Targeted quantitative analysis of superoxide dismutase 1 in cisplatin-sensitive and cisplatin-resistant human ovarian cancer cells

Protein quantification in a complex protein mixture presents a daunting task in biochemical analysis. Antibody-based immunoassays are traditional methods for protein quantification. However, there are issues associated with accuracy and specificity in these assays, especially when the changes are small (e.g., <2-fold). With recent developments in mass spectrometry, monitoring a selected peptide, thus protein, in a complex biological sample has become possible. In this study, we demonstrate a simple mass spectrometry-based method for selective measurement of a moderately low abundant protein, superoxide dismutase 1 (SOD1), in cisplatin-sensitive and cisplatin-resistant human ovarian cancer cells. Selected-reaction-monitoring (SRM) technology was employed to specifically analyze the target peptides in a pair of human ovarian cancer cell lines: 2008/2008-C13*5.25 (cisplatin-sensitive/cisplatin-resistant, respectively). The observed 1.47-fold higher expression in the resistant cell line is consistent with findings by other approaches. This robust liquid chromatography/mass spectrometry (LC/MS) method provides a powerful tool for targeted proteomic verification and/or validation studies.     

Species Composition of Bacterial Communities Influences Attraction of Mosquitoes to Experimental Plant Infusions

In the container habitats of immature mosquitoes, catabolism of plant matter and other organic detritus by microbial organisms produces metabolites that mediate the oviposition behavior of Aedes aegypti and Aedes albopictus. Public health agencies commonly use oviposition traps containing plant infusions for monitoring populations of these mosquito species, which are global vectors of dengue viruses. In laboratory experiments, gravid females exhibited significantly diminished responses to experimental infusions made with sterilized white oak leaves, showing that attractive odorants were produced through microbial metabolic activity. We evaluated effects of infusion concentration and fermentation time on attraction of gravid females to infusions made from senescent bamboo or white oak leaves. We used plate counts of heterotrophic bacteria, total counts of 4′,6-diamidino-2-phenylindole-stained bacterial cells, and 16S ribosomal DNA (rDNA) polymerase chain reaction–denaturing gradient gel electrophoresis (DGGE) to show that changes in the relative abundance of bacteria and the species composition of bacterial communities influenced attraction of gravid A. aegypti and A. albopictus mosquitoes to infusions. DGGE profiles showed that bacterial species composition in infusions changed over time. Principal components analysis indicated that oviposition responses to plant infusions were in general most affected by bacterial diversity and abundance. Analysis of bacterial 16S rDNA sequences derived from DGGE bands revealed that Proteobacteria (Alpha-, Beta-, Delta-, and Gamma-) were the predominant bacteria detected in both types of plant infusions. Gravid A. aegypti were significantly attracted to a mix of 14 bacterial species cultured from bamboo leaf infusion. The oviposition response of gravid mosquitoes to plant infusions is strongly influenced by abundance and diversity of bacterial species, which in turn is affected by plant species, leaf biomass, and fermentation time.     

Ribosomal Protein Genes RPS10 and RPS26 Are Commonly Mutated in Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA), an inherited bone marrow failure syndrome characterized by anemia that usually presents before the first birthday or in early childhood, is associated with birth defects and an increased risk of cancer. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital malformations, in particular craniofacial, upper limb, heart, and urinary system defects that are present in ∼30%–50% of patients. DBA has been associated with mutations in seven ribosomal protein (RP) genes, RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, and RPS7, in about 43% of patients. To continue our large-scale screen of RP genes in a DBA population, we sequenced 35 ribosomal protein genes, RPL15, RPL24, RPL29, RPL32, RPL34, RPL9, RPL37, RPS14, RPS23, RPL10A, RPS10, RPS12, RPS18, RPL30, RPS20, RPL12, RPL7A, RPS6, RPL27A, RPLP2, RPS25, RPS3, RPL41, RPL6, RPLP0, RPS26, RPL21, RPL36AL, RPS29, RPL4, RPLP1, RPL13, RPS15A, RPS2, and RPL38, in our DBA patient cohort of 117 probands. We identified three distinct mutations of RPS10 in five probands and nine distinct mutations of RPS26 in 12 probands. Pre-rRNA analysis in lymphoblastoid cells from patients bearing mutations in RPS10 and RPS26 showed elevated levels of 18S-E pre-rRNA. This accumulation is consistent with the phenotype observed in HeLa cells after knockdown of RPS10 or RPS26 expression with siRNAs, which indicates that mutations in the RPS10 and RPS26 genes in DBA patients affect the function of the proteins in rRNA processing.     

