No effects of intermittent 50 Hz EMF on cytoplasmic free calcium and on the mitochondrial membrane potential in human diploid fibroblasts

The recently described increase in DNA strand breaks of cultured human diploid fibroblasts after intermittent exposure to extremely-low-frequency electromagnetic fields (ELF-EMF) of more than about 70 µT ELF-EMF is difficult to explain by a direct induction of covalent bond disruption. Therefore the hypothesis has been tested that ELF-EMF-induced DNA strand breaks might be mediated by cellular processes that cause alteration of the intracellular concentration of free calcium ([Ca²⁺]_i) and/or the membrane potential (_m). [Ca²⁺]_i was determined by the ratiometric fura-2 technique. Changes in _m were assessed by using the potential-dependent lipophilic cationic probe JC-1. Human fibroblasts were exposed to intermittent ELF-EMF (50 Hz, 1000 µT). Although exposure of fiboblasts to ELF-EMF resulted in a highly significant increase in DNA strand breaks as determined by the comet assay, no effect on JC-1 fluorescence emission or on [Ca²⁺]_i has been observed when comparing exposed with sham-exposed cells. Therefore, it is suggested that ELF-EMF-induced DNA strand breaks are unlikely to be caused by intracellular changes that affect [Ca²⁺]_i and/or _m.     

Semi-synthetic salinomycin analogs exert cytotoxic activity against human colorectal cancer stem cells

Salinomycin, a polyether antibiotic, is a well-known inhibitor of human cancer stem cells. Chemical modification of the allylic C20 hydroxyl of salinomycin has enabled access to synthetic analogs that display increased cytotoxic activity compared to the native structure. The aim of this study was to investigate the activity of a cohort of C20-O-acyl analogs of salinomycin on human colorectal cancer cell lines in vitro. Two human colorectal cancer cell lines (SW480 and SW620) were exposed to three C20-O-acylated analogs and salinomycin. The impact of salinomycin and its analogs on tumor cell number, migration, cell death, and cancer stem cell specifity was analyzed. Exposure of human colorectal cancer cells to the C20-O-acylated analogs of salinomycin resulted in reduced tumor cell number and impaired tumor cell migration at lower concentrations than salinomycin. When used at higher (micromolar) concentrations, these effects were accompanied by induction of apoptotic cell death. Salinomycin analogs further expose improved activity against cancer stem cells compared to salinomycin.     

Crystallization and preliminary characterization of CheY, a chemotaxis control protein from Escherichia coli

Single crystals of the 14.1-kDa cheY gene product from Escherichia coli have been grown from buffered ammonium sulfate solutions using the combined methods of microdialysis and pulsed diffusion. The crystals are of the monoclinic space group P2(l), have cell constants of a = 51.4 A, b = 112 A, c = 51.2 A, and beta = 107.3 degrees, and contain four molecules per asymmetric unit. They are stable to x-ray radiation and diffract beyond 3.0 A resolution.     

Mitochondrial DNA evolution in theobscura species subgroup ofDrosophila

Summary Mitochondrial DNA (mtDNA) restriction site maps for nine species of theDrosophila obscura subgroup and forDrosophila melanogaster were established. Taking into account all restriction enzymes (12) and strains (45) analyzed, a total of 105 different sites were detected, which corresponds to a sample of 3.49% of the mtDNA genome. Based on nucleotide divergences, two phylogenetic trees were constructed assuming either constant or variable rates of evolution. Both methods led to the same relationships. Five differentiated clusters were found for theobscura subgroup species, one Nearctic, represented byDrosophila pseudoobscura, and four Palearctic, two grouping the related triads of speciesDrosophila subobscura, Drosophila madeirensis, Drosophila guanche, andDrosophila ambigua, Drosophila obscura, Drosophila subsilvestris, and two more represented by one species each,Drosophila bifasciata, andDrosophila tristis. The different Palearctic clusters are as distant between themselves as with the Nearctic one. For the related speciesD. subobscura, D. madeirensis, andD. guanche, the pairD. subobscura-D. madeirensis is the closest one. The relationships found by nucleotide divergence were confirmed by differences in mitochondrial genome size, with related species sharing similar genome lengths and differing from the distant ones. The total mtDNA size range for theobscura subgroup species was from 15.5 kb forD. pseudoobscura to 17.1 forD. tristis.     

