Proteomics of human primary osteoarthritic chondrocytes exposed to extremely low-frequency electromagnetic fields (ELF EMFs) and to therapeutic application of musically modulated electromagnetic fields (TAMMEF).

Osteoarthritis (OA) is the most frequent joint disease, characterized by degradation of extracellular matrix and alterations in chondrocyte metabolism. Some authors reported that electromagnetic fields (EMFs) can positively interfere with patients affected by OA, even though the nature of the interaction is still debated. Human primary osteoarthritic chondrocytes isolated from the femoral heads of OA-patients undergoing to total hip replacement, were cultured in vitro and exposed 30 min/day for two weeks to extremely-low-frequency electromagnetic field (ELF) with fixed frequency (100 Hz) and to therapeutic application of musically modulated electromagnetic fields (TAMMEF) with variable frequencies, intensities and waveforms. Sham-exposed (S.E.) cells served as control group. Cell viability was measured at days 2, 7 and 14. After two weeks, cell lysates were processed using a proteomic approach. Chondrocyte exposed to ELF and TAMMEF system demonstrated different viability compared to untreated chondrocytes (S.E.). Proteome analysis of 2D-Electrophoresis and protein identification by mass spectrometry showed different expression of proteins derived from nucleus, cytoplasm and organelles. Function analysis of the identified proteins showed changes in related-proteins metabolism (glyceraldeyde-3-phosphate-dehydrogenase), stress response (Mn-superoxide-dismutase, heat-shock proteins), cytoskeletal regulation (actin), proteinase inhibition (cystatin-B) and inflammation regulatory functions (S100-A10, S100-A11) among the experimental groups (ELF, TAMMEF and S.E.). In conclusion, EMFs do not cause damage to chondrocytes, besides stimulate safely OA-chondrocytes and are responsible of different protein expression among the three groups. Furthermore, protein analysis of OA-chondrocytes treated with ELF and the new TAMMEF systems could be useful to clarify the pathogenetic mechanisms of OA by identifying biomarkers of the disease.

Human osteoarthritic chondrocytes exposed to extremely low-frequency electromagnetic fields (ELF) and therapeutic application of musically modulated electromagnetic fields (TAMMEF) systems: a comparative study.

Osteoarthritis (OA) is the most common joint disease, characterized by matrix degradation and changes in chondrocyte morphology and metabolism. Literature reported that electromagnetic fields (EMFs) can produce benefits in OA patients, even if EMFs mechanism of action is debated. Human osteoarthritic chondrocytes isolated from femoral heads were cultured in vitro in bidimensional (2-D) flasks and in three-dimensional (3-D) alginate beads to mimic closely cartilage environment in vivo. Cells were exposed 30 min/day for 2 weeks to extremely low-frequency electromagnetic field (ELF) with fixed frequency (100 Hz) and to therapeutic application of musically modulated electromagnetic field (TAMMEF) with variable frequencies, intensities, and waveforms. Cell viability was measured at days 7 and 14, while healthy-cell density, heavily vacuolized (hv) cell density, and cluster density were measured by light microscopy only for 3-D cultures after treatments. Cell morphology was observed for 2-D and 3-D cultures by transmission electron microscopy (TEM). Chondrocyte exposure to TAMMEF enhances cell viability at days 7 and 14 compared to ELF. Light microscopy analysis showed that TAMMEF enhances healthy-cell density, reduces hv-cell density and clustering, compared to ELF. Furthermore, TEM analysis showed different morphology for 2-D (fibroblast-like) and 3-D (rounded shape) cultures, confirming light microscopy results. In conclusion, EMFs are effective and safe for OA chondrocytes. TAMMEF can positively interfere with OA chondrocytes representing an innovative non-pharmacological approach to treat OA.

Analysis of aqueous humour proteins in patients with retinoblastoma.

