter their protein synthesis following exposure to 60 Hz magnetic fields at levels at 3 mT. © 1994 Wiley-Liss, Inc.     

The thyroliberin receptor interacts directly with a stimulatory guanine-nucleotide-binding protein in the activation of adenylyl cyclase in GH3 rat pituitary tumour cells. Evidence obtained by the use of antisense RNA inhibition and immunoblocking of the stimulatory guanine-nucleotide-binding protein.

The thyroliberin receptor in GH3 pituitary tumour cells is known to couple to phospholipase C via a guanine-nucleotide-binding protein (G protein). Thyroliberin is postulated also to activate adenylyl cyclase, via the stimulatory G protein (Gs). In order to study this coupling, we constructed an antisense RNA expression vector that contained part of the Gs alpha-subunit cDNA clone (Gs alpha) in an inverted orientation relative to the mouse metallothionein promoter. The cDNA fragment included part of the coding region and all of the 3' non-translated region. Transient expression of Gs alpha antisense RNA in GH3 cells resulted in the specific decrease of Gs alpha mRNA levels, followed by decreased Gs alpha protein levels. Thyroliberin-elicited adenylyl cyclase activation in membrane preparations showed a reduction of up to 85%, whereas phospholipase C stimulation remained unaffected. Activation of adenylyl cyclase by vasoactive intestinal peptide was reduced by 30-40%. Investigation of the effects of thyroliberin and vasoactive intestinal peptide on adenylyl cyclase in GH3 cell membranes pretreated with antisera against Gs alpha and Gi-1 alpha/Gi-2 alpha support the results obtained by the use of the antisense technique. We conclude that thyroliberin has a bifunctional effect on GH3 cells, in activating adenylyl cyclase via Gs or a Gs-like protein in addition to the coupling to phospholipase C.     

Altered expression of G-protein mRNA in spontaneously hypertensive rats.

We have recently demonstrated that the decreased ability of hormones, forskolin and GTP to stimulate adenylate cyclase in heart and aorta from spontaneously hypertensive rats (SHR), as compared to their age-matched Wistar-Kyoto control rats (WKY), was associated with enhanced levels of Gi- and not with Gs-regulatory proteins. In the present studies we have investigated the expression of Gi-regulatory proteins at the mRNA level by Northern blotting. Total RNA of heart ventricle and aorta from WKY and SHR was probed with radiolabeled cDNA inserts encoding Gi alpha-2 and Gi alpha-3. The Gi alpha-2 and Gi alpha-3 probes detected a message of 2-3 and 3-5 kb, respectively, in both WKY and SHR, however, the message was significantly enhanced in SHR, as compared by WKY. On the other hand the cDNA probe encoding Gs alpha detected a message of 1.8 kb in heart and aorta from both WKY and SHR, however, no difference in the levels of Gs alpha mRNA was detected in SHR and WKY tissues. These results indicate that the mRNA levels of Gi alpha-2 and Gi alpha-3 and not of Gs are overexpressed in heart and aorta from SHR, which may be responsible for the increased levels of Gi as shown earlier by immunoblotting techniques. It may be suggested that the enhanced vascular tone and impaired cardiac contractility in hypertension may partly be the consequences of increased levels of Gi in heart and aorta.     

Effect of heat shock on gene expression of Aedes albopictus cells infected with Mayaro virus

Three major Mayaro virus proteins of 62, 50 and 34 kDa were detected in Aedes albopictus cells after 48 h postinfection at 28°C. When the infected cells were shifted from 28 to 37°C for 90 min (heat shock conditions), the synthesis of two major heat shock proteins (HSP) 82 and 70 kDa was induced concomitantly with strong inhibition of virus and normal protein synthesis. Total cellular RNA was isolated from mock and infected cells incubated at 28°C or under heat shock. Northern blot analysis with HSP genomic probes from Drosophila sp showed that (1) the probe for HSP 82 hybridized with an RNA of 2.6 kb present only in heat-shocked cells, (2) the HSP 70 probe hybridized with RNA species of 2.5 kb, present only in RNA from heat-shocked cells. These results showed that Mayaro virus was not able to alter the reprogrammation of gene expression induced by heat shock in A. albopictus cells.     

Effect of high-density extremely low frequency magnetic field on sister chromatid exchanges in mouse m5S cells

The induction of sister chromatid exchanges (SCEs) was evaluated in the cultured mouse m5S cells after exposure to extremely low frequency magnetic field (ELFMF; 5, 50 and 400 mT). Exposure to 5 mT and 50 mT ELFMF led to a very small increase in the frequency of SCEs, but no significant difference was observed between exposed and unexposed control cells. The cells exposed to 400 mT ELFMF exhibited a significant elevation of the SCE frequencies. There was no significant difference between data from treatments with mitomycin-C (MMC) alone and from combined treatments of MMC plus ELFMF (400 mT) at any MMC concentrations from 4 to 40 nM. These results suggest that exposure to highest-density ELFMF of 400 mT may induce DNA damage, resulting in an elevation of the SCE frequencies. We suppose that there may be a threshold for the elevation of the SCE frequencies, that is at least over the magnetic density of 50 mT.     