What does it take to be a plant pathogen: genomic insights from <Emphasis Type="Italic">Streptomyces</Emphasis> species

Plant pathogenicity is rare in the genus Streptomyces, with only a dozen or so species possessing this trait out of the more than 900 species described. Nevertheless, such species have had a significant impact on agricultural economies throughout the world due to their ability to cause important crop diseases such as potato common scab, which is characterized by lesions that form on the potato tuber surface. All pathogenic species that cause common scab produce a family of phytotoxins called the thaxtomins, which function as cellulose synthesis inhibitors. In addition, the nec1 and tomA genes are conserved in several pathogenic streptomycetes, the former of which is predicted to function in the suppression of plant defense responses. Streptomyces scabies is the oldest plant pathogen described and has a world-wide distribution, whereas species such as S. turgidiscabies and S. acidiscabies are believed to be newly emergent pathogens found in more limited geographical locations. The genome sequence of S. scabies 87-22 was recently completed, and comparative genomic analyses with other sequenced microbial pathogens have revealed the presence of additional genes that may play a role in plant pathogenicity, an idea that is supported by functional analysis of one such putative virulence locus. In addition, the availability of multiple genome sequences for both pathogenic and nonpathogenic streptomycetes has provided an opportunity for comparative genomic analyses to identify the Streptomyces pathogenome. Such genomic analyses will contribute to the fundamental understanding of the mechanisms and evolution of plant pathogenicity and plant-microbe biology within this genus.     

Laser microdissection of conifer stem tissues: Isolation and analysis of high quality RNA,terpene synthase enzyme activity and terpenoid metabolites from resin ducts and cambial zone tissue of white spruce (<Emphasis Type="Italic">Picea glauca</Emphasis>)

Background  

Laser microdissection (LMD) has been established for isolation of individual tissue types from herbaceous plants. However, there are few reports of cell- and tissue-specific analysis in woody perennials. While microdissected tissues are commonly analyzed for gene expression, reports of protein, enzyme activity and metabolite analysis are limited due in part to an inability to amplify these molecules. Conifer stem tissues are organized in regular patterns with xylem, phloem and cortex development controlled by the activity of the cambial zone (CZ). Defense responses of conifer stems against insects and pathogens involve increased accumulation of terpenoids in cortical resin ducts (CRDs) and de novo formation of traumatic resin ducts from CZ initials. These tissues are difficult to isolate for tissue-specific molecular and biochemical characterization and are thus good targets for application of LMD.     

Phylogenetic analysis of the pPT23A plasmid family of Pseudomonas syringae

The pPT23A plasmid family of Pseudomonas syringae contains members that contribute to the ecological and pathogenic fitness of their P. syringae hosts. In an effort to understand the evolution of these plasmids and their hosts, we undertook a comparative analysis of the phylogeny of plasmid genes and that of conserved chromosomal genes from P. syringae. In total, comparative sequence and phylogenetic analyses were done utilizing 47 pPT23A family plasmids (PFPs) from 16 pathovars belonging to six genomospecies. Our results showed that the plasmid replication gene (repA), the only gene currently known to be distributed among all the PFPs, had a phylogeny that was distinct from that of the P. syringae hosts of these plasmids and from those of other individual genes on PFPs. The phylogenies of two housekeeping chromosomal genes, those for DNA gyrase B subunit (gyrB) and primary sigma factor (rpoD), however, were strongly associated with genomospecies of P. syringae. Based on the results from this study, we conclude that the pPT23A plasmid family represents a dynamic genome that is mobile among P. syringae pathovars.     

Directed evolution of the thermostable xylanase from Thermomyces lanuginosus

The thermostability of the endo-beta-1,4-xylanase from Thermomyces lanuginosus (xynA) was improved by directed evolution using error-prone PCR. Transformants expressing the variant xylanases were first selected on 0.4% Remazol Brilliant Blue-xylan and then exposed to 80 degrees C. Whereas the wild type XynA lost 90% activity after 10 min at 80 degrees C, five mutants displayed both higher stabilities and activities than XynA. Four mutants were subjected to further mutagenesis to improve the stability and activity of the xylanase. Subsequent screening revealed three mutants with enhanced thermostability. Mutant 2B7-10 retained 71% of its activity after treatment at 80 degrees C for 60 min and had a half-life of 215 min at 70 degrees C, which is higher than that attained by XynA. Sequence analysis of second generation mutants revealed that mutations were not concentrated in any particular region of the protein and exhibited much variation. The best mutant obtained from this study was variant 2B7-10, which had a single substitution (Y58F) in beta-sheet A of the protein, which is the hydrophilic, solvent-accessible outer surface of the enzyme. Most of the mutants obtained in this study displayed a compromise between stability and activity, the only exception being mutant 2B7-10. This variant showed increased activity and thermostability.