Keap1 perceives stress via three sensors for the endogenous signaling molecules nitric oxide, zinc, and alkenals.

Recognition and repair of cellular damage is crucial if organisms are to survive harmful environmental conditions. In mammals, the Keap1 protein orchestrates this response, but how it perceives adverse circumstances is not fully understood. Herein, we implicate NO, Zn(2+), and alkenals, endogenously occurring chemicals whose concentrations increase during stress, in this process. By combining molecular modeling with phylogenetic, chemical, and functional analyses, we show that Keap1 directly recognizes NO, Zn(2+), and alkenals through three distinct sensors. The C288 alkenal sensor is of ancient origin, having evolved in a common ancestor of bilaterans. The Zn(2+) sensor minimally comprises H225, C226, and C613. The most recent sensor, the NO sensor, emerged coincident with an expansion of the NOS gene family in vertebrates. It comprises a cluster of basic amino acids (H129, K131, R135, K150, and H154) that facilitate S-nitrosation of C151. Taken together, our data suggest that Keap1 is a specialized sensor that quantifies stress by monitoring the intracellular concentrations of NO, Zn(2+), and alkenals, which collectively serve as second messengers that may signify danger and/or damage.

Heterocellular OSM-OSMR signalling reprograms fibroblasts to promote pancreatic cancer growth and metastasis.

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy with a complex microenvironment. Dichotomous tumour-promoting and -restrictive roles have been ascribed to the tumour microenvironment, however the effects of individual stromal subsets remain incompletely characterised. Here, we describe how heterocellular Oncostatin M (OSM) - Oncostatin M Receptor (OSMR) signalling reprograms fibroblasts, regulates tumour growth and metastasis. Macrophage-secreted OSM stimulates inflammatory gene expression in cancer-associated fibroblasts (CAFs), which in turn induce a pro-tumourigenic environment and engage tumour cell survival and migratory signalling pathways. Tumour cells implanted in Osm-deficient (Osm-/-) mice display an epithelial-dominated morphology, reduced tumour growth and do not metastasise. Moreover, the tumour microenvironment of Osm-/- animals exhibit increased abundance of α smooth muscle actin positive myofibroblasts and a shift in myeloid and T cell phenotypes, consistent with a more immunogenic environment. Taken together, these data demonstrate how OSM-OSMR signalling coordinates heterocellular interactions to drive a pro-tumourigenic environment in PDA.

Fate of glycosylphosphatidylinositol (GPI)-less procyclin and characterization of sialylated non-GPI-anchored surface coat molecules of procyclic-form Trypanosoma brucei.

A Trypanosoma brucei TbGPI12 null mutant that is unable to express cell surface procyclins and free glycosylphosphatidylinositols (GPI) revealed that these are not the only surface coat molecules of the procyclic life cycle stage. Here, we show that non-GPI-anchored procyclins are N-glycosylated, accumulate in the lysosome, and appear as proteolytic fragments in the medium. We also show, using lectin agglutination and galactose oxidase-NaB(3)H(4) labeling, that the cell surface of the TbGPI12 null parasites contains glycoconjugates that terminate in sialic acid linked to galactose. Following desialylation, a high-apparent-molecular-weight glycoconjugate fraction was purified by ricin affinity chromatography and gel filtration and shown to contain mannose, galactose, N-acetylglucosamine, and fucose. The latter has not been previously reported in T. brucei glycoproteins. A proteomic analysis of this fraction revealed a mixture of polytopic transmembrane proteins, including P-type ATPase and vacuolar proton-translocating pyrophosphatase. Immunolocalization studies showed that both could be labeled on the surfaces of wild-type and TbGPI12 null cells. Neither galactose oxidase-NaB(3)H(4) labeling of the non-GPI-anchored surface glycoconjugates nor immunogold labeling of the P-type ATPase was affected by the presence of procyclins in the wild-type cells, suggesting that the procyclins do not, by themselves, form a macromolecular barrier.

Channel glass-based detection of human short insertion/deletion polymorphisms by tandem hybridization.

