Ultrasensitive detection of biomolecules with fluorescent dye-doped nanoparticles

Fluorescent-labeled molecules have been used extensively for a wide range of applications in biological detection and diagnosis. A new form of highly luminescent and photostable nanoparticles was generated by doping the fluorescent dye tris(2'2-bipyridyl)dichlororuthenium(II)hexahydrate (Rubpy) inside silica material. Because thousands of fluorescent dye molecules are encapsulated in the silica matrix that also serves to protect Rubpy dye from photodamaging oxidation, the Rubpy-dye-doped nanoparticles are extremely bright and photostable. We have used these nanoparticles successfully in various fluorescence labeling techniques, including fluorescent-linked immunosorbent assay, immunocytochemistry, immunohistochemistry, DNA microarray, and protein microarray. By combining the high-intensity luminescent nanoparticles with the specificity of antibody-mediated recognition, ultrasensitive target detection has been achieved. In all cases, assay results clearly demonstrated the superiority of the nanoparticles over organic fluorescent dye molecules and quantum dots in probe labeling for sensitive target detection. These results demonstrate the potential to apply these newly developed fluorescent nanoparticles in various biodetection systems.     

Crystal structures of the Apo and penicillin-acylated forms of the BlaR1 beta-lactam sensor of Staphylococcus aureus

Staphylococcus aureus is among the most prevalent and antibiotic-resistant of pathogenic bacteria. The resistance of S. aureus to prototypal beta-lactam antibiotics is conferred by two mechanisms: (i) secretion of hydrolytic beta-lactamase enzymes and (ii) production of beta-lactam-insensitive penicillin-binding proteins (PBP2a). Despite their distinct modes of resistance, expression of these proteins is controlled by similar regulation systems, including a repressor (BlaI/MecI) and a multidomain transmembrane receptor (BlaR1/MecR1). Resistance is triggered in response to a covalent binding event between a beta-lactam antibiotic and the extracellular sensor domain of BlaR1/MecR1 by transduction of the binding signal to an intracellular protease domain capable of repressor inactivation. This study describes the first crystal structures of the sensor domain of BlaR1 (BlaRS) from S. aureus in both the apo and penicillin-acylated forms. The structures show that the sensor domain resembles the beta-lactam-hydrolyzing class D beta-lactamases, but is rendered a penicillin-binding protein due to the formation of a very stable acyl-enzyme. Surprisingly, conformational changes upon penicillin binding were not observed in our structures, supporting the hypothesis that transduction of the antibiotic-binding signal into the cytosol is mediated by additional intramolecular interactions of the sensor domain with an adjacent extracellular loop in BlaR1.     

Keratins modulate colonocyte electrolyte transport via protein mistargeting

The function of intestinal keratins is unknown, although keratin 8 (K8)-null mice develop colitis, hyperplasia, diarrhea, and mistarget jejunal apical markers. We quantified the diarrhea in K8-null stool and examined its physiologic basis. Isolated crypt-units from K8-null and wild-type mice have similar viability. K8-null distal colon has normal tight junction permeability and paracellular transport but shows decreased short circuit current and net Na absorption associated with net Cl secretion, blunted intracellular Cl/HCO3-dependent pH regulation, hyperproliferation and enlarged goblet cells, partial loss of the membrane-proximal markers H,K-ATPase-beta and F-actin, increased and redistributed basolateral anion exchanger AE1/2 protein, and redistributed Na-transporter ENaC-gamma. Diarrhea and protein mistargeting are observed 1-2 d after birth while hyperproliferation/inflammation occurs later. The AE1/2 changes and altered intracellular pH regulation likely account, at least in part, for the ion transport defects and hyperproliferation. Therefore, colonic keratins have a novel function in regulating electrolyte transport, likely by targeting ion transporters to their cellular compartments.     

Expansion and functional relevance of high-avidity myelin-specific CD4+ T cells in multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease in which myelin-specific T cells are believed to play a crucial pathogenic role. Nevertheless, so far it has been extremely difficult to demonstrate differences in T cell reactivity to myelin Ag between MS patients and controls. We believe that by using unphysiologically high Ag concentrations previous studies have missed a highly relevant aspect of autoimmune responses, i.e., T cells recognizing Ag with high functional avidity. Therefore, we focused on the characterization of high-avidity myelin-specific CD4+ T cells in a large cohort of MS patients and controls that was matched demographically and with respect to expression of MHC class II alleles. We demonstrated that their frequency is significantly higher in MS patients while the numbers of control T cells specific for influenza hemagglutinin are virtually identical between the two cohorts; that high-avidity T cells are enriched for previously in vivo-activated cells and are significantly skewed toward a proinflammatory phenotype. Moreover, the immunodominant epitopes that were most discriminatory between MS patients and controls differed from those described previously and were clearly biased toward epitopes with lower predicted binding affinities to HLA-DR molecules, pointing at the importance of thymic selection for the generation of the autoimmune T cell repertoire. Correlations between selected immunological parameters and magnetic resonance imaging markers indicate that the specificity and function of these cells influences phenotypic disease expression. These data have important implications for autoimmunity research and should be considered in the development of Ag-specific therapies in MS.     

