Examining ubiquitinated peptide enrichment efficiency through an epitope labeled protein.

Ubiquitination is a dynamic process that is responsible for regulation of cellular responses to stimuli in a number of biological systems. Previous efforts to study this post-translational modification have focused on protein enrichment; however, recent research utilizes the presence of the di-glycine (Gly-Gly) remnants following trypsin digestion to immuno-enrich ubiquitinated peptides. Monoclonal antibodies developed to the cleaved ubiquitin modification epitope, (tert-butoxycarbonyl) glycylglycine (Boc-Gly-Gly-NHS)(1), are used to identify the Gly-Gly signature. Here, we have successfully generated the Boc-Gly-Gly-NHS modification and showed that when conjugated to a lysine containing protein, such as lysozyme, it can be applied as a standard protein to examine ubiquitinated peptide enrichment within a complex background.

The influence of matrix type, diurnal rhythm and sample collection and processing on the measurement of plasma beta-amyloid isoforms using the INNO-BIA plasma Abeta forms multiplex assay.

OBJECTIVE: The aim of the study was to determine the extent to which plasma matrix types, diurnal rhythm and sample collection and processing procedures contribute to overall variability of measurements with the INNO-BIA plasma Abeta forms assay. METHODS: Plasma samples from healthy volunteers were collected at BARC-CRI. Analyte concentrations from various plasma matrix types (EDTA, heparin, fluoride) were compared to serum after collection of blood in commercial plastic and glass tubes. Sample processing variables including time and temperature before and after centrifugation, centrifugal force and plasma dilution factor were also investigated. Diurnal variability in plasma Abeta isoforms was determined in 29 healthy volunteers by analysis of EDTA plasma specimens serially collected over 24 hours and stored frozen following oral administration of a placebo treatment. All plasma samples from a given individual and experiment were analyzed in a single analytical run. RESULTS: Highest Abeta levels were obtained using EDTA-plasma samples (in contrast to serum, heparin, citrate, or fluoride). Addition of aprotinin to EDTA plasma had no effect on Abeta peptide recovery. The elapsed time and temperature exposure, before and after sample processing affects the recovery of Abeta isoforms. Analyte recovery was not significantly affected by the presence of platelets in plasma samples. At the subject level, analysis of serially collected EDTA plasma specimens from healthy volunteers revealed no evidence of diurnal variation in any of the Abeta isoforms investigated and results from samples collected on a monthly basis showed only very limited intra-individual variation. CONCLUSIONS: Optimal recovery of Abeta peptides was obtained from blood drawn into EDTA tubes and processed within 4 h. Plasma that was refrigerated after separation and analysed within 4 h gave comparable results to samples immediately processed and frozen at -70 degrees C.

Global gene expression during nitrogen starvation in the rice blast fungus, Magnaporthe grisea.

Efficient regulation of nitrogen metabolism likely plays a role in the ability of fungi to exploit ecological niches. To learn about regulation of nitrogen metabolism in the rice blast pathogen Magnaporthe grisea, we undertook a genome-wide analysis of gene expression under nitrogen-limiting conditions. Five hundred and twenty genes showed increased transcript levels at 12 and 48 h after shifting the fungus to media lacking nitrate as a nitrogen source. Thirty-nine of these genes have putative functions in amino acid metabolism and uptake, and include the global nitrogen regulator in M. grisea, NUT1. Evaluation of seven nitrogen starvation-induced genes revealed that all were expressed during rice infection. Targeted gene replacement on one such gene, the vacuolar serine protease, SPM1, resulted in decreased sporulation and appressorial development as well as a greatly attenuated ability to cause disease. Data are discussed in the context of nitrogen metabolism under starvation conditions, as well as conditions potentially encountered during invasive growth in planta.

Construction of a watermelon BAC library and identification of SSRs anchored to melon or Arabidopsis genomes.

