Pseudomonas aeruginosa ExoS ADP-ribosyltransferase inhibits ERM phosphorylation

Pseudomonas aeruginosa causes life-threatening infections in compromised and cystic fibrosis patients. Pathogenesis stems from a number of virulence factors, including four type III translocated cytotoxins: ExoS, ExoT, ExoY and ExoU. ExoS is a bifunctional toxin: the N terminus (amino acids 96-219) encodes a Rho GTPase Activating Protein (GAP) domain. The C terminus (amino acids 234-453) encodes a 14-3-3-dependent ADP-ribosyltransferase domain which transfers ADP-ribose from NAD onto substrates such as the Ras GTPases and vimentin. Ezrin/radixin/moesin (ERM) proteins have recently been identified as high-affinity substrates for ADP-ribosylation by ExoS. Expression of ExoS in HeLa cells led to a loss of phosphorylation of ERM proteins that was dependent upon the expression of ADP-ribosyltransferase activity. MALDI-MS and site-directed mutagenesis studies determined that ExoS ADP-ribosylated moesin at three C-terminal arginines (Arg553, Arg560 and Arg563), which cluster Thr558, the site of phosphorylation by protein kinase C and Rho kinase. ADP-ribosylated-moesin was a poor target for phosphorylation by protein kinase C and Rho kinase, which showed that ADP-ribosylation directly inhibited ERM phosphorylation. Expression of dominant active-moesin inhibited cell rounding elicited by ExoS, indicating that moesin is a physiological target in cultured cells. This is the first demonstration that a bacterial toxin inhibits the phosphorylation of a mammalian protein through ADP-ribosylation. These data explain how the expression of the ADP-ribosylation of ExoS modifies the actin cytoskeleton and indicate that ExoS possesses redundant enzymatic activities to depolymerize the actin cytoskeleton.     

Covalent functionalization of multiwalled carbon nanotubes with a low molecular weight chitosan

Covalent functionalization of shortened multiwalled carbon nanotubes (MWNTs) with a natural low molecular weight chitosan (LMCS) was accomplished by a nucleophilic substitution reaction. Amino and primary hydroxyl groups of the LMCS contributed mainly to the formation of MWNT-LMCS conjugates. The LMCS content in the MWNT-LMCS is approximately 58 wt %, and approximately four molecular chains of the LMCS are attached to 1000 carbon atoms of the nanotube sidewalls. Most interestingly, the amorphous packing structure of the LMCS changed dramatically when it attached to the MWNTs. The MWNTs might induce the crystalline character of the LMCS. As a novel derivative of MWNTs, the MWNT-LMCS is soluble in dimethylformamide, dimethyl acetamide, dimethylsulfoxide, and acetic acid aqueous solution. The confirmation of the chitosan-based covalent functionalization route might lead to further studies aiming for potential applications in catalysis and environmental protection.     

Conformational specificity of the lac repressor coiled-coil tetramerization domain

Predictive understanding of how the folded, functional shape of a native protein is encoded in the linear sequence of its amino acid residues remains an unsolved challenge in modern structural biology. Antiparallel four-stranded coiled coils are relatively simple protein structures that embody a heptad sequence repeat and rich diversity for tertiary packing of alpha-helices. To explore specific sequence determinants of the lac repressor coiled-coil tetramerization domain, we have engineered a set of buried nonpolar side chains at the a-, d-, and e-positions into the hydrophobic interior of the dimeric GCN4 leucine zipper. Circular dichroism and equilibrium ultracentrifugation studies show that this core variant (GCN4-pAeLV) forms a stable tetrameric structure with a reversible and highly cooperative thermal unfolding transition. The X-ray crystal structure at 1.9 A reveals that GCN4-pAeLV is an antiparallel four-stranded coiled coil of the lac repressor type in which the a, d, and e side chains associate by means of combined knobs-against-knobs and knobs-into-holes packing with a characteristic interhelical offset of 0.25 heptad. Comparison of the side chain shape and packing in the antiparallel tetramers shows that the burial of alanine residues at the e positions between the neighboring helices of GCN4-pAeLV dictates both the antiparallel orientation and helix offset. This study fills in a gap in our knowledge of the determinants of structural specificity in antiparallel coiled coils and improves our understanding of how specific side chain packing forms the teritiary structure of a functional protein.     

