Biomedical subjects

Eric Blanc

Publications and source records attributed to Eric Blanc.

7 recordsLinked to original sources

PubMed2005

Genome-wide gene expression in response to parasitoid attack in Drosophila.

BACKGROUND: Parasitoids are insect parasites whose larvae develop in the bodies of other insects. The main immune defense against parasitoids is encapsulation of the foreign body by blood cells, which subsequently often melanize. The capsule sequesters and kills the parasite. The molecular processes involved are still poorly understood, especially compared with insect humoral immunity. RESULTS: We explored the transcriptional response to parasitoid attack in Drosophila larvae at nine time points following parasitism, hybridizing five biologic replicates per time point to whole-genome microarrays for both parasitized and control larvae. We found significantly different expression profiles for 159 probe sets (representing genes), and we classified them into 16 clusters based on patterns of co-expression. A series of functional annotations were nonrandomly associated with different clusters, including several involving immunity and related functions. We also identified nonrandom associations of transcription factor binding sites for three main regulators of innate immune responses (GATA/srp-like, NF-kappaB/Rel-like and Stat), as well as a novel putative binding site for an unknown transcription factor. The appearance or absence of candidate genes previously associated with insect immunity in our differentially expressed gene set was surveyed. CONCLUSION: Most genes that exhibited altered expression following parasitoid attack differed from those induced during antimicrobial immune responses, and had not previously been associated with defense. Applying bioinformatic techniques contributed toward a description of the encapsulation response as an integrated system, identifying putative regulators of co-expressed and functionally related genes. Genome-wide studies such as ours are a powerful first approach to investigating novel genes involved in invertebrate immunity.

Animals↗

PubMed2005

Evolutionary and mechanistic relationships between glycosidases acting on alpha- and beta-bonds.

Because of the fast accumulation of sequences derived from genome sequencing efforts, the sampling of the sequence space in glycosidase and related enzyme families is such that sensitive sequence similarity detection methods like PSI-BLAST are now able to reveal distant, but clear, structural and evolutionary relations between glycosidases acting on alpha- and beta-bonds. We have observed this trend within groups of glycosidases with completely different folds. We postulate that the evolutionary interconversion between alpha- and beta-acting glycosidases was greatly facilitated by the fact that both types share a similar axial orientation of the glycosidic bond in the reactive bound substrate. Glycosides in the beta anomeric configuration, require a sugar ring distortion, resulting in an axial orientation of the glycosidic bond, equivalent to that of an alpha glycosidic bond, prior to displacement by nucleophilic substitution.

Evolution, Molecular↗

PubMed2004

Shared transcriptional signature in Caenorhabditis elegans Dauer larvae and long-lived daf-2 mutants implicates detoxification system in longevity assurance.

In the nematode Caenorhabditis elegans, formation of the long-lived dauer larva and adult aging are both controlled by insulin/insulin-like growth factor-1 signaling. Potentially, increased adult life span in daf-2 insulin/insulin-like growth factor-1 receptor mutants results from mis-expression in the adult of a dauer larva longevity program. By using oligonucleotide microarray analysis, we identified a dauer transcriptional signature in daf-2 mutant adults. By means of a nonbiased statistical approach, we identified gene classes whose expression is altered similarly in dauers and daf-2 mutants, which represent potential determinants of life span. These include known determinants of longevity; the small heat shock protein/alpha-crystallins are up-regulated in both milieus. The cytochrome P450, short-chain dehydrogenase/reductase, UDP-glucuronosyltransferase, and glutathione S-transferase (in daf-2 mutants) gene classes were also up-regulated. These four gene classes act together in metabolism and excretion of toxic endobiotic and xenobiotic metabolites. This suggests that diverse toxic lipophilic and electrophilic metabolites, disposed of by phase 1 and phase 2 drug metabolism, may be the major determinants of the molecular damage that causes aging. In addition, we observed down-regulation of genes linked to nutrient uptake, including nhx-2 and pep-2. These work together in the uptake of dipeptides in the intestine, implying dietary restriction in daf-2 mutants. Some gene groups up-regulated in dauers and/or daf-2 were enriched for certain promoter elements as follows: the daf-16-binding element, the heat shock-response element, the heat shock-associated sequence, or the hif-1-response element. By contrast, the daf-16-associated element was enriched in genes down-regulated in dauers and daf-2 mutants. Thus, particular promoter elements appear longevity-associated or aging associated.