Effect of high-density extremely low frequency magnetic field on sister chromatid exchanges in mouse m5S cells

The induction of sister chromatid exchanges (SCEs) was evaluated in the cultured mouse m5S cells after exposure to extremely low frequency magnetic field (ELFMF; 5, 50 and 400 mT). Exposure to 5 mT and 50 mT ELFMF led to a very small increase in the frequency of SCEs, but no significant difference was observed between exposed and unexposed control cells. The cells exposed to 400 mT ELFMF exhibited a significant elevation of the SCE frequencies. There was no significant difference between data from treatments with mitomycin-C (MMC) alone and from combined treatments of MMC plus ELFMF (400 mT) at any MMC concentrations from 4 to 40 nM. These results suggest that exposure to highest-density ELFMF of 400 mT may induce DNA damage, resulting in an elevation of the SCE frequencies. We suppose that there may be a threshold for the elevation of the SCE frequencies, that is at least over the magnetic density of 50 mT.     

Social and immunological differences among uninfected Brazilians exposed or unexposed to human immunodeficiency virus-infected partners

Understanding the social conditions and immunological characteristics that allow some human immunodeficiency virus (HIV)-exposed patients to remain uninfected represents an on-going challenge. In this study, the socio-demographic and sexual behaviour characteristics and immune activation profiles of uninfected individuals exposed to HIV-infected partners were investigated. A confidential and detailed questionnaire was administered and venous blood was tested using HIV-1/enzyme immunoassays, plasma HIV-1 RNA levels/bDNA and immunophenotyping/flow cytometry to determine the frequencies of CD4 and CD8 T cells expressing activation markers. The data analysis showed significant differences (p < 0.05) for immune parameters in individuals who were uninfected, albeit exposed to HIV-infected partners, compared with unexposed individuals. In particular, the exposed, uninfected individuals had a higher frequency (median, minimum-maximum) of CD4⁺HLA-DR⁺ (4.2, 1.8-6.1), CD8⁺HLA-DR⁺ (4.6, 0.9-13.7), CD4⁺CD45RO⁺ (27.5, 14.2-46.6), CD4⁺CD45RO⁺CD62L⁺ (46.7, 33.9-67.1), CD8⁺CD45RA⁺HLA-DR⁺ (12.1, 3.4-35.8) and CD8⁺CD45RO⁺HLA-DR⁺ (9.0, 3.2-14.8) cells, a decreased percentage of CD8⁺CD28⁺ cells (11.7, 4.5-24.0) and a lower cell-surface expression of Fcγ-R/CD16 on monocytes (56.5, 22.0-130.0). The plasma HIV-1 RNA levels demonstrated detectable RNA virus loads in 57% of the HIV-1⁺ female partners. These findings demonstrate an activation profile in both CD4 and CD8 peripheral T cells from HIV-1 exposed seronegative individuals of serodiscordant couples from a referral centre in Belo Horizonte, state of Minas Gerais.     

Mouse testes contain two size classes of actin mRNA that are differentially expressed during spermatogenesis.

Using several actin isotype-specific cDNA probes, we found actin mRNA of two size classes, 2.1 and 1.5 kilobases (kb), in extracts of polyadenylated and nonpolyadenylated RNA from sexually mature CD-1 mouse testes. Although the 2.1-kb sequence was present in both meiotic and postmeiotic testicular cell types, it decreased manyfold in late haploid cells. The 1.5-kb actin sequence was not detectable in meiotic pachytene spermatocytes (or in liver or kidney cells), but was present in round and elongating spermatids and residual bodies. To differentiate between the beta- and gamma-actin mRNAs, we isolated a cDNA, pMGA, containing the 3' untranslated region of a mouse cytoplasmic actin that has homology to the 3' untranslated region of a human gamma-actin cDNA but not to the 3' untranslated regions of human alpha-, beta-, or cardiac actins. Dot blot hybridizations with pMGA detected high levels of presumptive gamma-actin mRNA in pachytene spermatocytes and round spermatids, with lower amounts found in elongating spermatids. Hybridization with the 3' untranslated region of a rat beta-actin probe revealed that round spermatids contained higher levels of beta-actin mRNA than did pachytene spermatocytes or residual bodies. Both probes hybridized to the 2.1-kb actin mRNA but failed to hybridize to the 1.5-kb mRNA.     

Fluorescein-labeled oligonucleotides for real-time pcr: using the inherent quenching of deoxyguanosine nucleotides

Fluorescein-labeled oligonucleotide probes can be used to continuously monitor the polymerase chain reaction. Depending on the sequence, the fluorescence intensity of the probe is either increased or decreased by hybridization. The greatest effect is probe quenching by hybridization to amplicons containing deoxyguanosine nucleotides (Gs), giving a sequence-specific decrease in fluorescence as product accumulates. Quenching of the probes by Gs is position dependent. A 25% decrease in fluorescence of 5'-labeled probes was observed with a G at the first position of the 3'-dangling end. Additional Gs can increase quenching to about 40%. This change in fluorescence with hybridization allows real-time quantification and mutation detection with a simple single labeled probe. Quantification of the initial template copy number is possible by monitoring fluorescence at each cycle at a constant temperature. Mutation detection by Tm estimates from melting curve analysis for factor V Leiden, hemoglobin C, hemoglobin S, the thermolabile mutation of methylenetetrahydrofolate reductase, and the cystic fibrosis-associated deletion F508del is demonstrated. By using the inherent quenching of deoxyguanosine nucleotides in the amplicon, complicated probe designs involving internal quenching can be avoided.