BACKGROUND: To investigate aqueous humour protein composition from retinoblastoma patients. DESIGN: Prospective, hospital-based study. PARTICIPANTS: Eighteen retinoblastoma patients (Reese-Ellsworth stage V or ABC classification group E RB) undergoing ocular enucleation, and 10 normal subjects undergoing cataract surgery. Five of 18 patients presented with associated secondary glaucoma whereas 13 had no secondary glaucoma; 5 of 13 patients with no secondary glaucoma received chemotherapeutical treatment with melphalan. METHODS: Aqueous humour samples were collected by limbal paracentesis of the anterior chamber after ocular enucleation in patients and after the stab peripheral corneal incision in controls. Total protein concentration according to Bradford method and sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the samples were performed. MAIN OUTCOME MEASURE: Aqueous humour protein concentration. RESULTS: Aqueous humour protein concentration was significantly higher in retinoblastoma patients than controls (P < 0.01); patients with secondary glaucoma presented the highest values (P < 0.05 vs. controls); patients treated with melphalan presented a significant decrease (P < 0.01) versus non-treated; controls did not significantly differ from treated patients. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis pattern in retinoblastoma patients who did not receive any treatment was very different either from treated or from controls. CONCLUSION: This study represents a preliminary step towards a more accurate two dimensional electrophoresis (2DE) pattern, which will be combined with mass spectrometry analysis to clarify the potential role of specific proteins in tumour development and progression; although these results suggest that aqueous humour protein pattern in retinoblastoma is characteristic, several aspects of the study are still under investigation.

Alterations in adenylate kinase activity in human PBMCs after in vitro exposure to electromagnetic field: comparison between extremely low frequency electromagnetic field (ELF) and therapeutic application of a musically modulated electromagnetic field (TAMMEF).

This study investigated the effects of electromagnetic fields on enzymes involved in purine metabolism in human peripheral blood mononuclear cells in vitro. Cells were obtained from 20 volunteers. We tested both low-energy, extremely low frequency (ELF; 100-Hz) electromagnetic fields and the Therapeutic Application of Musically Modulated Electromagnetic Fields (TAMMEFs); the latter is characterized by variable frequencies, intensities, and wave shapes. Adenylate kinase activity was increased after ELF field exposure but decreased slightly after TAMMEF exposure. Neither of the two electromagnetic field affected the activities of the purine metabolism enzymes ecto-5'-nucleotidase, adenosine deaminase, and adenosine kinase. We concluded that ELF fields may influence cellular electrical charge stability; stimulation of adenylate kinase activity could restore the cell to a state of equilibrium. In contrast, TAMMEF fields may be useful for maintaining and regulating the cellular electrical charge.

Evidence of a new phosphoryl transfer system in nucleotide metabolism.

Crude rat liver extract showed AMP-AMP phosphotransferase activity which, on purification, was ascribed to a novel interaction between adenylate kinase, also known as myokinase (EC 2.7.4.3), and adenosine kinase (EC 2.7.1.20). The activity was duplicated using the same enzymes purified from recombinant sources. The reaction requires physical contact between myokinase and adenosine kinase, and the net reaction is aided by the presence of adenosine deaminase (EC 3.5.4.4), which fills the gap in the energy balance of the phosphoryl transfer and shifts the equilibrium towards ADP and inosine synthesis. The proposed mechanism involves the association of adenosine kinase and myokinase through non-covalent, transient interactions that induce slight conformational changes in the active site of myokinase, bringing two already bound molecules of AMP together for phosphoryl transfer to form ADP. The proposed mechanism suggests a physiological role for the enzymes and for the AMP-AMP phosphotransferase reaction under conditions of extreme energy drain (such as hypoxia or temporary anoxia, as in cancer tissues) when the enzymes cannot display their conventional activity because of substrate deficiency.

Structure and function correlations between the rat liver threonine deaminase and aminotransferases.

The rat liver threonine deaminase is a cytoplasmic enzyme that catalyses the pyridoxal-phosphate-dependent dehydrative deamination of L-threonine and L-serine to ammonia and alpha-ketobutyrate and pyruvate, respectively, in vivo. During deamination, a molecule of the cofactor is converted to pyridoxamine phosphate. Recently, the ability of this enzyme to accomplish an inverse half-reaction, restoring pyridoxal-phosphate and L-alanine or L-aminobutyrate, respectively, from pyruvate or 2-oxobutyrate, was reported. In order to investigate the molecular mechanisms of this transaminating activity, a molecular model of rat liver threonine deaminase was constructed on the basis of sequence homology with the biosynthetic threonine deaminase of Escherichia coli, the crystal structure of which is known. The model has structural features shared by aminotransferases, suggesting that tertiary structural elements may be responsible for the transaminating activity observed for rat liver threonine deaminase.