Aging↗

PubMed2004

On the interpretation and use of <|E|2>(d*) profiles.

Profiles of squared normalized structure factors, <|E|(2)>(d*), have been computed for a large number of proteins and nucleic acids. These are interpreted in terms of their underlying structural features. It is also shown that the 'solvent dip' at around 6.3 A resolution is to a large extent a protein secondary-structure effect that is enhanced by the water structure. A hierarchical classification of protein structures based on their <|E|(2)>(d*) profiles is briefly outlined, together with the use of <|E|(2)>(d*) profiles as an improvement over Wilson absolute scale estimation and as a novel solvent-modelling method.

Cluster Analysis↗

PubMed2003

Why are there so many carbohydrate-active enzyme-related genes in plants?

Plants contain far more carbohydrate-active enzyme-encoding genes than any other organism sequenced to date. The extremely large number of glycosidase and glycosyltransferase-related genes in plant genomes can be explained by the complex structure of the plant cell wall, by ancient genome duplication and by recent local duplications, but also by the recent emergence of novel and unrelated protein functions based on widely available pre-existing scaffolds.

Carbohydrate Metabolism↗

PubMed2002

A medium-throughput crystallization approach.

The first results of a medium-scale structural genomics program clearly demonstrate the value of using a medium-throughput crystallization approach based on a two-step procedure: a large screening step employing robotics, followed by manual or automated optimization of the crystallization conditions. The structural genomics program was based on cloning in the Gateway vectors pDEST17, introducing a long 21-residue tail at the N-terminus. So far, this tail has not appeared to hamper crystallization. In ten months, 25 proteins were subjected to crystallization; 13 yielded crystals, of which ten led to usable data sets and five to structures. Furthermore, the results using a robot dispensing 50-200 nl drops indicate that smaller protein samples can be used for crystallization. These still partial results might indicate present and future directions for those who have to make crucial choices concerning their crystallization platform in structural genomics programs.

Crystallization↗

PubMedDate not supplied

Synthesis, characterization, and application of cy-dye- and alexa-dye-labeled hongotoxin(1) analogues. The first high affinity fluorescence probes for voltage-gated K+ channels.

Hongotoxin(1) (HgTX(1)), a 39-residue peptide recently isolated from the venom of Centruroides limbatus, blocks the voltage-gated K+ channels K(v)1.1, K(v)1.2, and K(v)1.3 at picomolar toxin concentrations (Koschak, A., Bugianesi, R. M., Mitterdorfer, J., Kaczorowski, G. J., Garcia, M. L., and Knaus, H. G. (1998) J. Biol. Chem. 273, 2639-2644). In this report, we determine the three-dimensional structure of HgTX(1) using NMR spectroscopy (PDB-code: 1HLY). HgTX(1) was found to possess a structure similar to previously characterized K+ channel toxins (e.g. margatoxin) consisting of a three-stranded antiparallel beta-sheet (residues 2-4, 26-30, and 33-37) and a helical conformation (part 3(10) helix and part alpha helix; residues 10-20). Due to the importance of residue Lys-28 for high-affinity interaction with the respective channels, lysine-reactive fluorescence dyes cannot be used to label wild-type HgTX(1). On the basis of previous studies (see above) and our NMR data, a HgTX(1) mutant (HgTX(1)-A19C) was engineered, expressed, and purified. HgTX(1)-A19C-SH was labeled using sulfhydryl-reactive Cy3-, Cy5-, and Alexa-dyes. Pharmacological characterization of fluorescently labeled HgTX(1)-A19C in radioligand binding studies indicated that these hongotoxin(1) analogues retain high-affinity for voltage-gated K+ channels and a respective pharmacological profile. Cy3- and Alexa-dye-labeled hongotoxin(1) analogues were used to investigate the localization of K+ channels in brain sections. The distribution of toxin binding closely follows the distribution of K(v)1.2 immunoreactivity with the highest expression levels in the cerebellar Purkinje cell layer. Taken together, these results demonstrate that fluorescently labeled HgTX(1) analogues comprise novel probes to characterize a subset of voltage-gated K+ channels.

Animals↗