Brain antioxidant systems in human methamphetamine users

Animal data suggest that the widely abused psychostimulant methamphetamine can damage brain dopamine neurones by causing dopamine-dependent oxidative stress; however, the relevance to human methamphetamine users is unclear. We measured levels of key antioxidant defences [reduced (GSH) and oxidized (GSSG) glutathione, six major GSH system enzymes, copper-zinc superoxide dismutase (CuZnSOD), uric acid] that are often altered after exposure to oxidative stress, in autopsied brain of human methamphetamine users and matched controls. Changes in the total (n = 20) methamphetamine group were limited to the dopamine-rich caudate (the striatal subdivision with the most severe dopamine loss) in which only activity of CuZnSOD (+ 14%) and GSSG levels (+ 58%) were changed. In the six methamphetamine users with severe (- 72 to - 97%) caudate dopamine loss, caudate CuZnSOD activity (+ 20%) and uric acid levels (+ 63%) were increased with a trend for decreased (- 35%) GSH concentration. Our data suggest that brain levels of many antioxidant systems are preserved in methamphetamine users and that GSH depletion, commonly observed during severe oxidative stress, might occur only with severe dopamine loss. Increased CuZnSOD and uric acid might reflect compensatory responses to oxidative stress. Future studies are necessary to establish whether these changes are associated with oxidative brain damage in human methamphetamine users.     

Plastid transformation in the monocotyledonous cereal crop, rice (Oryza sativa) and transmission of transgenes to their progeny

The plastid transformation approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering, transgene containment, and a lack of gene silencing and position effects. The extension of plastid transformation technology to monocotyledonous cereal crops, including rice, bears great promise for the improvement of agronomic traits, and the efficient production of pharmaceutical or nutritional enhancement. Here, we report a promising step towards stable plastid transformation in rice. We produced fertile transplastomic rice plants and demonstrated transmission of the plastid-expressed green fluorescent protein (GFP) and aminoglycoside 3'-adenylyltransferase genes to the progeny of these plants. Transgenic chloroplasts were determined to have stably expressed the GFP, which was confirmed by both confocal microscopy and Western blot analyses. Although the produced rice plastid transformants were found to be heteroplastomic, and the transformation efficiency requires further improvement, this study has established a variety of parameters for the use of plastid transformation technology in cereal crops.     

Secramine inhibits Cdc42-dependent functions in cells and Cdc42 activation in vitro

Inspired by the usefulness of small molecules to study membrane traffic, we used high-throughput synthesis and phenotypic screening to discover secramine, a molecule that inhibits membrane traffic out of the Golgi apparatus by an unknown mechanism. We report here that secramine inhibits activation of the Rho GTPase Cdc42, a protein involved in membrane traffic, by a mechanism dependent upon the guanine dissociation inhibitor RhoGDI. RhoGDI binds Cdc42 and antagonizes its membrane association, nucleotide exchange and effector binding. In vitro, secramine inhibits Cdc42 binding to membranes, GTP and effectors in a RhoGDI-dependent manner. In cells, secramine mimics the effects of dominant-negative Cdc42 expression on protein export from the Golgi and on Golgi polarization in migrating cells. RhoGDI-dependent Cdc42 inhibition by secramine illustrates a new way to inhibit Rho GTPases with small molecules and provides a new means to study Cdc42, RhoGDI and the cellular processes they mediate.     

Chloroplast DNA variation and population structure in the widespread forest tree, Eucalyptus grandis

Recognition of genetic structure of populations and the ability to identify vulnerable populations is useful for the formation of conservation management strategies for plants. Eucalyptus grandis is a tall forest tree that has a major area of occurrence in subtropical eastern Australia, with smaller populations located in the east coast tropics. Many widespread forest species exhibit population differentiation that corresponds to geographic regions. However, Eucalyptus grandis appears to be an exception based on isozyme and morphological data. This is intriguing given a large discontinuity between northern populations and those in the southern part of the species range. In this study, the distribution of a maternally inherited chloroplast locus was examined because it was more likely to reveal genetic structure due to the slower evolution of the chloroplast genome and limited dispersal of seed in eucalypts. As expected, the G _ST for chloroplast DNA was higher than that for nuclear DNA but indicated low population differentiation for a forest tree species. Phylogeographic analysis indicated that the 15 populations grouped into three broad geographical regions; however, overall population structure was weak suggesting that the large geographical disjunction in the distribution of E. grandis may be relatively recent. A paradigm for conservation management of E. grandis based on chloroplast DNA haplotype distribution would take into account the low differentiation among populations.     

N-Glycosylation is Required for Secretion-Competent Human Plasma Phospholipid Transfer Protein

Human plasma phospholipid transfer protein (PLTP) contains six potential N-glycosylation sites (Asn-X-Ser). To study the role of these sites on PLTP structure and function, seven variants in which asparagine (N) residues were converted to glycine (G) were prepared by site-directed mutagenesis. These were N₄₇G, N₇₇G, N₁₀₀G, N₁₂₆G, N₂₂₈G, N₃₈₁G and N_{47, 77, 100, 126, 228, 381}G (N_nullG). These variants and wild-type (WT) PLTP were expressed in COS-7 cells. Intracellular and secreted PLTP mass was analyzed by Western blots and quantitative enzyme-linked immunosorbent assay; PLTP activities in cellular lysates and media were based on the transfer of [³H]dipalmitoylphosphatidylcholine from phospholipid single bilayer vesicles to HDL. N_nullG was not detected intracellularly. N₃₈₁G was similar to WT PLTP with respect to specific activity and secretion efficiency. The specific activities of N₄₇G, N₇₇G, N₁₀₀G, N₁₂₆G, N₂₂₈G and N₃₈₁G were similar in cell lysate (range = 67–90% WT) and medium (range = 65–77% WT). Intracellular masses of these PLTP variants were similar to that of WT (Mean = 103% WT); mean secreted mass was 88% WT. These results suggest that secretion-competent PLTP requires glycosylation but that no single glycosylation site is required.