The development and critical evaluation of new technologies for identifying genetic polymorphisms will rapidly accelerate the discovery and diagnosis of disease-related genes. We report a novel way for distinguishing a new class of human DNA polymorphisms, short insertion/deletion polymorphisms (indels). A sensor with cylindrical pores named channel glass in combination with tandem hybridization, which uses a 5'-fluorescent labeled stacking probe and microarray-based short allele-specific oligonucleotide (capture probe) was investigated. This methodology allows indels to be detected individually and rapidly with small quantities of target DNA. This establishes a reliable quantitative test. Approaches for simultaneously hybridizing different targets to arrayed probes, designed to detect various indels in parallel, were examined. Five markers were consistently detected in a single hybridization. Possible factors impeding the hybridization reaction process are discussed.

A comparison of hybridization efficiency between flat glass and channel glass solid supports.

Two different solid supports, channel glass and flat glass, were compared for their affect on the sensitivity and efficiency of DNA hybridization reactions. Both solid supports were tested using a set of arrayed, synthetic oligonucleotides that are designed to detect short insertion/deletion polymorphisms (SIDPs). A total of 13 different human SIDPs were chosen for analysis. Capture probes, designed for this test set, were covalently immobilized on substrates. Hybridization efficiency was assessed using fluorescently labeled stacking probes which were preannealed to the target and then hybridized to the support-bound oligonucleotide array; the hybridization pattern was detected by fluorescence imaging. It was found that structural features of nucleic acid capture probes tethered to a solid support and the molecular basis of their interaction with targets in solution have direct implications on the hybridization process. Our results demonstrate that channel glass has a number of practical advantages over flat glass including higher sensitivity and a faster hybridization rate.

Comparative analysis of the excretory-secretory proteome of the muscle larva of Trichinella pseudospiralis and Trichinella spiralis.

The nematodes Trichinella spiralis and Trichinella pseudospiralis are both intracellular parasites of skeletal muscle cells and induce profound alterations in the host cell resulting in a re-alignment of muscle-specific gene expression. While T. spiralis induces the production of a collagen capsule surrounding the host-parasite complex, T. pseudospiralis exists in a non-encapsulated form and is also characterised by suppression of the host inflammatory response in the muscle. These observed differences between the two species are thought to be due to variation in the proteins excreted or secreted (ES proteins) by the muscle larva. In this study, we use a global proteomics approach to compare the ES protein profiles from both species and to identify individual T. pseudospiralis proteins that complement earlier studies with T. spiralis. Following two-dimensional gel electrophoresis, tandem mass spectrometry was used to identify the peptide spots. In many cases identification was aided by the determination of partial peptide sequence from selected mass ions. The T. pseudospiralis spots identified included the major secreted glycoproteins and the secreted 5'-nucleotidase. Furthermore, two major groups of T. spiralis-specific proteins and several T. pseudospiralis-specific proteins were identified. Our results demonstrate the value of proteomics as a tool for the identification of ES proteins that are differentially expressed between Trichinella species and as an aid to identifying key parasite proteins that are involved in the host-parasite interaction. The value of this approach will be further enhanced by data arising out the current T. spiralis genome sequencing project.

Proteins interacting with Saccharomyces cerevisiae type 1 protein phosphatase catalytic subunit identified by single-step affinity purification and mass spectrometry.

The catalytic subunit of type 1 protein phosphatase (PP1C) interacts with a large number of polypeptides in eukaryotic cells from yeast to man and these regulatory subunits can both modulate the activity of PP1C and target it to different subcellular locations. Thus, PP1 is really a family of protein phosphatases that share a common catalytic subunit, and identifying and characterizing the PP1-associated proteins is therefore critical to understanding the cellular roles of PP1 and its ability to dephosphorylate specific substrates. Here we describe methods for affinity isolation of PP1C-containing protein complexes in the yeast Saccharomyces cerevisiae and the identification of the associated polypeptides by mass spectrometry. The basic method we describe could be easily adapted to study PP1C-associated proteins in other lower or higher eukaryotes and for characterizing the protein-protein interactions of other protein phosphatases in yeast.

Fingerprinting of prokaryotic 16S rRNA genes using oligodeoxyribonucleotide microarrays and virtual hybridization.