A bacterial artificial chromosome (BAC) library was constructed for watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus) with an average insert-size of 106 kb, providing 21 haploid genome equivalents. The library was used to identify BAC clones that are anchored to probes evenly distributed on the genomes of melon or Arabidopsis. Twenty eight probes (representing 66% of the tested probes) from melon and 30 probes (65%) from Arabidopsis identified positive BAC clones. Two methods were implemented to identify SSRs from the positively hybridizing BAC clones. First, analysis of BAC end sequences revealed 37 SSRs. For the second method, pooled DNA of BACs identified by the melon probes was used to develop a shotgun library. The library was then screened with synthetic SSR oligonucleotides by hybridization. Sequence analysis of positively hybridizing shotgun clones revealed 142 different SSRs. Thirty eight SSRs were characterized using three watermelon cultivars, five plant introduction (PI) accessions of C. lanatus var lanatus and four PIs of C. lanatus var citroides. Of these, 36 (95%) were found to be polymorphic with up to six alleles per marker. Polymorphism information content values for polymorphic markers varied between 0.22 and 0.79 with an average of 0.53. The methods described herein will be valuable for the construction of a watermelon linkage map with SSRs evenly distributed on its genome that is anchored to the genomes of melon and Arabidopsis.

Effects of a gamma-secretase inhibitor in a randomized study of patients with Alzheimer disease.

LY450139 dihydrate, a gamma-secretase inhibitor, was studied in a randomized, controlled trial of 70 patients with Alzheimer disease. Subjects were given 30 mg for 1 week followed by 40 mg for 5 weeks. Treatment was well tolerated. Abeta(1-40) in plasma decreased by 38.2%; in CSF, Abeta(1-40) decreased by 4.42 +/- 9.55% (p = not significant). Higher drug doses may result in additional decreases in plasma Abeta concentrations and a measurable decrease in CSF Abeta.

Whole genome comparison of Aspergillus flavus and A. oryzae.

Aspergillus flavus is a plant and animal pathogen that also produces the potent carcinogen aflatoxin. Aspergillus oryzae is a closely related species that has been used for centuries in the food fermentation industry and is Generally Regarded As Safe (GRAS). Whole genome sequences for these two fungi are now complete, providing us with the opportunity to examine any genomic differences that may explain the different ecological niches of these two fungi, and perhaps to identify pathogenicity factors in A. flavus. These two fungi are very similar in genome size and number of predicted genes. The estimated genome size (36·8 Mb) and predicted number of genes (12 197) for A. flavus is similar to that of A. oryzae (36·7 Mb and 12 079, respectively). These two fungi have significantly larger genomes than Aspergillus nidulans (30·1) and Aspergillus fumigatus (29·4). The A. flavus and A. oryzae genomes are enriched in genes for secondary metabolism, but do not differ greatly from one another in the predicted number of polyketide synthases, nonribosomal peptide synthases or the number of genes coding for cytochrome P450 enzymes. A micro-scale analysis of the two fungi did show differences in DNA correspondence between the two species and in the number of transposable elements. Each species has approximately 350 unique genes. The high degree of sequence similarity between the two fungi suggests that they may be ecotypes of the same species and that A. oryzae has resulted from the domestication of A. flavus.

Radiation induced MMP expression from rectal cancer is short lived but contributes to in vitro invasion.

AIMS: Matrix metalloproteinase (MMP) activity is increased after radiation. The aims of this study were to assess the time course of this increase and its effects on malignant cell invasion. METHODS: Colorectal cancer (HCT 116, LoVo, C 170 HM 2, CaCO-2), fibroblast (46-BR.IGI, CCD-18 Co) and fibrosarcoma (HT1080) cell lines were irradiated at 4 gray (4 Gy) and matrix metalloproteinase gene and protein expression examined over a 96 h period by real time polymerase chain reaction and gelatin zymography. Invasion was assessed on Matrigel. Human rectal tumour MMP expression was compared before and after long course radiotherapy. RESULTS: Radiation increased MMP gene expression of tumour cell lines, and resulted in increased MMP protein activity in the HT1080 line. HT1080 and HCT 116 in monoculture and LoVo in co-culture were more invasive after radiation at 48 h in vitro, but long course radiotherapy did not result in a consistent increase in MMP expression from human rectal tumour biopsies. CONCLUSIONS: Radiation results in increased MMP expression for a limited time period. This results in an early increase in cell line invasion. Further clinical research is required to clarify if MMP inhibition given perioperatively following radiotherapy decreases local recurrence rates.

