Uptake kinetics of 99Tc in common duckweed

The uptake of the nuclear waste product technetium-99 was studied in common duckweed (Lemna minor). In addition to measurements, a model involving two compartments in duckweed with different chemical forms of technetium was derived. The model was tested by chemical speciation, i.e. differentiating between reduced Tc-compounds and Tc(VII)O(4)(-). The TcO(4)(-) concentrations measured were in good agreement with those predicted by the model. Two processes determine technetium uptake: (1) transport of Tc(VII)O(4)(-) across the cell membrane, and (2) reduction of Tc(VII). The TcO(4)(-) concentration in duckweed reaches a steady state within 2 h while reduced Tc-compounds are stored, as a result of absence of release or re-oxidation processes. Bioaccumulation kinetic properties were derived by varying 99Tc concentration, temperature, nutrient concentrations, and light intensity. The reduction of technetium in duckweed was highly correlated with light intensity and temperature. At 25 degrees C the maximum reduction rate was observed at light intensities above 200 μmol m(-2) s(-1) while half of the maximum transformation rate was reached at 41 μmol m(-2) s(-1). Transport of TcO(4)(-) over the cell membrane requires about 9.4 kJ mol(-1), indicating an active transport mechanism. However, this mechanism behaved as first-order kinetics instead of Michaelis-Menten kinetics between 1x10(-14) and 2.5x10(-5) mol l(-1) TcO(4)(-). Tc uptake could not be inhibited by 10(-3) mol l(-1) nitrate, phosphate, sulphate or chloride.     

Hec1 and nuf2 are core components of the kinetochore outer plate essential for organizing microtubule attachment sites

A major goal in the study of vertebrate mitosis is to identify proteins that create the kinetochore-microtubule attachment site. Attachment sites within the kinetochore outer plate generate microtubule dependent forces for chromosome movement and regulate spindle checkpoint protein assembly at the kinetochore. The Ndc80 complex, comprised of Ndc80 (Hec1), Nuf2, Spc24, and Spc25, is essential for metaphase chromosome alignment and anaphase chromosome segregation. It has also been suggested to have roles in kinetochore microtubule formation, production of kinetochore tension, and the spindle checkpoint. Here we show that Nuf2 and Hec1 localize throughout the outer plate, and not the corona, of the vertebrate kinetochore. They are part of a stable "core" region whose assembly dynamics are distinct from other outer domain spindle checkpoint and motor proteins. Furthermore, Nuf2 and Hec1 are required for formation and/or maintenance of the outer plate structure itself. Fluorescence light microscopy, live cell imaging, and electron microscopy provide quantitative data demonstrating that Nuf2 and Hec1 are essential for normal kinetochore microtubule attachment. Our results indicate that Nuf2 and Hec1 are required for organization of stable microtubule plus-end binding sites in the outer plate that are needed for the sustained poleward forces required for biorientation at kinetochores.     

Heme-regulated inhibitor kinase-mediated phosphorylation of eukaryotic translation initiation factor 2 inhibits translation, induces stress granule formation, and mediates survival upon arsenite exposure

Exposure to arsenite inhibits protein synthesis and activates multiple stress signaling pathways. Although arsenite has diverse effects on cell metabolism, we demonstrated that phosphorylation of eukaryotic translation initiation factor 2 at Ser-51 on the alpha subunit was necessary to inhibit protein synthesis initiation in arsenite-treated cells and was essential for stress granule formation. Of the four protein kinases known to phosphorylate eukaryotic translation initiation factor 2alpha, only the heme-regulated inhibitor kinase (HRI) was required for the translational inhibition in response to arsenite treatment in mouse embryonic fibroblasts. In addition, HRI expression was required for stress granule formation and cellular survival after arsenite treatment. In vivo studies elucidated a fundamental requirement for HRI in murine survival upon acute arsenite exposure. The results demonstrated an essential role for HRI in mediating arsenite stress-induced phosphorylation of eukaryotic translation initiation factor 2alpha, inhibition of protein synthesis, stress granule formation, and survival.     

