Detection of cellular responses to toxicants by dielectrophoresis

The dielectrophoretic (DEP) crossover method has been applied to the detection of cell responses to toxicants. Time and dose responses of the human cultured leukemia (HL-60) line were measured for paraquat, styrene oxide (SO), N-nitroso-N-methylurea (NMU) and puromycin. These toxicants were chosen because of their different predominant mechanisms of action, namely membrane free radical attack, simultaneous membrane and nucleic acid attack, nucleic acid alkylation, and protein synthesis inhibition, respectively. For all treatments, the specific membrane capacitance (C_mem) of the cells decreased while the specific membrane conductance (G_mem) increased in dose- and time-dependent manners. The DEP responses correlated sensitively with alterations in cell surface morphology, especially folds, microvilli, and blebs, observed by scanning electron microscopy. The DEP method was more sensitive to agents that had a direct action on the membrane than to agents for which membrane alterations were secondary. The responses to paraquat and SO, which directly damaged the cell membrane, could be detected 15 min after exposure, while those for puromycin and NMU, which acted on intracellular targets, could be detected after 30 min. The detection times and dose sensitivity results showed that the DEP method is much faster and more sensitive than conventional cell and higher organism viability testing techniques. The feasibility of producing small instruments for toxicity detection and screening based on cellular dielectric responses is discussed.     

Mechanism of rat liver DNA methyltransferase interaction with anti-benzo[a]pyrenediol epoxide modified DNA templates

We investigated the methylation reaction catalyzed by 1500-fold purified rat liver DNA methyltransferase (DMase) on native Micrococcal luteus DNA (ML-DNA) and poly(dC-dG) templates containing covalently bound (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), the strongly carcinogenic, principal metabolite of benzo[a]pyrene. Since eukaryotic DNA methyltransferases recognize the dinucleotide 5'd[CG] in DNA as a substrate for methylation, the model polynucleotide poly(dC-dG) was used to study in more detail the mode of interaction and effect on incorporation. With either of these BPDE-modified templates, a progressive inhibition of methylation was correlated with increasing amount of BPDE substitution. The effect of BPDE-dG adducts did not alter the apparent km with respect to the concentration of d[CG] in either unmodified or BPDE-modified poly(dC-dG) (km = 10 microM) but lowered the relative apparent Vmax. In assays in which perturbation by salt of preformed enzyme-DNA complex is measured, no change in the relative stability to either unsubstituted or the carcinogen-modified template was noted, thus, excluding any change in the ionic component of this interaction. However, in competition-type experiments, BPDE-DNA is an inhibitor of the methylation reaction on native DNA. When BPDE-DNA is allowed to interact with the enzyme before the addition of native competitor DNA, the methylation rate is not stimulated, suggesting very tight hydrophobic binding of the enzyme to BPDE-DNA and an inhibition in the dissociation of DMase from the template following a methylation event.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three new species of the carnivorous snail genus Perrottetia Kobelt, 1905 from Thailand (Pulmonata,?Streptaxidae)

Three new species of the streptaxid snail genus Perrottetia are described from north and northeastern Thailand, Perrottetia aquilonaria sp. n., Perrottetia dermapyrrhosa sp. n. and Perrottetia phuphamanensis sp. n. Each species is endemic to a single or a few limestone mountain ranges. The species are characterized by the morphology of their genital organs, as well as by shell characters. Perrottetia aquilonaria sp. n. has a club shaped distal penis and large penial hooks are present and penial papillae cover almost the entire penial hook portion; adjacent areas possess low reticulated folds. Perrottetia dermapyrrhosa sp. n. has a long genital atrium and the penial sheath is about two-thirds of the penis length. Penial hooks are long, scattered and sunken into deep ovate hollows; vaginal hooks are present. Perrottetia phuphamanensis sp. n. has a rounded and protruded shell periphery. The aperture is subcircular, peristome is thick and the second parietal lamella is adjacent to the first parietal lamella; a basal lamella is the smaller than in the other Thai species.     

