Platinum toxicity and gene expression in Xenopus embryos: analysis by FETAX and differential display

Since the level of platinum in the environment is destined to increase, because of its use in vehicle catalytic converters, the toxicity of platinum needs further investigation. In this study, the frog embryo teratogenesis assay-Xenopus (FETAX) was used to compare the embryotoxicity and teratogenicity of two common platinum species, (NH4)2PtCl4 and (NH4)2PtCl6. The uptake rates of the two platinum species were studied, and also their effects on the expression of genes encoding metallothionein and heat-shock protein 70, which are known to be induced by several stress factors. In addition, the differential display technique was used to search for genes that were specifically induced by platinum. A gene for the type I collagen alpha-chain and a novel gene were identified.     

Graphite-epoxy composites as a new transducing material for electrochemical genosensing

The use of a rigid carbon-polymer composite material as an electrochemical transducer in hybridisation genosensors is reported. Graphite-epoxy composites (GEC) have an uneven surface where DNA can be adsorbed using a simple dry-adsorption procedure. Single-stranded-DNA binds strongly to GEC in a way that prevents the strands from self-associating, while permitting hybridisation with complementary DNA. Hybridisation has been detected through biotin-streptavidin interaction using a streptavidin conjugated to horseradish peroxidase. Non-specific adsorption onto GEC is almost non-existent even when the surface has not been treated by blocking reagents. The analytical signal obtained was higher when compared with other electrochemical genosensors. Results can be achieved in 150 min, and the detection limit is in the order of fmol. Additionally, surface regeneration is possible using a simple polishing procedure, allowing for multiple use. The new genosensor based on GEC fulfils the requirements desired for these devices: ease of preparation as dry-adsorption of DNA is very simple and easily automated, robustness, sensitivity, low cost of production, ease of miniaturisation and simple use and fast response. Additionally, it can be used for field measurements and can be produced as a genosensor kit. Also, this material can be implemented for screen-printing procedures for the mass production of genosensors. The utility of the genosensor based on GEC is also illustrated with the detection of a sequence related to novel determinant of beta-lactamase resistance in Staphylococcus aureus.     

NMR structure of free RGS4 reveals an induced conformational change upon binding Galpha

Heterotrimeric guanine nucleotide-binding proteins (G-proteins) are transducers in many cellular transmembrane signaling systems where regulators of G-protein signaling (RGS) act as attenuators of the G-protein signal cascade by binding to the Galpha subunit of G-proteins (G(i)(alpha)(1)) and increasing the rate of GTP hydrolysis. The high-resolution solution structure of free RGS4 has been determined using two-dimensional and three-dimensional heteronuclear NMR spectroscopy. A total of 30 structures were calculated by means of hybrid distance geometry-simulated annealing using a total of 2871 experimental NMR restraints. The atomic rms distribution about the mean coordinate positions for residues 5-134 for the 30 structures is 0.47 +/- 0.05 A for the backbone atoms, 0. 86 +/- 0.05 A for all atoms, and 0.56 +/- 0.04 A for all atoms excluding disordered side chains. The NMR solution structure of free RGS4 suggests a significant conformational change upon binding G(i)(alpha)(1) as evident by the backbone atomic rms difference of 1. 94 A between the free and bound forms of RGS4. The underlying cause of this structural change is a perturbation in the secondary structure elements in the vicinity of the G(i)(alpha)(1) binding site. A kink in the helix between residues K116-Y119 is more pronounced in the RGS4-G(i)(alpha)(1) X-ray structure relative to the free RGS4 NMR structure, resulting in a reorganization of the packing of the N-terminal and C-terminal helices. The presence of the helical disruption in the RGS4-G(i)(alpha)(1) X-ray structure allows for the formation of a hydrogen-bonding network within the binding pocket for G(i)(alpha)(1) on RGS4, where RGS4 residues D117, S118, and R121 interact with residue T182 from G(i)(alpha)(1). The binding pocket for G(i)(alpha)(1) on RGS4 is larger and more accessible in the free RGS4 NMR structure and does not present the preformed binding site observed in the RGS4-G(i)(alpha)(1) X-ray structure. This observation implies that the successful complex formation between RGS4 and G(i)(alpha)(1) is dependent on both the formation of the bound RGS4 conformation and the proper orientation of T182 from G(i)(alpha)(1). The observed changes for the free RGS4 NMR structure suggest a mechanism for its selectivity for the Galpha-GTP-Mg(2+) complex and a means to facilitate the GTPase cycle.     