A new method for the histochemical demonstration of O-acyl sugars in human colonic epithelial glycoproteins

Summary A new general method has been developed for the specific histochemical identification ofO-acyl sugars in any epithelial glycoprotein. These sugars include hexose, 6-deoxyhexose andN-acetylhexosamine with an ester substituenent(s) located on a potentialvicinal diol(s). In the procedure reported [the periodic acid-borohydride reduction-saponification-selective periodate oxidation-borohydride reduction-periodic acid-Schiff (PA-Bh-KOH-PA-Bh*-Bh-PAS) method] the initial PA-Bh treatment rendersvicinal diols located on either sialic acid or neutral sugars PAS unreactive. In the subsequent steps ester substituents are removed from bothO-acyl sugars andO-acyl sialic acids by saponification (KOH), sialic acidvicinal diols are selectively removed by the PA^*-Bh sequence andO-acyl sugars are stained with the PAS technique. This method has the advantage that the results are obtained with a single section and the results are either positive or negative. Consequently, it is superior to the three indirect methods investigated because it does not require an observer to compare the intensity or the shade of the staining obtained with serial sections.Using the PA-Bh-KOH-PA^*-Bh-PAS method we have demonstrated, for the first time, thatO-acyl sugars occur in the epithelial goblet cell glycoproteins of adult human colon. The effect of the presence ofO-acyl sugars on the interpretation of a number of other methods for the histochemical investigation of glycoproteins is discussed. It is recommended that the results obtained with the PA-Bh-KOH-PA^*-Bh-PAS method be evaluated before histochemical procedures for the investigation of neutral sugars andO-acyl sialic acids are selected.     

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Genetic architecture of fruit quality traits in Malus x domestica (Borkh.) compared between own-rooted seedlings and vegetative propagules on ��M. 9�� rootstock

The comparative performance of vegetative propagules (VP) grafted on ‘M. 9’ rootstock and own-rooted (OR) seedlings of the same genotypes was investigated for apple [Malus x domestica (Borkh.)] fruit quality traits using offspring from 22 control-pollinated families. Fifteen fruit quality traits including fruit size and shape, skin colour, russet and sensory eating characteristics were evaluated for individual seedlings. Estimates of genetic variance–covariance matrices (G) were obtained and compared between the two treatments (OR versus VP) using Mantel’s test and regression approaches. Empirical estimates of correlations, at the individual-tree and family-mean levels, between the two treatments were compared with those obtained from deterministic simulations. Estimates of narrow-sense heritability (h ²) were higher in the VP population than in the OR population for 11 of the 15 traits. The estimated G matrices were significantly different between the two treatments. Simulation study showed that with an increase in the total genetic variance, the correspondence between the two treatments at the family-mean level improved dramatically for different family sizes. As the ratio of dominance/additive variance increased from 0 to 1, the family-mean correlations between the two treatments decreased for all family sizes. The average estimated correlations between the two treatments at the family-mean level were higher than at the individual-tree level (0.78 and 0.42, respectively). These observed correlations were very similar to our theoretical expectations. In the light of these results, caution is required when comparing apple seedlings tested on their own roots with those tested on ‘M. 9’ rootstock, as potential cultivar breeding parents.     

Synthesis and pharmacological evaluation of coumarin derivatives as cannabinoid receptor antagonists and inverse agonists

In the present study we synthesized 36 coumarin and 2H-chromene derivatives applying a recently developed umpoled domino reaction using substituted salicylaldehyde and α,β-unsaturated aldehyde derivatives as starting compounds. In radioligand binding studies 5-substituted 3-benzylcoumarin derivatives showed affinity to cannabinoid CB₁ and CB₂ receptors and were identified as new lead structures. In further GTPγS binding studies selected compounds were shown to be antagonists or inverse agonists.     

Characteristics of tumor necrosis factor production in rheumatoid arthritis

The biological effects of tumor necrosis factor (TNF) include the enhancement of fibroblast proliferation, the secretion of collagenase and prostaglandin E2 (PGE2) by fibroblasts, and the resorption of bone and cartilage, suggesting a role for this cytokine in arthritic conditions. To investigate this, we measured the levels of TNF in synovial fluids and evaluated its secretion by synovial fluid mononuclear cells and tissues from patients with rheumatoid arthritis, osteoarthritis, and seronegative arthritis and normals. TNF was found to be secreted in all arthritic conditions but not in normals. The levels of TNF were highest in synovial fluid and correlated with interferon-gamma (IFN-gamma) levels but not PGE2. The production of TNF was stable in a single joint for 3 to 6 months. Using immunohistochemical staining, TNF was localized to mononuclear cells in the lining layer, sublining, and perivascular areas of synovial tissue. The secretion of TNF by rheumatoid synovial fluid mononuclear cells was inhibited by PGE2, while IFN-gamma enhanced its production in those cells which were spontaneously secreting TNF. Our data suggest that TNF may play a role in various arthritic diseases.