An oligonucleotide microarray hybridization system to differentiate microbial species was designed and tested. Seven microbial species were studied, including one Bacillus and six Pseudomonas strains. DNA sequences near the 5' end of 16S rRNA genes were aligned and two contiguous regions of high variability, flanked by highly conserved sequences, were found. The conserved sequences were used to design PCR primers which efficiently amplified these polymorphic regions from all seven species. The amplicon sequences were used to design 88 9mer hybridization probes which were arrayed onto glass slides. Single-stranded, fluorescence-tagged PCR products were hybridized to the microarrays at 15 degrees C. The experimental results were compared with the DeltaG(0) values for all matched and mismatched duplexes possible between the synthetic probes and the 16S target sequences of the seven test species, calculated using a 'virtual hybridization' software program. Although the observed hybridization patterns differed significantly from patterns predicted solely on the basis of perfect sequence matches, a unique hybridization fingerprint was obtained for each of the species, including closely related Pseudomonas species, and there was a reasonable correlation between the intensity of observed hybridization signals and the calculated DeltaG(0) values. The results suggest that both perfect and mismatched pairings can contribute to microbial identification by hybridization fingerprinting.

Low density DNA microarray for detection of most frequent TP53 missense point mutations.

BACKGROUND: We have developed an oligonucleotide microarray (genosensor) utilizing a double tandem hybridization technique to search for 9 point mutations located in the most frequently altered codons of the TP53 gene. Isolated and multiplexed PCR products, 108 and 92 bp long, from exons 7 and 8, respectively, were obtained from 24 different samples. Single-stranded target DNA was then prepared from isolated or multiplexed PCR products, through cyclic DNA synthesis. Independent ssDNA's were annealed with the corresponding pairs of labeled stacking oligonucleotides to create partially duplex DNA having a 7-nt gap, which contains the sequence that will be interrogated by the capture probes forming double tandem hybridization. In the case of multiplexed ssPCR products, only two stacking oligonucleotides were added per target, therefore the gap for the PCR products having two consecutive codons to be interrogated in exon 7 was 12 nt long, so only single tandem hybridization was produced with these respective probes. RESULTS: 18 codon substitutions were found by DNA sequencing. In 13 of them a perfect correlation with the pattern of hybridization was seen (In 5 no signal was seen with the wt probe while a new signal was seen with the appropriate mutant probe, and in 8 more, as expected, no signal was seen with any probe due to the absence of the corresponding probe in the array). In 3 other cases a mutation was falsely suggested by the combination of the absence of the wild type signal along with a false signal in the other probe. In the other 2 cases the presence of the mutation was not detected due to the production of a false hybridization signal with the wild type probe. In both cases (false mutation or no mutation detected) relatively stable mismatched target/probe duplexes should be formed. These problems could be avoided by the addition of probes to improve the performance of the array. CONCLUSION: Our results demonstrate that a simple TP53 microarray employing short (7-mer) probes, used in combination with single or double tandem hybridization approach and a simple or multiplex target preparation method, can identify common TP53 missense mutations from a variety of DNA sources with good specificity.

Detection of mutations in RET proto-oncogene codon 634 through double tandem hybridization.

We developed a procedure to detect the 7 point mutations at Cys634 of the proto-oncogene RET, which is responsible for medullary thyroid carcinoma (MTC). Genomic DNA was prepared from blood samples obtained from normal and MTC-affected individuals belonging to a family with a history of the disease. The RET genotype for each individual was first established by performing restriction and sequencing analyses. Single-stranded target DNA was prepared by asymmetric polymerase chain reaction (PCR) amplification of a 93-bp fragment containing Cys634. The target was annealed with pairs of prelabeled stacking oligonucleotides designed to create appropriate 7-nucleotide gaps, which served as the sites of subsequent hybridization with glass-immobilized 7-mer probes. The target-stacking oligonucleotide duplexes were hybridized with DNA chips containing a set of eight 7-mer probes designed to detect the wild-type sequence and the seven point mutations described. We tested two sets of immobilized probes containing internal or 5'-terminal codon-634 single-base variations. Both groups of probes were able to discriminatively identify the mutations. The hybridization patterns indicated that the disease in this family was due to the C634Y mutation, in accord with the original sequence analysis. The hybridization-based mutation assignment was additionally supported by determination of the control homozygous and heterozygous hybridization patterns produced with synthetic targets having the normal or codon 634 mutant sequences. The effects of mismatch type and nearest-neighbor sequences on the occurrence of false-positive (mismatched) hybridizations are discussed.

