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Biomedical subjects

R A Dean

Publications and source records attributed to R A Dean.

At least 19 recordsLinked to original sources
PubMed2006

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.

Amino Acids↗
PubMed2006

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.

Alleles↗
PubMed2006

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.

Alanine↗
PubMed2005

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.

Adenocarcinoma↗
PubMed2004

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.

Computational Biology↗
PubMed2003

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.

Adenylyl Cyclases↗
PubMed2001

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.

Amino Acid Motifs↗
PubMed2001

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.

Chromosome Mapping↗
PubMed2001

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.

Chromosomes, Artificial, Bacterial↗
PubMed2000

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.

Adaptor Proteins, Signal Transducing↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed2000

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.

Arabidopsis↗
PubMed2000

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.

Aged↗
PubMed1999

A novel method for increasing the transformation efficiency of Escherichia coli-application forbacterial artificial chromosome library construction.

Bacterial artificial chromosome (BAC) libraries play a pivotal role in genomics studies. A crucial step in BAC library construction is the transformation of Escherichia coli by electroporation. Absolute efficiency (cfu/microgram DNA) is affected by a number of factors including the topological form and treatment of DNA samples. Here we report a simple new protocol using tRNA assisted precipitation that increased transformation efficiency by 70-fold for BAC ligations and up to 400-fold for plasmid ligations. The mechanism may involve altering or stabilizing the topographical form of the DNA molecules.

Chromosomes, Bacterial↗
PubMed1999

Physical map and gene survey of the Ochrobactrum anthropi genome using bacterial artificial chromosome contigs.

Bacterial artificial chromosome (BAC) clones are effective mapping and sequencing reagents for use with a wide variety of small and large genomes. This report describes research aimed at determining the genome structure of Ochrobactrum anthropi, an opportunistic human pathogen that has potential applications in biodegradation of hazardous organic compounds. A BAC library for O. anthropi was constructed that provides a 70-fold genome coverage based on an estimated genome size of 4.8 Mb. The library contains 3072 clones with an average insert size of 112 kb. High-density colony filters of the library were made, and a physical map of the genome was constructed using a hybridization without replacement strategy. In addition, 1536 BAC clones were fingerprinted with HindIII and analyzed using IMAGE and Fingerprint Contig software (FPC, Sanger Centre, U.K.). The FPC results supported the hybridization data, resulting in the formation of two major contigs representing the two major replicons of the O. anthropi genome. After determining a reduced tiling path, 138 BAC ends from the reduced tile were sequenced for a preliminary gene survey. A search of the public databases with the BLASTX algorithm resulted in 77 strong hits (E-value < 0.001), of which 89% showed similarity to a wide variety of prokaryotic genes. These results provide a contig-based physical map to assist the cloning of important genomic regions and the potential sequencing of the O. anthropi genome.

Chromosomes, Bacterial↗
PubMed1999

Physical map and organization of chromosome 7 in the rice blast fungus, Magnaporthe grisea.

The rice blast fungus Magnaporthe grisea is a highly destructive plant pathogen and one of the most important for studying various aspects of host-plant interactions. It has been widely adopted as a model organism because it is ideally suited for genetic and biological studies. To facilitate map-based cloning, chromosome walking, and genome organization studies of M. grisea, a complete physical map of chromosome 7 was constructed using a large-insert (130 kb) bacterial artificial chromosome (BAC) library. Using 147 chromosome 7-specific single-copy BAC clones and 20 RFLP markers on chromosome 7, 625 BAC clones were identified by hybridization. BAC clones were digested with HindIII, and fragments were size separated on analytical agarose gels to create DNA fingerprints. Hybridization contigs were constructed using a random cost algorithm, whereas fingerprinting contigs were constructed using the software package FPC. Results from both methods were generally in agreement, but numerous anomalies were observed. The combined data produced five robust anchored contigs after gap closure by chromosomal walking. The genetic and physical maps agreed closely. The final physical map was estimated to cover >95% of the 4.2 Mb of chromosome 7. Based on the contig maps, a minimum BAC tile containing 42 BAC clones was created, and organization of repetitive elements and expressed genes of the chromosome was investigated.

Algorithms↗
PubMed1998

A novel lipid-anchored A-kinase Anchoring Protein facilitates cAMP-responsive membrane events.

Compartmentalization of protein kinases with substrates is a mechanism that may promote specificity of intracellular phosphorylation events. We have cloned a low-molecular weight A-kinase Anchoring Protein, called AKAP18, which targets the cAMP-dependent protein kinase (PKA) to the plasma membrane, and permits functional coupling to the L-type calcium channel. Membrane anchoring is mediated by the first 10 amino acids of AKAP18, and involves residues Gly1, Cys4 and Cys5 which are lipid-modified through myristoylation and dual palmitoylation, respectively. Transient transfection of AKAP18 into HEK-293 cells expressing the cardiac L-type Ca2+ channel promoted a 34 9% increase in cAMP-responsive Ca2+ currents. In contrast, a targeting-deficient mutant of AKAP18 had no effect on Ca2+ currents in response to the application of a cAMP analog. Further studies demonstrate that AKAP18 facilitates GLP-1-mediated insulin secretion in a pancreatic beta cell line (RINm5F), suggesting that membrane anchoring of the kinase participates in physiologically relevant cAMP-responsive events that may involve ion channel activation.

A Kinase Anchor Proteins↗