2.45 GHz radiofrequency fields alter gene expression in cultured human cells

The biological effect of radiofrequency (RF) fields remains controversial. We address this issue by examining whether RF fields can cause changes in gene expression. We used the pulsed RF fields at a frequency of 2.45 GHz that is commonly used in telecommunication to expose cultured human HL-60 cells. We used the serial analysis of gene expression (SAGE) method to measure the RF effect on gene expression at the genome level. We observed that 221 genes altered their expression after a 2-h exposure. The number of affected genes increased to 759 after a 6-h exposure. Functional classification of the affected genes reveals that apoptosis-related genes were among the upregulated ones and the cell cycle genes among the downregulated ones. We observed no significant increase in the expression of heat shock genes. These results indicate that the RF fields at 2.45 GHz can alter gene expression in cultured human cells through non-thermal mechanism.     

Inhibition of HIV-1 envelope glycoprotein-mediated cell fusion by a DL-amino acid-containing fusion peptide: possible recognition of the fusion complex

The N-terminal fusion peptide (FP) of human immunodeficiency virus-1 (HIV-1) is a potent inhibitor of cell-cell fusion, possibly because of its ability to recognize the corresponding segments inside the fusion complex within the membrane. Here we show that a fusion peptide in which the highly conserved Ile(4), Phe(8), Phe(11), and Ala(14) were replaced by their d-enantiomers (IFFA) is a potent inhibitor of cell-cell fusion. Fourier transform infrared spectroscopy confirmed that despite these drastic modifications, the peptide preserved most of its structure within the membrane. Fluorescence energy transfer studies demonstrated that the diastereomeric peptide interacted with the wild type FP, suggesting this segment as the target site for inhibition of membrane fusion. This is further supported by the similar localization of the wild type and IFFA FPs to microdomains in T cells and the preferred partitioning into ordered regions within sphingomyelin/phosphatidyl-choline/cholesterol giant vesicles. These studies provide insight into the mechanism of molecular recognition within the membrane milieu and may serve in designing novel HIV entry inhibitors.     

Hetero-assembly between all-L- and all-D-amino acid transmembrane domains: forces involved and implication for inactivation of membrane proteins

Protein-protein interactions within the membrane, partially or fully mediated by transmembrane (TM) domains, are involved in many vital cellular processes. Since the unique feature of the membrane environment enables protein-protein assembly that otherwise is not energetically favored in solution, the structural restrictions involved in the assembly of soluble proteins are not necessarily valid for the assembly of TM domains. Here we used the N-terminal TM domain (Tar-1) of the Escherichia coli aspartate receptor as a model system for examining the stereospecificity of TM-TM interactions in vitro and in vivo in isolated systems, and in the context of the full receptor. For this propose, we synthesized Tar-1 all-l and all-d amino acid TM peptides, a mutant TM peptide and an unrelated TM peptide. The data revealed: (i) Tar-1 all-d specifically associated with Tar-1 all-l within a model lipid membrane, as determined by using fluorescence energy transfer experiments; (ii) Tar-1 all-l and all-d, but not the control peptides, demonstrated a dose-dependant dominant negative effect on the Tar-1 TM homodimerization in the bacterial ToxR assembly system, suggesting a wild-type-like interaction; and most interestingly, (iii) both Tar-1 all-l and all-d showed a remarkable ability to inhibit the chemotaxis response of the full-length receptor, in vivo. Peptide binding to the bacteria was confirmed through confocal imaging, and Western blotting confirmed that ToxR Tar-1 chimera protein levels are not affected by the presence of the exogenous peptides. These findings present the first evidence that an all-d TM domain peptide acts in vivo similarly to its parental all-l peptide and suggest that the dimerization of the TM domains is mainly mediated by side-chain interactions, rather than geometrically fitted conformations. In addition, the study provides a new approach for modifying the function of membrane proteins by proteolysis-free peptides.     

Clindamycin is neuroprotective in experimental Streptococcus pneumoniae meningitis compared with ceftriaxone

In animal models of Streptococcus pneumoniae meningitis, rifampin is neuroprotective in comparison to ceftriaxone. So far it is not clear whether this can be generalized for other protein synthesis-inhibiting antimicrobial agents. We examined the effects of the bactericidal protein synthesis-inhibiting clindamycin (n = 12) on the release of proinflammatory bacterial components, the formation of neurotoxic compounds and neuronal injury compared with the standard therapy with ceftriaxone (n = 12) in a rabbit model of pneumococcal meningitis. Analysis of the CSF and histological evaluation were combined with microdialysis from the hippocampal formation and the neocortex. Compared with ceftriaxone, clindamycin reduced the release of lipoteichoic acids from the bacteria (p = 0.004) into the CSF and the CSF leucocyte count (p = 0.011). This led to lower extracellular concentrations of hydroxyl radicals (p = 0.034) and glutamate (p = 0.016) in the hippocampal formation and a subsequent reduction of extracellular glycerol levels (p = 0.018) and neuronal apoptosis in the dentate gyrus (p = 0.008). The present data document beneficial effects of clindamycin compared with ceftriaxone on various parameters linked with the pathophysiology of pneumococcal meningitis and development of neuronal injury. This study suggests neuroprotection to be a group effect of bactericidal protein synthesis-inhibiting antimicrobial agents compared with the standard therapy with beta-lactam antibiotics in meningitis.     