Characterization of the protein processing and secretion pathways in a comprehensive set of expressed sequence tags from Trichoderma reesei.

Trichoderma reesei is a filamentous fungus widely used as an efficient protein producer and known to secrete large quantities of biomass degrading enzymes. Much work has been done aimed at improving the secretion efficiency of this fungus. It is generally accepted that the major bottlenecks in secretion are protein folding and ornamentation steps in this pathway. In an attempt to identify genes involved in these steps, the 5' ends of 21888 cDNA clones were sequenced from which a unique set of over 5000 were also 3' sequenced. Using annotation tools Gene Ontology terms were assigned to 2732 of the sequences. Homologs to the majority of Aspergillus niger's Srg genes as well as a number of homologs to genes involved in protein folding and ornamentation pathways were identified.

Identification of proteins that interact with two regulators of appressorium development, adenylate cyclase and cAMP-dependent protein kinase A, in the rice blast fungus Magnaporthe grisea.

Adenylate cyclase (MAC1) and the catalytic subunit of cAMP-dependent protein kinase A (CPKA) are required for appressorium development and pathogenesis in the rice blast pathogen Magnaporthe grisea. To identify new components in the cAMP signal transduction pathway, we used the yeast two-hybrid system to screen MAC1 and CPKA against an appressorium cDNA library. The cDNA library was constructed by GATEWAY recombinational cloning, enabling transfer of the library to various alternative vectors. The protein phosphatase domain in MAC1, which is unique to fungal adenylate cyclases, interacted with a MAP kinase kinase and a Ser/Thr kinase. Interactions of MAC1 with the kinases may prove to be part of feedback loops between the corresponding signaling pathways. A predicted membrane protein, ACI1, which is highly expressed under conditions that are conducive to appressorium formation, also interacted with MAC1. ACI1 has an extracellular domain containing eight-cysteines, which is also present in other fungal proteins implicated in pathogenesis. The N-terminal half of CPKA, which includes a glutamine-rich sequence unique to a group of fungal sequences, interacted with a putative transcriptional regulator and two different glycosyl hydrolases. Phosphorylation motifs in these sequences suggest that they could be CPKA substrates. The protein interaction assay employed here can now be scaled up to identify interactions between a larger set of proteins in the M. grisea interactome.

Development of PCR-ELISA for Detection and Differentiation of Didymella bryoniae from Related Phoma species.

The causal agent of gummy stem blight, Didymella bryoniae, often is isolated from infected cucurbits together with other Phoma spp. Polymerase chain reaction (PCR) primers specific to D. bryoniae and Phoma were used to develop and evaluate a microtiter-based PCR-enzyme-linked immunosorbent assay (ELISA) technique. Primers were modified by addition of a fluorescein and a biotin label to the 5' ends of the forward and reverse primers, respectively. After amplification, PCR products were detected in an ELISA using horseradish peroxidase-conjugated antifluorescein antibody and three substrates that yielded three colored products, one for each fungal group. The most sensitive substrate (highest signal:noise ratio) was 2,2' -azino-bis[3-ethylbenz-thiazoline-6-sulfonic acid]. PCR-ELISA successfully detected 45 of 46 D. bryoniae and all 13 Phoma isolates that were used. Results were comparable to those obtained with gel electrophoresis. Only one D. bryoniae isolate could not be detected with PCR-ELISA; this isolate also produced a fragment larger than other D. bryoniae isolates on agarose gels. PCR-ELISA was used successfully on crude extracts of "blind" fungal samples and identified seven of seven isolates as D. bryoniae or Phoma. Although less sensitive than gel electrophoresis, PCR-ELISA was a highly specific, yet simple, rapid and convenient assay for detection of D. bryoniae and Phoma sp.