Laboratory studies of dissolved radiolabelled microcystin-LR in lake water.

The fate of dissolved microcystin-LR was studied in laboratory experiments using surface water taken from a eutrophic lake. Based on initial range finding, a concentration of 50 microg l(-1) dissolved 14C-microcystin-LR was selected for subsequent time-course experiments. The first was performed in May before the cyanobacterial bloom season and low increases in the radioactivity of particulate fractions occurred with an approx. halving of the cyano-toxin during 4 days. The radioactivity of the dissolved fraction remained stable and there was no significant formation of radiolabelled inorganic carbon. A second time-course experiment was performed in September during the cyanobacterial bloom season. At the end of the four-day incubation period, the microcystin-LR concentration had decreased to an undetectable level and 24% of the added radiolabelled substance was found in different particulate fractions. The study demonstrated that biodegradation of dissolved microcystin-LR occurred in water collected at a lake surface with carbon dioxide as a major end-product.

Comparative effects and metabolism of two microcystins and nodularin in the brine shrimp Artemia salina.

The toxicity and metabolism of the cyanobacterial toxins microcystin-LR (MCLR), Dhb-microcystin-HtyR and nodularin were investigated in the cysts, nauplii and adults of the brine shrimp Artemia salina. The presence of the phase II detoxication system glutathione S-transferase (sGST) in these stages was shown using different substrates. Exposure of adult A. salina to the toxins led to an elevation of GST activity in vivo. All three toxins were conjugated to glutathione via GST, which has been shown as an initial step of microcystin and nodularin detoxication.

Bacterial classification using genomic fingerprints obtained by virtual hybridization.

In silico genomic fingerprints were produced by virtual hybridization of 191 fully sequenced bacterial genomes using a set of 15,264 13-mer probes specially designed to produce universal whole genome fingerprints. A novel approach for constructing phylogenetic trees, based on comparative analysis of genomic fingerprints, was developed. The resultant bacterial phylogenetic tree had strong similarities to those produced from the alignment of conserved sequences. Notably, the trees derived from the alignment of other conserved COG genes divided the Bacillus and Corynebacterium genera into the same subgroups produced by the novel bacterial tree. A number of discrepancies between both techniques were observed for the grouping of some Lactobacillus species. However, a detailed analysis of the alignment of these genomes using other bioinformatics tools revealed that the grouping of these organisms in the novel tree was more satisfactory than the groupings from previous classifications, which used only a few conserved genes. All these data suggest that the bacterial taxonomy produced by genomic fingerprints is satisfactory, but sometimes different from classical taxonomies. Discrepancies probably arise because the fingerprinting technique analyzes genomic sequences and reveals more information than previously used approaches.

LifePrint: a novel k-tuple distance method for construction of phylogenetic trees.

PURPOSE: Here we describe LifePrint, a sequence alignment-independent k-tuple distance method to estimate relatedness between complete genomes. METHODS: We designed a representative sample of all possible DNA tuples of length 9 (9-tuples). The final sample comprises 1878 tuples (called the LifePrint set of 9-tuples; LPS9) that are distinct from each other by at least two internal and noncontiguous nucleotide differences. For validation of our k-tuple distance method, we analyzed several real and simulated viroid genomes. Using different distance metrics, we scrutinized diverse viroid genomes to estimate the k-tuple distances between these genomic sequences. Then we used the estimated genomic k-tuple distances to construct phylogenetic trees using the neighbor-joining algorithm. A comparison of the accuracy of LPS9 and the previously reported 5-tuple method was made using symmetric differences between the trees estimated from each method and a simulated "true" phylogenetic tree. RESULTS: The identified optimal search scheme for LPS9 allows only up to two nucleotide differences between each 9-tuple and the scrutinized genome. Similarity search results of simulated viroid genomes indicate that, in most cases, LPS9 is able to detect single-base substitutions between genomes efficiently. Analysis of simulated genomic variants with a high proportion of base substitutions indicates that LPS9 is able to discern relationships between genomic variants with up to 40% of nucleotide substitution. CONCLUSION: Our LPS9 method generates more accurate phylogenetic reconstructions than the previously proposed 5-tuples strategy. LPS9-reconstructed trees show higher bootstrap proportion values than distance trees derived from the 5-tuple method.