Phy tunes: phosphorylation status and phytochrome-mediated signaling

Phytochromes are photoreceptors that regulate various aspects of plant growth and development. In this issue of Cell, Ryu et al. (2005) show that PAPP5, a type 5 protein phosphatase, acts on a biologically active phytochrome, increasing its stability and affinity for a downstream signal transducer and thus enhancing plant photoresponses.     

Amelioration of inflammatory arthritis by targeting the pre-ligand assembly domain of tumor necrosis factor receptors

Tumor necrosis factor (TNF)-alpha has an important role in the pathogenesis of autoimmune and inflammatory diseases such as rheumatoid and septic arthritis. The biological effects of TNF-alpha are mediated by binding to TNF receptors TNFR1 (also known as P60) or TNFR2 (also known as P80). The pre-ligand assembly domain (PLAD) is a portion of the extracellular region of TNFRs that mediates receptor-chain association essential for signaling. We found that soluble versions of PLAD, especially those derived from P60, block the biochemical effects of TNF-alpha in vitro and potently inhibit arthritis in animal models. Thus, targeting the PLAD may have clinical value in the treatment of human arthritis and other disorders involving receptors of the TNFR superfamily.     

Gas chromatography-mass spectrometric analysis of hexanal and heptanal in human blood by headspace single-drop microextraction with droplet derivatization

In this work, we developed a new approach to the analysis of the lung cancer biomarkers, hexanal and heptanal in human blood that was based on headspace single-drop microextraction (HS-SDME) with droplet derivatization, followed by gas chromatography-mass spectrometry (GC-MS). Aldehydes in blood were headspace extracted, concentrated, and derivatized by a suspended microdrop solvent containing the derivatization agent O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride. The aldehyde oximes formed in the microdrop solvent were analyzed by GC-MS. The optimal HS-SDME with droplet derivatization parameters extraction solvent of decane, sample temperature of 40 degrees C, extraction time of 6 min, stirring rate of 1100 rpm, and solvent volume of 2.0 microL were obtained and used for analysis of hexanal and heptanal in blood. The method reproducibility, linearity, recovery, and detection limit were studied and the obtained results demonstrated the method feasibility. Finally, the proposed method was applied to the quantification of hexanal and heptanal in cancer blood and normal blood. Due to sample extraction, concentration, and derivatization being performed in a single step, the method provided a simple, rapid, low-cost, and efficient approach to analysis of aldehydes in blood samples.     

Colorimetric approach to high-throughput mutation analysis

High-throughput genomic mutation screening for primary tumors has characteristically been expensive, labor-intensive, and inadequate to detect low levels of mutation in a background of wild-type signal. We present a new, combined PCR and colorimetric approach that is inexpensive, simple, and can detect the presence of 1% mutation in a background of wild-type. We compared manual dideoxy sequencing of p53 for eight lung cancer samples to a novel assay combining a primer extension step and an enzymatic colorimetric step in a 96-well plate with covalently attached oligonucleotide sequences. For every sample, we were able to detect the presence or absence of the specific mutation with a statistically significant difference between the sample optical density (OD) and the background OD, with a sensitivity and specificity of 100%. This assay is straightforward, accurate, inexpensive, and allows for rapid, high-throughput analysis of samples, making it ideal for genomic mutation or polymorphism screening studies in both clinical and research settings.     

Effect of incubation temperature on green sturgeon embryos, <Emphasis Type="Italic">Acipenser medirostris</Emphasis>

Regulation of river flow and the amount of winter rainfall are the major factors affecting the water temperature of the spawning grounds, for green sturgeon in the Klamath River. During the primary spawning period of green sturgeon, mid-April to June, the water temperature may vary from 8 to 21°C. To estimate the potential implications of this modified thermal regime, we examined the survival and development in three progeny groups of green sturgeon embryos from zygote to hatch, at constant incubation temperatures (11–26°C). Temperatures 23–26°C affected cleavage and gastrulation and all died before hatch. Temperatures 17.5–22°C were suboptimal as an increasing number of embryos developed abnormally and hatching success decreased at 20.5–22°C, although the tolerance to these temperatures varied between progenies. The lower temperature limit was not evident from this study, although hatching rate decreased at 11°C and hatched embryos were shorter, compared to 14°C. The mean total length of hatched embryos decreased with increasing temperature, although their wet and dry weight remained relatively constant. We concluded that temperatures 17–18°C may be the upper limit of the thermal optima for green sturgeon embryos, and that the river thermal regime during dry years may affect green sturgeon reproduction.     

