Electromagnetic field exposure and health among RF plastic sealer operators

Operators of RF plastic sealers (RF operators) are an occupational category highly exposed to radiofrequency electromagnetic fields. The aim of the present study was to make an appropriate exposure assessment of RF welding and examine the health status of the operators. In total, 35 RF operators and 37 controls were included. The leakage fields (electric and magnetic field strength) were measured, as well as induced and contact current. Information about welding time and productivity was used to calculate time integrated exposure. A neurophysiological examination and 24 h ECG were also carried out. The participants also had to answer a questionnaire about subjective symptoms. The measurements showed that RF operators were exposed to rather intense electric and magnetic fields. The mean values of the calculated 6 min, spatially averaged E and H field strengths, in line with ICNIRP reference levels, are 107 V/m and 0.24 A/m, respectively. The maximum measured field strengths were 2 kV/m and 1.5 A/m, respectively. The induced current in ankles and wrists varied, depending on the work situation, with a mean value of 101 mA and a maximum measured value of 1 A. In total, 11 out of 46 measured RF plastic sealers exceeded the ICNIRP reference levels. RF operators, especially the ready made clothing workers had a slightly disturbed two-point discrimination ability compared to a control group. A nonsignificant difference between RF operators and controls was found in the prevalence of subjective symptoms, but the time integrated exposure parameters seem to be of importance to the prevalence of some subjective symptoms: fatigue, headaches, and warmth sensations in the hands. Further, RF operators had a significantly lower heart rate (24 h registration) and more episodes of bradycardia compared to controls.     

Synthesis and biological evaluation of 6-aryl-6H-pyrrolo[3,4-d]pyridazine derivatives: high-affinity ligands to the alpha 2 delta subunit of voltage gated calcium channels

A novel class of 6-aryl-6H-pyrrolo[3,4-d]pyridazine ligands for the alpha2delta subunit of voltage-gated calcium channels has been described. Substitutions in the aryl ring of the molecule were generally not tolerated, and resulted in diminished binding to the alpha2delta subunit. Modifications to the pyridazine ring revealed numerous permissive substitutions, and detailed SAR studies were carried out in this portion of the molecule. Replacement of the pyridazine ring methyl group with an aminomethyl functionality provided greatly improved potency over the initial lead. The initial lead compound displayed good rat pharmacokinetic properties, and was shown to be efficacious in the Chung model for neuropathic pain in rats.     

Quantitative mass spectrometric immunoassay of insulin like growth factor 1

Reported in this work are the development of mass spectrometric immunoassay (MSIA) devices and methods for the qualitative analysis of IGF-1 and -2, and the rigorous quantification of IGF-1 from human plasma. A method involving addition of SDS in moderate concentration to unfractionated plasma for disrupting IGF/IGFBP complexes was initially developed. The method is suitable for the direct extraction of the IGFs and subsequent mass spectrometric analysis. Rat plasma, containing IGF-1 that is mass shifted from human IGF-1, was used as an internal reference standard (IRS) for the quantification of IGF-1 directly from human plasma. A standard curve with linear dynamic range of at least 2 orders of magnitude was constructed from serially diluted IGF-1 standards containing equal amounts of rat plasma. Using the standard curve, IGF-1 levels in plasma samples from eight individuals were determined. The limit of detection for the IGF-1 MSIA was also evaluated and established to be approximately 15 pM. The assay is rapid and can be performed in parallel via high-throughput robotics processing. Furthermore, the mass spectrometry aspect of the developed IGF-1 immunoassay offers a new dimension in the ongoing study of IGF-1 and related diseases.     

Diverse substrate recognition mechanisms for rhomboids; thrombomodulin is cleaved by Mammalian rhomboids

The rhomboids are a recently discovered family of intramembrane proteases that are conserved across evolution. Drosophila was the first organism in which they were characterized, where at least Rhomboids 1-3 activate EGF receptor signaling by releasing the active forms of EGF-like growth factors. Subsequent work has begun to shed light on the role of these proteases in bacteria and yeast, but nothing is known about the function of rhomboids in vertebrates beyond evidence that the subclass of mitochondrial rhomboids is conserved. Here, we report that the anticoagulant cell-surface protein thrombomodulin is the first mammalian protein to be a rhomboid substrate in a cell culture assay. The thrombomodulin transmembrane domain (TMD) is cleaved only by vertebrate RHBDL2-like rhomboids. Thrombomodulin TMD cleavage is directed not by sequences within the TMD, as is the case with Spitz but by its cytoplasmic domain, which, at least in some contexts, is necessary and sufficient to determine cleavage by RHBDL2. These data suggest that thrombomodulin could be a physiological substrate for rhomboid. Moreover, the discovery of a second mode of substrate recognition by rhomboids implies mechanistic diversity in this family of intramembrane proteases.     

