Mutagenicity,sister chromatid exchange inducibility andin vitro cell transforming ability of particulates from Athens air

Airborne particulates were collected over a period of twelve months by the use of Hi-Vol samplers in the basin of Athens, Greece. N-Hexane extracts were tested in a battery ofin vitro tests for their ability to induce mutation in bacteria as well as mutation, sister chromatid exchange and morphological transformation in cultured mammalian cells. Positive results were found for mutagenicity withSalmonella strain TA98 in the Ames assay, for sister chromatid exchange induction in CHO cells and for transformation in BALB/c 3T3 cells in culture. They also showed weak non-doserelated induction of ouabain resistance in BALB/c 3T3 cells. The contribution of oxidizing and nitrating agents found in the Athens atmosphere, together with sunlight UV irradiation in the formation of direct acting mutagens and potential carcinogens from ambient polycyclic aromatic hydrocarbons, is suggested.Abbreviations FCS fetal calf serum - FPG fluorescent-plus-Giemsa technique - oua^R ouabain resistant - PAH polycyclic aromatic hydrocarbon - SCE sister chromatid exchange - TSP total suspended particulate     

Paradoxical activation of Leptomonas NAD-linked alpha-glycerophosphate dehydrogenase by ethidium and antrycide.

Antrycide and ethidium bromide — 2 cationic trypanocides — inhibited NAD-linked α-glycerophosphate dehydrogenase from $\text{Leptomonas}$ sp. The kinetics of enzyme inhibition was determined by Lineweaver-Burk, Dixon, or direct linear plots. Inhibition by Antrycide was noncompetitive for dihydroxyacetone phosphate in the presence of saturating Mg²⁺ or spermidine. With dihydroxyacetone phosphate at saturation, Antrycide inhibition was also noncompetitive with respect to Mg²⁺ (K_i = 115 μM) and spermidine (K_i = 85 μM). Inhibition by ethidium in the presence of saturating dihydroxyacetone phosphate, was noncompetitive for Mg²⁺ (K_i = 400 μM) but mixed for spermidine (K_i = 495 μM); inhibition was noncompetitive for dihydroxyacetone phosphate in the presence of saturating Mg²⁺ or spermidine. Rabbit-muscle α-glycerophosphate dehydrogenase was inhibited at all concentrations of Antrycide and ethidium tested, but the $\text{Leptomonas}$ enzyme was stimulated up to 3.5-fold by low concentrations of inhibitors in the absence of polyamine. New chemotherapeutic possibilities may thus be opened and an evolutionary distinction between trypanosomatid and mammalian enzyme.     

Characterization and localization of short-specific polygalacturonase in distylous Turnera subulata (Turneraceae)

We describe for distylous Turnera subulata a polygalacturonase specific to short-styled plants that is localized to the style transmitting tissue (the tissue through which pollen tubes grow). The polygalacturonase gene is linked to and may be upregulated by the S allele of the distyly locus. Because of its tissue-specific location, the polygalacturonase may be involved in the self-incompatibility response, acting in a complementary or antagonistic manner, or possibly in signalling downstream events. A pollen-specific polygalacturonase was also identified and may be a member of a small multigene family of pollen polygalacturonases. The role, if any, played by the pollen polygalacturonase in distyly, is presently unknown.     

DNA damage and apoptosis in hydrogen peroxide-exposed Jurkat cells: bolus addition versus continuous generation of H(2)O(2)

Aspects of the molecular mechanism(s) of hydrogen peroxide-induced DNA damage and cell death were studied in the present investigation. Jurkat T-cells in culture were exposed either to low rates of continuously generated H(2)O(2) by the action of glucose oxidase or to a bolus addition of the same agent. In the first case, steady state conditions were prevailing, while in the latter, H(2)O(2) was removed by the cellular defense systems following first order kinetics. By using single-cell gel electrophoresis (also called comet assay), an initial increase in the formation of DNA single-strand breaks was observed in cells exposed to a bolus of 150 microM H(2)O(2). As the H(2)O(2) was exhausted, a gradual decrease in DNA damage was apparent, indicating the existence of an effective repair of single-strand breaks. Addition of 10 ng glucose oxidase in 100 microl growth medium (containing 1.5 x 10(5) cells) generated 2.0 +/- 0.2 microM H(2)O(2) per min. This treatment induced an increase in the level of single-strand breaks reaching the upper limit of detection by the methodology used and continued to be high for the following 6 h. However, when a variety of markers for apoptotic cell death (DNA cell content, DNA laddering, activation of caspases, PARP cleavage) were examined, only bolus additions of H(2)O(2) were able to induce apoptosis, while the continuous presence of this agent inhibited the execution of the apoptotic process no matter whether the inducer was H(2)O(2) itself or an anti-Fas antibody. These observations stress that, apart from the apparent genotoxic and proapoptotic effects of H(2)O(2), it can also exert antiapoptotic actions when present, even at low concentrations, during the execution of apoptosis.     