Global analysis of protein activities using proteome chips.

To facilitate studies of the yeast proteome, we cloned 5800 open reading frames and overexpressed and purified their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with proteins and phospholipids. We identified many new calmodulin- and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. The microarrays can also be used to screen protein-drug interactions and to detect posttranslational modifications.

A framework for sequencing the rice genome.

Rice is an important food crop and a model plant for other cereal genomes. The Clemson University Genomics Institute framework project, begun two years ago in anticipation of the now ongoing international effort to sequence the rice genome, is nearing completion. Two bacterial artificial chromosome (BAC) libraries have been constructed from the Oryza sativa cultivar Nipponbare. Over 100,000 BAC end sequences have been generated from these libraries and, at a current total of 28 Mbp, represent 6.5% of the total rice genome sequence. This sequence information has allowed us to draw first conclusions about unique and redundant rice genomic sequences. In addition, more than 60,000 clones (19 genome equivalents) have been successfully fingerprinted and assembled into contigs using FPC software. Many of these contigs have been anchored to the rice chromosomes using a variety of techniques. Hybridization experiments have shown these contigs to be very robust. Contig assembly and hybridization experiments have revealed some surprising insights into the organization of the rice genome, which will have significant repercussions for the sequencing effort. Integration of BAC end sequence data with anchored contig information has provided unexpected revelations on sequence organization at the chromosomal level.

Melon bacterial artificial chromosome (BAC) library construction using improved methods and identification of clones linked to the locus conferring resistance to melon Fusarium wilt (Fom-2).

Utilizing improved methods, two bacterial artificial chromosome (BAC) libraries were constructed for the multidisease-resistant line of melon MR-1. The HindIII library consists of 177 microtiter plates in a 384-well format, while the EcoRI library consists of 222 microtiter plates. Approximately 95.6% of the HindIII library clones contain nuclear DNA inserts with an average size of 118 kb, providing a coverage of 15.4 genome equivalents. Similarly, 96% of the EcoRI library clones contain nuclear DNA inserts with an average size of 114 kb, providing a coverage of 18.7 genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBac536 vector, and organellar DNA sequences. High-density filters were screened with two genetic markers FM and AM that co-segregate with Fom-2, a gene conferring resistance to races 0 and 1 of Fusarium wilt. Fourteen and 18 candidate BAC clones were identified for the FM and AM probes, respectively, from the HindIII library, while 34 were identified for the AM probe from filters A, B, and C of the EcoRI library.

Phenotype Instability in Botrytis cinerea in the Absence of Benzimidazole and Dicarboximide Fungicides.