Analysis by NMR spectroscopy of the structural homology between the linear and the cyclic peptide recognized by anti-human leukocyte antigen class I monoclonal antibody TP25.99*

The anti-human leukocyte antigen (HLA) class I monoclonal antibody (mAb) TP25.99 has a unique specificity since it recognizes both a conformational and a linear determinant expressed on the beta(2)-mu-associated and beta(2)-mu-free HLA class I heavy chains, respectively. Previously, we reported the identification of a cyclic and a linear peptide that inhibits mAb TP25.99 binding to the beta(2)-mu-associated and beta(2)-mu-free HLA class I heavy chains (S. A. Desai, X. Wang, E. J. Noronha, Q. Zhou, V. Rebmann, H. Grosse-Wilde, F. J. Moy, R. Powers, and S. Ferrone, submitted for publication). The linear X(19) and cyclic LX-8 peptides contain sequence homologous to residues 239-242, 245, and 246 and to residues 194-198, respectively, of HLA class I heavy chain alpha(3) domain. Analysis by two-dimensional transfer nuclear Overhauser effect spectroscopy of the induced solution structures of the linear X(19) and cyclic LX-8 peptides in the presence of mAb TP25.99 showed that the two peptides adopt a similar structural motif despite the lack of sequence homology. The backbone fold is suggestive of a short helical segment followed by a tight turn, reminiscent of the determinant loop region (residues 194-198) on beta(2)-mu-associated HLA class I heavy chains. The structural similarity between the linear X(19) and cyclic LX-8 peptides and the lack of sequence homology suggests that mAb TP25.99 predominantly recognizes a structural motif instead of a consensus sequence.     

Tests of continuum theories as models of ion channels. I. Poisson-Boltzmann theory versus Brownian dynamics

Continuum theories of electrolytes are widely used to describe physical processes in various biological systems. Although these are well-established theories in macroscopic situations, it is not clear from the outset that they should work in small systems whose dimensions are comparable to or smaller than the Debye length. Here, we test the validity of the mean-field approximation in Poisson-Boltzmann theory by comparing its predictions with those of Brownian dynamics simulations. For this purpose we use spherical and cylindrical boundaries and a catenary shape similar to that of the acetylcholine receptor channel. The interior region filled with electrolyte is assumed to have a high dielectric constant, and the exterior region representing protein a low one. Comparisons of the force on a test ion obtained with the two methods show that the shielding effect due to counterions is overestimated in Poisson-Boltzmann theory when the ion is within a Debye length of the boundary. As the ion gets closer to the boundary, the discrepancy in force grows rapidly. The implication for membrane channels, whose radii are typically smaller than the Debye length, is that Poisson-Boltzmann theory cannot be used to obtain reliable estimates of the electrostatic potential energy and force on an ion in the channel environment.     

The Evolutionary History of the Genus Acanthamoeba and the Identification of Eight New 18S rRNA Gene Sequence Types

ABSTRACT The 18S rRNA gene ( Rns ) phylogeny of Acanthamoeba is being investigated as a basis for improvements in the nomenclature and taxonomy of the genus. We previously analyzed Rns sequences from 18 isolates from morphological groups 2 and 3 and found that they fell into four distinct evolutionary lineages we called sequence types T1-T4. Here, we analyzed sequences from 53 isolates representing 16 species and including 35 new strains. Eight additional lineages (sequence types T5-T12) were identified. Four of the 12 sequence types included strains from more than one nominal species. Thus, sequence types could be equated with species in some cases or with complexes of closely related species in others. The largest complex, sequence type T4, which contained six closely related nominal species, included 24 of 25 keratitis isolates. Rns sequence variation was insufficient for full phylogenetic resolution of branching orders within this complex, but the mixing of species observed at terminal nodes confirmed that traditional classification of isolates has been inconsistent. One solution to this problem would be to equate sequence types and single species. Alternatively, additional molecular information will be required to reliably differentiate species within the complexes. Three sequence types of morphological group 1 species represented the earliest divergence in the history of the genus and, based on their genetic distinctiveness, are candidates for reclassification as one or more novel genera.