Cell homeostasis in a Leishmania major mutant overexpressing the spliced leader RNA is maintained by an increased proteolytic activity.

Although several stage-specific genes have been identified in Leishmania, the molecular mechanisms governing developmental gene regulation in this organism are still not well understood. We have previously reported an attenuation of virulence in Leishmania major and L. braziliensis carrying extra-copies of the spliced leader RNA gene. Here, we surveyed the major differences in proteome and transcript expression profiles between the spliced leader RNA overexpressor and control lines using two-dimensional gel electrophoresis and differential display reverse transcription PCR, respectively. Thirty-nine genes related to stress response, cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, gene transcription, RNA processing and post-transcriptional regulation have abnormal patterns of expression in the spliced leader RNA overexpressor line. The evaluation of proteolytic pathways in the mutant revealed a selective increase of cysteine protease activity and an exacerbated ubiquitin-labeled protein population. Polysome profile analysis and measurement of cellular protein aggregates showed that protein translation in the spliced leader RNA overexpressor line is increased when compared to the control line. We found that L. major promastigotes maintain homeostasis in culture when challenged with a metabolic imbalance generated by spliced leader RNA surplus through modulation of intracellular proteolysis. However, this might interfere with a fine-tuned gene expression control necessary for the amastigote multiplication in the mammalian host.

Specific mutation screening of TP53 gene by low-density DNA microarray.

TP53 is the most commonly mutated gene in human cancers. Approximately 90% of mutations in this gene are localized between domains encoding exons 5 to 8. The aim of this investigation was to examine the ability of the low density DNA microarray with the assistance of double tandem hybridization platform to characterize TP53 mutational hotspots in exons 5, 7, and 8 of the TP53. Nineteen capture probes specific to each potential mutation site were designed to hybridize to specific site. Virtual hybridization was used to predict the stability of hybridization of each capture probe with the target. Thirty-three DNA samples from different sources were analyzed for mutants in these exons. A total of 32 codon substitutions were found by DNA sequencing. 24 of them a showed a perfect correlation with the hybridization pattern system and DNA sequencing analysis of the regions scanned. Although in this work we directed our attention to some of the most representative mutations of the TP53 gene, the results suggest that this microarray system proved to be a rapid, reliable, and effective method for screening all the mutations in TP53 gene.

Novel interactions of Saccharomyces cerevisiae type 1 protein phosphatase identified by single-step affinity purification and mass spectrometry.

The catalytic subunit of Saccharomyces cerevisiae type 1 protein phosphatase (PP1(C)) is encoded by the essential gene GLC7 and is involved in regulating diverse cellular processes. To identify potential regulatory or targeting subunits of yeast PP1(C), we tagged Glc7p at its amino terminus with protein A and affinity-purified Glc7p protein complexes from yeast. The purified proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and identified by peptide mass fingerprint analysis using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. To confirm the accuracy of our identifications, peptides from some of the proteins were also sequenced using high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry. Only four of the Glc7p-associated proteins that we identified (Mhp1p, Bni4p, Ref2p, and Sds22p) have previously been shown to interact with Glc7p, and multiple components of the CPF (cleavage and polyadenylation factor) complex involved in messenger RNA 3'-end processing were present as major components in the Glc7p-associated protein fraction. To confirm the interaction of Glc7p with this complex, we used the same approach to purify and characterize the components of the yeast CPF complex using protein A-tagged Pta1p. Six known components of the yeast (CPF) complex, together with Glc7p, were identified among the Pta1p-associated polypeptides using peptide mass fingerprint analysis. Thus Glc7p is a novel component of the CPF complex and may therefore be involved regulating mRNA 3'-end processing.