ABSTRACT Stability of phenotypes of isolates of Botrytis cinerea that were sensitive or resistant to benzimidazole and dicarboximide fungicides was examined in the absence of fungicides in laboratory and growth room experiments. Twelve greenhouse isolates of B. cinerea were subcultured on potato dextrose agar (PDA) for 20 generations and on geranium seedlings for 15 generations. Three isolates of each of the following four phenotypes were used: sensitive to the fungicides thiophanate-methy1 (a benzimidazole) and vinclozolin (a dicarboximide) (S(T)S(V)), resistant to both fungicides (R(T)R(V)), resistant to thiophanate-methy1 and sensitive to vinclozolin (R(T)S(V)), and sensitive to thiophanate-methy1 and resistant to vinclozolin (S(T)R(V)). In three trials on PDA, 36 populations were subcultured; 8 populations changed phenotypes by the end of 20 generations, as determined by conidium germination on fungicide-amended medium. Five of the eight initially were S(T)R(V); the resulting phenotypes were S(T)S(V), R(T)S(V), and R(T)R(V). Populations from eight other isolates exhibited temporary changes in phenotype during intermediate generations on PDA but reverted to initial phenotypes by the twentieth generation; five of these populations changed to phenotype R(T)R(V). In two geranium seedling trials, each of the 12 greenhouse isolates was inoculated onto a set of three seedlings for each generation, and diseased tissue that developed was used to initiate the next generation. Therefore, a total of 72 populations of B. cinerea were subcultured in the two trials; 5 of these populations changed phenotype at the end of 15 generations. Three of the five initially were S(T)R(V); these changed to phenotypes S(T)S(V) or R(T)R(V). In each of the two trials on geranium seedlings, a population subcultured from one S(T)S(V) isolate changed phenotype one to phenotype R(T)R(V) and one to phenotype R(T)S(V). In all trials, no population resistant to thiophanate-methy1 changed to a thiophanate-methy1-sensitive phenotype, and no population changed to phenotype S(T)R(V). Random amplified polymorphic DNA (RAPD) fingerprints were generated with the 12 initial isolates and 49 isolates subcultured on PDA or geranium seedlings. Cluster analyses of RAPD markers showed that subcultured isolates exhibiting the same phenotype clustered together and that subcultured isolates derived from a common greenhouse isolate but with different phenotypes were in different clusters. Some populations that did not change phenotype exhibited considerable differences in RAPD marker patterns. The results of this study indicate that, in the absence of fungicides, sensitive populations of B. cinerea can develop resistance to thiophanate-methy1 and vinclozolin, and this resistance can be maintained in populations through multiple generations. Populations resistant only to vinclozolin (S(T)R(V)) exhibited a high frequency of phenotype change, and populations resistant to both fungicides (R(T)R(V)) were stable.

Genes expressed during early stages of rice infection with the rice blast fungus Magnaporthe grisea.

summary A system-wide approach was adopted to further elucidate mechanisms regulating disease outcome between rice and the fungal pathogen Magnaporthe grisea. First, a cDNA library was constructed from M. grisea infected rice at 48 h post-inoculation. The 5' end-sequencing of 619 randomly selected clones revealed 359 expressed sequence tags (ESTs) that had not previously been described. A total of 124 from 260 ESTs with high and moderate similarity scores, based on BlastX, were organized into categories according to their putative function. The largest category of sequences (21%) contained stress or defence response genes. Eleven per cent of identified ESTs were redundant. In a second approach, differential hybridization analysis of the cDNA library using high-density filters resulted in the identification of novel genes and previously characterized M. grisea genes, including several that had previously been implicated in the infection process. A survey of up-regulated cDNA clones revealed clone 29003, which corresponded to the rice peroxidase POX22.3. This gene is known to be expressed in rice upon infection with Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen. Importantly, this approach demonstrates the utility of gene discovery, through ESTs, for revealing novel genes in addition to those previously characterized as being potentially implicated in host-pathogen interactions.

Site-directed mutagenesis of the magB gene affects growth and development in Magnaporthe grisea.

G protein signaling is commonly involved in regulating growth and differentiation of eukaryotic cells. We previously identified MAGB, encoding a Galpha subunit, from Magnaporthe grisea, and disruption of MAGB led to defects in a number of cellular responses, including appressorium formation, conidiation, sexual development, mycelial growth, and surface sensing. In this study, site-directed mutagenesis was used to further dissect the pleiotropic effects controlled by MAGB. Conversion of glycine 42 to arginine was predicted to abolish GTPase activity, which in turn would constitutively activate G protein signaling in magB(G42R). This dominant mutation caused autolysis of aged colonies, misscheduled melanization, reduction in both sexual and asexual reproduction, and reduced virulence. Furthermore, magB(G42R) mutants were able to produce appressoria on both hydrophobic and hydrophilic surfaces, although development on the hydrophilic surface was delayed. A second dominant mutation, magB(G203R) (glycine 203 converted to arginine), was expected to block dissociation of the Gbetagamma from the Galpha subunit, thus producing a constitutively inactive G protein complex. This mutation did not cause drastic phenotypic changes in the wild-type genetic background, other than increased sensitivity to repression of conidiation by osmotic stress. However, magB(G203R) is able to complement phenotypic defects in magB mutants. Comparative analyses of the phenotypical effects of different magB mutations are consistent with the involvement of the Gbetagamma subunit in the signaling pathways regulating cellular development in M. grisea.