The de novo centriole assembly pathway in HeLa cells: cell cycle progression and centriole assembly/maturation

It has been reported that nontransformed mammalian cells become arrested during G1 in the absence of centrioles (Hinchcliffe, E., F. Miller, M. Cham, A. Khodjakov, and G. Sluder. 2001. Science. 291:1547-1550). Here, we show that removal of resident centrioles (by laser ablation or needle microsurgery) does not impede cell cycle progression in HeLa cells. HeLa cells born without centrosomes, later, assemble a variable number of centrioles de novo. Centriole assembly begins with the formation of small centrin aggregates that appear during the S phase. These, initially amorphous "precentrioles" become morphologically recognizable centrioles before mitosis. De novo-assembled centrioles mature (i.e., gain abilities to organize microtubules and replicate) in the next cell cycle. This maturation is not simply a time-dependent phenomenon, because de novo-formed centrioles do not mature if they are assembled in S phase-arrested cells. By selectively ablating only one centriole at a time, we find that the presence of a single centriole inhibits the assembly of additional centrioles, indicating that centrioles have an activity that suppresses the de novo pathway.     

Trends of reproductive hormones in male rats during psychosocial stress: role of glucocorticoid metabolism in behavioral dominance

Stress in socially subordinate male rats, associated with aggressive attacks by dominant males, was studied in a group-housing context called the visible burrow system (VBS). It has been established that subordinate males have reduced serum testosterone (T) and higher corticosterone (CORT) relative to dominant and singly housed control males. The relationship of the decreased circulating T levels in subordinate males to changes in serum LH concentrations has not been evaluated previously. Since decreases in LH during stress may cause reductions in Leydig cell steroidogenic activity, the present study defined the temporal profiles of serum LH, T, and CORT in dominant and subordinate males on Days 4, 7, and 14 of a 14-day housing period in the VBS. The same parameters were followed in serum samples from single-housed control males. Leydig cells express glucocorticoid receptors and may also be targeted for direct inhibition of steroidogenesis by glucocorticoid. We hypothesize that Leydig cells are protected from inhibition by CORT at basal concentrations through oxidative inactivation of glucocorticoid by 11beta-hydroxysteroid dehydrogenase (11betaHSD). However, Leydig cell steroidogenesis is inhibited when 11betaHSD metabolizing capacity is exceeded. Therefore, 11betaHSD enzyme activity levels were measured in Leydig cells of VBS-housed males at the same time points. Significant increases in LH and T relative to control were observed in the dominant animals on Day 4, which were associated with the overt establishment of behavioral dominance as evidenced by victorious agonistic encounters. Serum LH and T were lower in subordinate males on Day 7, but T alone was lower on Day 14, suggesting that lowered LH secretion in subordinates may gradually be reversed by declines in androgen-negative feedback. Serum CORT levels were higher in subordinate males compared to control at all three time points. In contrast, oxidative 11betaHSD activity in Leydig cells of dominant males was higher relative to control and unchanged in subordinates. These results suggest the following: 1) failure of Leydig cells of subordinate males to compensate for increased glucocorticoid action during stress, by increasing 11betaHSD oxidative activity, potentiates stress-mediated reductions in T secretion; and 2) an inhibition of the reproductive axis in subordinate males at the level of the pituitary.     

Antibiotic Use in Animal Agriculture: What Have We Learned and Where are We Going?

Antibiotics are enormously important for the humane and efficient production of food animals. These benefits are somewhat offset by the human and animal health antibiotic resistance risks posed by their use in animals. This article provides an overview of what we have learned about antibiotic resistance as an issue in animal agriculture and where that knowledge could lead us in the future. To preserve the effectiveness of antibiotics, more action is needed to ensure their prudent use, particularly in the case of antibiotic growth promoters and antibiotics deemed critically important for human and animal health.     

Sensing Biophysical Alterations of Human Lung Epithelial Cells (A549) in the Context of Toxicity Effects of Diesel Exhaust Particles

Diesel exhaust particles (DEP) in urban air are associated with numerous respiratory diseases. The role of underlying biomechanics in cytotoxicity of individual lung cells relating to DEP exposure is unclear. In this study, atomic force microscopy (AFM), confocal Raman microspectroscopy (RM), and fluorescence (FL) microscopy were used to monitor alterations of single A549 cells exposed to DEP. Results revealed a significant decrease in membrane surface adhesion force and a significant change in cell elasticity as a function of DEP–cell interaction time, and the dynamic changes in cellular biocomponents which were reflected by changes of characteristic Raman bands: 726 cm^?1 (adenine), 782 cm^?1 (uracil, cytosine, thymine), 788 cm^?1 (O–P–O), 1006 cm^?1 (phenylalanine), and 1320 cm^?1 (guanine) after DEP exposure. These findings suggest that the combination of multi-instruments (e.g., AFM/FL) may offer an exciting platform for investigating the roles of biophysical and biochemical responses to particulate matter-induced cell toxicity.