Optimization and use of peptide mass measurement accuracy in shotgun proteomics

Mass spectrometers that provide high mass accuracy such as FT-ICR instruments are increasingly used in proteomic studies. Although the importance of accurately determined molecular masses for the identification of biomolecules is generally accepted, its role in the analysis of shotgun proteomic data has not been thoroughly studied. To gain insight into this role, we used a hybrid linear quadrupole ion trap/FT-ICR (LTQ FT) mass spectrometer for LC-MS/MS analysis of a highly complex peptide mixture derived from a fraction of the yeast proteome. We applied three data-dependent MS/MS acquisition methods. The FT-ICR part of the hybrid mass spectrometer was either not exploited, used only for survey MS scans, or also used for acquiring selected ion monitoring scans to optimize mass accuracy. MS/MS data were assigned with the SEQUEST algorithm, and peptide identifications were validated by estimating the number of incorrect assignments using the composite target/decoy database search strategy. We developed a simple mass calibration strategy exploiting polydimethylcyclosiloxane background ions as calibrant ions. This strategy allowed us to substantially improve mass accuracy without reducing the number of MS/MS spectra acquired in an LC-MS/MS run. The benefits of high mass accuracy were greatest for assigning MS/MS spectra with low signal-to-noise ratios and for assigning phosphopeptides. Confident peptide identification rates from these data sets could be doubled by the use of mass accuracy information. It was also shown that improving mass accuracy at a cost to the MS/MS acquisition rate substantially lowered the sensitivity of LC-MS/MS analyses. The use of FT-ICR selected ion monitoring scans to maximize mass accuracy reduced the number of protein identifications by 40%.     

Activation of a novel long-chain free fatty acid generation and export system in mitochondria of diabetic rat hearts

A number of reports indicate that a long-chain free fatty acid export system may be operating in mitochondria. In this study, we sought evidence of its existence in rat heart mitochondria. To determine its potential role, we also sought evidence of its activation or inhibition in streptozotocin (STZ)-induced diabetic rat heart mitochondria. If confirmed, it could be a novel mechanism for regulation of long-chain fatty acid oxidation (FAO) in mitochondria. To obtain evidence of its existence, we tested whether heart mitochondria presented with palmitoyl-carnitine can generate and export palmitate. We found that intact mitochondria indeed generate and export palmitate. We have also found that the rates of these processes are markedly higher in STZ-diabetic rat heart mitochondria, in which palmitoyl-carnitine oxidation is also increased. Since mitochondrial thioesterase-1 (MTE-1) hydrolyzes acyl-CoA to CoA-SH + free fatty acid, and uncoupling protein-3 (UCP-3), reconstituted in liposomes, transports free fatty acids, we examined whether these proteins are also increased in STZ-diabetic rat heart mitochondria. We found that both of these proteins are indeed increased. Gene expression profile analysis revealed striking expression of mitochondrial long-chain fatty acid transport and oxidation genes, accompanying overexpression of MTE-1 and UCP-3 in STZ-diabetic rat hearts. Our findings provide the first direct evidence for the existence of a long-chain free fatty acid generation and export system in mitochondria and its activation in STZ-diabetic rat hearts in which FAO is enhanced. We suggest that its activation may facilitate, and inhibition may limit, enhancement of FAO. fatty acid oxidation; diabetes; lipotoxic cardiomyopathy; gene array     

Development of a spectrophotometric assay for cyclase activity

We describe the development of a rapid colorimetric assay for soluble guanylate cyclase (sGC) activity adapted for a 96-well microplate. The assay greatly decreases the analysis time and cost over traditional methodologies based on radio- and immunoassays and high-performance liquid chromatography (HPLC) separations. The method does not demonstrate any significant interference with chemicals commonly used for sGC purification and reaction kinetics. The assay converts the inorganic pyrophosphate produced in the cyclase reaction to inorganic phosphate, which is then measured using a modified Fiske-Subbarow assay. We used the assay to compare the reaction kinetics of preparations of sGC from a commercial source with those from our lab with Mg(2+)-guanosine 5'-triphosphate (GTP) or Mn(2+)-GTP as a substrate. The commercial preparation was found to have a specific activity of around 1.5 micromol/min/mg, which is significantly lower than expected, as was the fold-activation upon addition of nitric oxide (NO). Our laboratory preparation had a higher specific activity that was consistent with results from HPLC assays. We determined that the human isoform of sGC is more active in the basal and NO forms with Mn(2)-GTP as a substrate than Mg(2+)-GTP, a feature more similar to rat lung sGC than the more commonly studied bovine lung.     

Determination of functional strength imbalance of the lower extremities

The purposes of this study were (a) to determine whether a significant strength imbalance existed between the left and right or dominant (D) and nondominant (ND) legs and (b) to investigate possible correlations among various unilateral and bilateral closed kinetic chain tests, including a field test, and traditional isokinetic dynamometry used to determine strength imbalance. Fourteen Division I collegiate women softball players (20.2 +/- 1.4 years) volunteered to undergo measures of average peak torque for isokinetic flexion and extension at 60 degrees .s(-1) and 240 degrees .s(-1); in addition, measures of peak and average force of each leg during parallel back squat, 2-legged vertical jump, and single-leg vertical jump and performance in a 5-hop test were examined. Significant differences of between 4.2% and 16.0% were evident for all measures except for average force during single-leg vertical jump between the D and ND limbs, thus revealing a significant strength imbalance. The 5-hop test revealed a significant difference between D and ND limbs and showed a moderate correlation with more sophisticated laboratory tests, suggesting a potential use as a field test for the identification of strength imbalance. The results of this study indicate that a significant strength imbalance can exist even in collegiate level athletes, and future research should be conducted to determine how detrimental these imbalances could be in terms of peak performance for athletes, as well as the implications for injury risk.