Structural and Dynamical Properties of a Full-length HIV-1 Integrase: Molecular Dynamics Simulations

Abstract

The structural and dynamical properties of the complete full-length structure of HIV-1 integrase were investigated using Molecular Dynamics approach. Simulations were carried out for the three systems, core domain only (CORE), full-length structure without (FULL) and with a Mg²⁺ (FULL+ION) in its active site, aimed to investigate the difference in the molecular properties of the full-length models due to their different construction procedures as well as the effects of the two ends, C- and N-terminal, on those properties in the core domain. The full-length structure was prepared from the two experimental structures of two-domain fragment. The following properties were observed to differ significantly from the previous reports: (i) relative topology formed by an angle between the three domains; (ii) the cavity size defined by the catalytic triad, Asp64, Asp116, and Glul52; (iii) distances and solvation of the Mg²⁺; and (iv) conformation of the catalytic residues. In addition, the presence of the two terminal domains decreases the mobility of the central core domain significantly.     

Tubeufia asiana, the teleomorph of Aquaphila albicans in the tubeufiaceae, pleosporales, based on cultural and molecular data

The teleomorph of Aquaphila albicans was discovered on submerged wood collected in Thailand. Its black, soft-textured, setose ascomata, bitunicate asci and hyaline to pale brown, multiseptate ascospores indicated an affinity to Tubeufiaceae (Dothideomycetes). After morphological or molecular comparisons with related species in Tubeufia, Acanthostigma and Taphrophila, it is described and illustrated as a new species, T. asiana Sivichai & K.M. Tsui, sp. nov. Finding this Tubeufia teleomorph was surprising, given the falcate conidia of its A. albicans anamorph, which superficially resemble the conidia of Fusarium and not the coiled, helicosporous conidia of other species in Tubeufiaceae. We assessed the phylogenetic relationships of A. albicans-T. asiana with ribosomal sequences from SSU and ITS and partial LSU regions by parsimony and Bayesian analysis. An initial set of 40 taxa representing a wide range of ascomycete families and their SSU sequences from GenBank showed A. albicans-T. asiana to be nested within the Tubeufiaceae with 100% bootstrap support. Their placement was inferred with ITS and partial LSU ribosomal sequences. The nearly identical ITS sequences of two isolates of A. albicans and one isolate of Tubeufia asiana united these fungi as a monophyletic group with 100% bootstrap support and further nested them, with 88% bootstrap support, in a clade containing Helicoon gigantisporum and Helicoma chlamydosporum. This is the first molecular phylogenetic study to place a nonhelicosporous species within the Tubeufiaceae and to show that helical conidia were lost at least once within the family.     

Stone-tool usage by Thai long-tailed macaques (Macaca fascicularis)

In January and March of 2005, we conducted surveys of long-tailed macaques at Piak Nam Yai Island, Laem Son National Park (9 degrees N 34-35', 98 degrees E 28'), Ranong Province, situated in southern Thailand. Two of the three troops of long-tailed macaques found on the island were observed using axe-shaped stones to crack rock oysters, detached gastropods (Thais tissoti, Petit, 1852), bivalves (Gafrarium divaricatum, Gmelin, 1791), and swimming crabs (Thalamita danae, Stimpson, 1858). They smashed the shells with stones that were held in either the left or right hand, while using the opposite hand to gather the oyster meat. Some monkeys used both hands to handle the stones. According to Matsuzawa's 1996 hierarchical classification of tool usage (levels 0-3), the tool usage by Thai long-tailed macaques could be characterized as either level 1 (cracking rock oysters with stones) or level 2 (cracking drifting mollusks and crabs with stones by placing them on a rock). Our discovery of stone-tool usage by Thai long-tailed macaques provides a new point of reference for discussions regarding the evolution of tool usage and the material culture of primates.