Development of a Common Oligonucleotide Reference Standard for Microarray Data Normalization and Comparison across Different Microbial Communities

High-density functional gene arrays have become a powerful tool for environmental microbial detection and characterization. However, microarray data normalization and comparison for this type of microarray remain a challenge in environmental microbiology studies because some commonly used normalization methods (e.g., genomic DNA) for the study of pure cultures are not applicable. In this study, we developed a common oligonucleotide reference standard (CORS) method to address this problem. A unique 50-mer reference oligonucleotide probe was selected to co-spot with gene probes for each array feature. The complementary sequence was synthesized and labeled for use as the reference target, which was then spiked and cohybridized with each sample. The signal intensity of this reference target was used for microarray data normalization and comparison. The optimal amount or concentration were determined to be ca. 0.5 to 2.5% of a gene probe for the reference probe and ca. 0.25 to 1.25 fmol/μl for the reference target based on our evaluation with a pilot array. The CORS method was then compared to dye swap and genomic DNA normalization methods using the Desulfovibrio vulgaris whole-genome microarray, and significant linear correlations were observed. This method was then applied to a functional gene array to analyze soil microbial communities, and the results demonstrated that the variation of signal intensities among replicates based on the CORS method was significantly lower than the total intensity normalization method. The developed CORS provides a useful approach for microarray data normalization and comparison for studies of complex microbial communities.Microarray-based technology has become a robust genomic tool to detect, track, and profile hundreds to thousands of different microbial populations simultaneously in complex environments such as soils and sediments. For example, GeoChip, a comprehensive functional gene array, has been developed for investigating biogeochemical, ecological, and environmental processes (12, 18, 23, 27, 29, 32). Although a massive amount of microarray data can be generated rapidly, one of the bottlenecks in using microarrays for environmental microbial community studies is the lack of an appropriate standard for data comparison and normalization (6). Currently, it is difficult to compare microarray data across different sites, experiments, laboratories, and/or time periods (10). This limits the power of the technology to address ecological and environmental questions.In pure culture-based functional genomics studies, genomic DNAs (gDNAs) have been used as a common reference for hybridizations in which the same amount of gDNAs are used to cohybridize with each target cDNA sample and then to normalize different target cDNAs based on the gDNA standard (4, 5, 8, 9, 19, 21, 23). Several normalization methods such as scale normalization, quantile normalization, and Lowess normalization have been used for gene expression studies (2). Using the gDNA standard method can minimize or eliminate differences in target cDNA quantity, spot morphology, uneven hybridization, labeling, and sequence-specific hybridization behaviors (5), and this allows the comparison of microarray data across different sites, laboratories, experiments, and/or times. The main rationale for gDNA as a common reference is that it provides complete coverage for all genes represented on the array because the DNA composition from a particular organism should be identical across different treatment samples even though RNA expression is different (8). However, this approach is not applicable to microbial community studies because not all communities have identical DNA compositions. Pooling of equal amounts of gDNA or RNA from every target sample to make a common sample could be used as an alternative reference for cohybridization (1, 22). However, the disadvantage of the sample pooling approach is that samples do not provide large amounts of DNA or RNA in a reliable and reproducible way. For example, groundwater samples usually have a very low biomass and thus would not provide enough DNA for pooling. In addition, the sample pool itself is uncharacterized, and gene abundance may be diluted out so that insufficient DNA is present to result in a positive signal some array features, especially for those genes in low abundance. Moreover, a new sample pool would be required for every new experiment, making comparison across experiments difficult. Thus, other approaches need to be developed for microbial community studies.Dudley et al. (7) used a 25-mer oligonucleotide that matched a small portion of the parental EST clone vector contained in every PCR product printed on the array for normalization of pure culture RNA expression. Although the oligonucleotide generated a stable hybridization signal on every array feature, this method requires a universal sequence tag as a “capture” sequence, limiting its general use in microbial community studies. Thus, in the present study, we developed a common oligonucleotide reference standard (CORS) approach by co-spotting a common oligonucleotide with each array feature to improve the accuracy and comparability of microarray data for microbial community studies. This method was evaluated by using a pilot array, a whole-genome array, and a functional gene array, and all results demonstrate that the developed CORS is a reliable and reproducible method for microarray data normalization and comparison for microbial community studies.