Targeting of PKA to glutamate receptors through a MAGUK-AKAP complex.

Compartmentalization of glutamate receptors with the signaling enzymes that regulate their activity supports synaptic transmission. Two classes of binding proteins organize these complexes: the MAGUK proteins that cluster glutamate receptors and AKAPs that anchor kinases and phosphatases. In this report, we demonstrate that glutamate receptors and PKA are recruited into a macromolecular signaling complex through direct interaction between the MAGUK proteins, PSD-95 and SAP97, and AKAP79/150. The SH3 and GK regions of the MAGUKs mediate binding to the AKAP. Cell-based studies indicate that phosphorylation of AMPA receptors is enhanced by a SAP97-AKAP79 complex that directs PKA to GluR1 via a PDZ domain interaction. As AMPA receptor phosphorylation is implicated in regulating synaptic plasticity, these data suggest that a MAGUK-AKAP complex may be centrally involved.

Rice transposable elements: a survey of 73,000 sequence-tagged-connectors.

As part of an international effort to sequence the rice genome, the Clemson University Genomics Institute is developing a sequence-tagged-connector (STC) framework. This framework includes the generation of deep-coverage BAC libraries from O. sativa ssp. japonica c.v. Nipponbare and the sequencing of both ends of the genomic DNA insert of the BAC clones. Here, we report a survey of the transposable elements (TE) in >73,000 STCs. A total of 6848 STCs were found homologous to regions of known TE sequences (E<10(-5)) by FASTX search of STCs against a set of 1358 TE protein sequences obtained from GenBank. Of these TE-containing STCs (TE-STCs), 88% (6027) are related to retroelements and the remaining are transposase homologs. Nearly all DNA transposons known previously in plants were present in the STCs, including maize Ac/Ds, En/Spm, Mutator, and mariner-like elements. In addition, 2746 STCs were found to contain regions homologous to known miniature inverted-repeat transposable elements (MITEs). The distribution of these MITEs in regions near genes was confirmed by EST comparisons to MITE-containing STCs, and our results showed that the association of MITEs with known EST transcripts varies by MITE type. Unlike the biased distribution of retroelements in maize, we found no evidence for the presence of gene islands when we correlated TE-STCs with a physical map of the CUGI BAC library. These analyses of TEs in nearly 50 Mb of rice genomic DNA provide an interesting and informative preview of the rice genome.

The effects of hormone replacement therapy and raloxifene on C-reactive protein and homocysteine in healthy postmenopausal women: a randomized, controlled trial.

C-Reactive protein and homocysteine are independent risk factors for the development of cardiovascular disease. This study compared the effects of hormone replacement therapy (HRT) and raloxifene on serum C-reactive protein and homocysteine levels as markers of cardiovascular risk in healthy postmenopausal women. Healthy postmenopausal women (n = 390) were enrolled in a double blind, randomized, placebo-controlled, 6-month trial at eight out-patient sites in the United States. Women were randomly assigned to receive continuous combined HRT (0.625 mg/day conjugated equine estrogen and 2.5 mg/day medroxyprogesterone acetate), raloxifene (60 or 120 mg/day), or placebo for 6 months. C-Reactive protein and homocysteine were measured in baseline and 6-month serum samples. HRT increased C-reactive protein levels by 84% (P<0.001), whereas raloxifene (60 and 120 mg/day) had no significant effect (-6% and -4%;, respectively; P>0.2). Raloxifene (60 and 120 mg/day) significantly lowered serum levels ofhomocysteine by 8% (P = 0.014) and 6% (P = 0.024), respectively, similar to the 7% (P = 0.014) reduction obtained with HRT. We conclude that HRT and raloxifene lower serum homocysteine levels to a comparable extent in postmenopausal women. Whereas cardiovascular risk predicted by C-reactive protein in healthy postmenopausal women is not influenced by raloxifene, the relationship between elevated C-reactive protein levels with HRT and cardiovascular disease events requires further study.