The role of OX40 ligand interactions in the development of the Th2 response to the gastrointestinal nematode parasite Heligmosomoides polygyrus

In these studies, we examined the effects of OX40 ligand (OX40L) deficiency on the development of Th2 cells during the Th2 immune response to the intestinal nematode parasite Heligmosomoides polygyrus. Elevations in IL-4 production and total and Ag-specific serum IgE levels were partially inhibited during both the primary and memory immune responses to H. polygyrus in OX40L(-/-) mice. The host-protective memory response was compromised in OX40L(-/-) mice, as decreased worm expulsion and increased egg production were observed compared with H. polygyrus-inoculated OX40L(+/+) mice. To further examine the nature of the IL-4 defect during priming, adoptively transferred DO11.10 T cells were analyzed in the context of the H. polygyrus response. Although Ag-specific T cell IL-4 production was reduced in the OX40L(-/-) mice following immunization with OVA peptide plus H. polygyrus, Ag-specific T cell expansion, cell cycle progression, CXCR5 expression, and migration were comparable between OX40L(+/+) and OX40L(-/-) mice inoculated with OVA and H. polygyrus. These studies suggest an important role for OX40/OX40L interactions in specifically promoting IL-4 production, as well as associated IgE elevations, in Th2 responses to H. polygyrus. However, OX40L interactions were not required for serum IgG1 elevations, increases in germinal center formation, and Ag-specific Th2 cell expansion and migration to the B cell zone.     

Charting the Drosophila neuropile: a strategy for the standardised characterisation of genetically amenable neurites

Insect neurons are individually identifiable and have been used successfully to study principles of the formation and function of neuronal circuits. In the fruitfly Drosophila, studies on identifiable neurons can be combined with efficient genetic approaches. However, to capitalise on this potential for studies of circuit formation in the CNS of Drosophila embryos or larvae, we need to identify pre- and postsynaptic elements of such circuits and describe the neuropilar territories they occupy. Here, we present a strategy for neurite mapping, using a set of evenly distributed landmarks labelled by commercially available anti-Fasciclin2 antibodies which remain comparatively constant between specimens and over developmental time. By applying this procedure to neurites labelled by three Gal4 lines, we show that neuritic territories are established in the embryo and maintained throughout larval life, although the complexity of neuritic arborisations increases during this period. Using additional immunostainings or dye fills, we can assign Gal4-targeted neurites to individual neurons and characterise them further as a reference for future experiments on circuit formation. Using the Fasciclin2-based mapping procedure as a standard (e.g., in a common database) would facilitate studies on the functional architecture of the neuropile and the identification of candiate circuit elements.     

A microscope-based screening platform for large-scale functional protein analysis in intact cells

A modular microscope-based screening platform, with applications in large-scale analysis of protein function in intact cells is described. It includes automated sample preparation, image acquisition, data management and analysis, and the genome-wide automated retrieval of bioinformatic information. The modular nature of the system ensures that it is rapidly adaptable to new biological questions or sets of proteins. Two automated functional assays addressing protein secretion and the integrity of the Golgi complex were developed and tested. This shows the potential of the system in large-scale, cell-based functional proteomic projects.     

Stage-specific inductive signals in the Drosophila neuroectoderm control the temporal sequence of neuroblast specification

One of the initial steps of neurogenesis in the Drosophila embryo is the delamination of a stereotype set of neural progenitor cells (neuroblasts) from the neuroectoderm. The time window of neuroblast segregation has been divided into five successive waves (S1-S5) in which subsets of neuroblasts with specific identities are formed. To test when identity specification of the various neuroblasts takes place and whether extrinsic signals are involved, we have performed heterochronic transplantation experiments. Single neuroectodermal cells from stage 10 donor embryos (after S2) were transplanted into the neuroectoderm of host embryos at stage 7 (before S1) and vice versa. The fate of these cells was uncovered by their lineages at stage 16/17. Transplanted cells adjusted their fate to the new temporal situation. Late neuroectodermal cells were able to take over the fate of early (S1/S2) neuroblasts. The early neuroectodermal cells preferentially generated late (S4/S5) neuroblasts, despite their reduced time of exposure to the neuroectoderm. Furthermore, neuroblast fates are independent from divisions of neuroectodermal progenitor cells. We conclude from these experiments that neuroblast specification occurs sequentially under the control of non-cell-autonomous and stage-specific inductive signals that act in the neuroectoderm.     

Conservation of intramembrane proteolytic activity and substrate specificity in prokaryotic and eukaryotic rhomboids

Rhomboid is an intramembrane serine protease responsible for the proteolytic activation of Drosophila epidermal growth factor receptor (EGFR) ligands. Although nothing is known about the function of the approximately 100 currently known rhomboid genes conserved throughout evolution, a recent analysis suggests that a Rhomboid from the pathogenic bacterium Providencia stuartii is involved in the production of a quorum-sensing factor. This suggests that an intercellular signaling mechanism may have been conserved between prokaryotes and metazoans. However, the function of prokaryotic Rhomboids is unknown. We have examined the ability of eight prokaryotic Rhomboids to cleave the three Drosophila EGFR ligands. Despite their striking sequence divergence, Rhomboids from one Gram-positive and four Gram-negative species, including Providencia, specifically cleaved Drosophila substrates, but not similar proteins such as Transforming Growth Factor alpha (TGFalpha) and Delta. Although the sequence similarity between these divergent Rhomboids is very limited, all contain the putative serine catalytic triad residues, and their specific mutation abolished protease activity. Therefore, despite low overall homology, the Rhomboids are a family of ancient, functionally conserved intramembrane serine proteases, some of which also have conserved substrate specificity. Moreover, a function for Rhomboids in activating intercellular signaling appears to have evolved early.