Noninvasive genetic studies of brown bears using power poles

One difficulty in the conservation of endangered wildlife is the lack of reliable information on its status. This lack of knowledge can often be attributed to financial and logistic constraints as well as the lack of trained personnel to collect data. We test a simple method to study bears in the southern Balkans by inspecting power poles, which are used by bears for marking and rubbing purposes. We created a network of barbed-wire fitted poles for the collection of hair samples, evenly distributed throughout six study areas. During 87 sampling sessions in the main study area, we collected 191 samples and identified six microsatellite loci that were variable enough for individual bear identification. The most and best-quality hair samples were collected during the mating period, and DNA was most successfully extracted from samples remaining <4 weeks in the field. In the six study areas, we identified 47 bears. An advantage of using power poles for hair sampling is their availability and accessibility; no bait is required, and the network can be easily set up. A drawback may be an unequal capture probability of sex and age classes of bears. Despite this limitation, using power poles proved to be a simple and cheap method for the noninvasive genetic study of bears that did not require any prior knowledge on habitat use and activity patterns. The method is suitable for large-scale surveys to estimate distribution and relative densities of bears and could also be applied for studying other species.     

Biomechanics of single chondrocytes under direct shear

Articular chondrocytes experience a variety of mechanical stimuli during daily activity. One such stimulus, direct shear, is known to affect chondrocyte homeostasis and induce catabolic or anabolic pathways. Understanding how single chondrocytes respond biomechanically and morphologically to various levels of applied shear is an important first step toward elucidating tissue level responses and disease etiology. To this end, a novel videocapture method was developed in this study to examine the effect of direct shear on single chondrocytes, applied via the controlled lateral displacement of a shearing probe. Through this approach, precise force and deformation measurements could be obtained during the shear event, as well as clear pictures of the initial cell-to-probe contact configuration. To further study the non-uniform shear characteristics of single chondrocytes, the probe was positioned in three different placement ranges along the cell height. It was observed that the apparent shear modulus of single chondrocytes decreased as the probe transitioned from being close to the cell base (4.1 ± 1.3 kPa), to the middle of the cell (2.6 ± 1.1 kPa), and then near its top (1.7 ± 0.8 kPa). In addition, cells experienced the greatest peak forward displacement (~30% of their initial diameter) when the probe was placed low, near the base. Forward cell movement during shear, regardless of its magnitude, continued until it reached a plateau at ~35% shear strain for all probe positions, suggesting that focal adhesions become activated at this shear level to firmly adhere the cell to its substrate. Based on intracellular staining, the observed height-specific variation in cell shear stiffness and plateau in forward cell movement appeared to be due to a rearrangement of focal adhesions and actin at higher shear strains. Understanding the fundamental mechanisms at play during shear of single cells will help elucidate potential treatments for chondrocyte pathology and loading regimens related to cartilage health and disease.     

Unique biomechanical interactions between myeloma cells and bone marrow stroma cells

We observed that BMSCs (bone marrow stromal cells) from myeloma patients (myeloma BMSCs) were significantly stiffer than control BMSCs using a cytocompression device. The stiffness of myeloma BMSCs and control BMSCs was further increased upon priming by myeloma cells. Additionally, myeloma cells became stiffer when primed by myeloma BMSCs. The focal adhesion kinase activity of myeloma cells was increased when cells were on stiffer collagen gels and on myeloma BMSCs. This change in myeloma stiffness is associated with increased colony formation of myeloma cells and FAK activation when co-cultured with stiffer myeloma BMSCs or stiffer collagen. Additionally, stem cells of RPMI8226 cells became stiffer after priming by myeloma BMSCs, with concomitant increases of stem cell colony formation. These results suggest the presence of a mechanotransduction loop between myeloma cells and myeloma BMSCs to increase the stiffness of both types of cells via FAK activation. The increase of stiffness may in turn support the growth of myeloma cells and myeloma stem cells.     

Cannabinoid receptor agonists are mitochondrial inhibitors: a unified hypothesis of how cannabinoids modulate mitochondrial function and induce cell death

Time-lapse microscopy of human lung cancer (H460) cells showed that the endogenous cannabinoid anandamide (AEA), the phyto-cannabinoid Δ-9-tetrahydrocannabinol (THC) and a synthetic cannabinoid HU 210 all caused morphological changes characteristic of apoptosis. Janus green assays of H460 cell viability showed that AEA and THC caused significant increases in OD 595 nm at lower concentrations (10-50 μM) and significant decreases at 100 μM, whilst HU 210 caused significant decreases at all concentrations. In rat heart mitochondria, all three ligands caused significant decreases in oxygen consumption and mitochondrial membrane potential. THC and HU 210 caused significant increases in mitochondrial hydrogen peroxide production, whereas AEA was without significant effect. All three ligands induced biphasic changes in either mitochondrial complex I activity and/or mitochondrial complex II-III activity. These data demonstrate that AEA, THC, and HU 210 are all able to cause changes in integrated mitochondrial function, directly, in the absence of cannabinoid receptors.