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P Gallant

Publications and source records attributed to P Gallant.

At least 19 recordsLinked to original sources
PubMed2006

Myc/Max/Mad in invertebrates: the evolution of the Max network.

The Myc proto-oncogenes, their binding partner Max and their antagonists from the Mad family of transcriptional repressors have been extensively analysed in vertebrates. However, members of this network are found in all animals examined so far. Several recent studies have addressed the physiological function of these proteins in invertebrate model organisms, in particular Drosophila melanogaster. This review describes the structure of invertebrate Myc/Max/Mad genes and it discusses their regulation and physiological functions, with special emphasis on their essential role in the control of cellular growth and proliferation.

Amino Acid Sequence↗
PubMed2005

Human c-Myc isoforms differentially regulate cell growth and apoptosis in Drosophila melanogaster.

The human c-myc proto-oncogene, implicated in the control of many cellular processes including cell growth and apoptosis, encodes three isoforms which differ in their N-terminal region. The functions of these isoforms have never been addressed in vivo. Here, we used Drosophila melanogaster to examine their functions in a fully integrated system. First, we established that the human c-Myc protein can rescue lethal mutations of the Drosophila myc ortholog, dmyc, demonstrating the biological relevance of this model. Then, we characterized a new lethal dmyc insertion allele, which permits expression of human c-Myc in place of dMyc and used it to compare physiological activities of these isoforms in whole-organism rescue, transcription, cell growth, and apoptosis. These isoforms differ both quantitatively and qualitatively. Most remarkably, while the small c-MycS form truncated for much of its N-terminal trans-activation domain efficiently rescued viability and cell growth, it did not induce detectable programmed cell death. Our data indicate that the main functional difference between c-Myc isoforms resides in their apoptotic properties and that the N-terminal region, containing the conserved MbI motif, is decisive in governing the choice between growth and death.

Alleles↗
PubMed2000

Detection of small-molecule enzyme inhibitors with peptides isolated from phage-displayed combinatorial peptide libraries.

BACKGROUND: The rapidly expanding list of pharmacologically important targets has highlighted the need for ways to discover new inhibitors that are independent of functional assays. We have utilized peptides to detect inhibitors of protein function. We hypothesized that most peptide ligands identified by phage display would bind to regions of biological interaction in target proteins and that these peptides could be used as sensitive probes for detecting low molecular weight inhibitors that bind to these sites. RESULTS: We selected a broad range of enzymes as targets for phage display and isolated a series of peptides that bound specifically to each target. Peptide ligands for each target contained similar amino acid sequences and competition analysis indicated that they bound one or two sites per target. Of 17 peptides tested, 13 were found to be specific inhibitors of enzyme function. Finally, we used two peptides specific for Haemophilus influenzae tyrosyl-tRNA synthetase to show that a simple binding assay can be used to detect small-molecule inhibitors with potencies in the micromolar to nanomolar range. CONCLUSIONS: Peptidic surrogate ligands identified using phage display are preferentially targeted to a limited number of sites that inhibit enzyme function. These peptides can be utilized in a binding assay as a rapid and sensitive method to detect small-molecule inhibitors of target protein function. The binding assay can be used with a variety of detection systems and is readily adaptable to automation, making this platform ideal for high-throughput screening of compound libraries for drug discovery.

Alcohol Dehydrogenase↗
PubMed2000

Heparin infusion prior to stenting (HIPS) trial: final results of a prospective, randomized, controlled trial evaluating the effects of local vascular delivery on intimal hyperplasia.

BACKGROUND: Local delivery of pharmacologic agents or genes at the site of angioplasty is a promising approach to reduce restenosis. However, there are unresolved questions concerning the safety and feasibility of local vascular delivery in clinical practice as well as the efficacy of delivered drug. To this end, the safety, feasibility, and efficacy of local delivery of heparin were evaluated in the Heparin Infusion Prior to Stenting (HIPS) trial. METHODS AND RESULTS: A total of 179 patients were enrolled in this multicenter, randomized, prospective, core laboratory-evaluated trial. Patients were randomly assigned to 5000 U heparin either administered to the coronary artery lumen or infused into the arterial wall immediately after angioplasty and before stent placement. End points included procedural events and clinical, angiographic, and intravascular ultrasound events at 6 months. Patient groups were evenly matched. There was no difference in the incidence of arterial injury, defined as an increase in arterial dissection, acute closure, or decrease in Thrombolysis In Myocardial Infarction grade blood flow in the group receiving local delivery. At follow-up there was no difference in the major adverse event rate between intraluminal (22.7%) and local groups (24.7%). There was no difference between intraluminal and local therapy in the angiographic in-stent restenosis rate (12.5%, 12.7%) or the in-stent volumetric analysis by intravascular ultrasound (IVUS) (37.19 +/- 20. 86 mm(3) vs 43.79 +/- 25.52 mm(3)). CONCLUSIONS: Local delivery of 5000 U heparin into the arterial wall before stent implantation is safe and feasible. There was not a favorable effect of locally delivered heparin on clinical, angiographic, or IVUS end points of restenosis. The use of IVUS to measure volume of intimal hyperplasia in a multicenter, core laboratory-controlled trial is feasible.

Blood Flow Velocity↗
PubMed1999

Drosophila myc regulates cellular growth during development.

Transcription factors of the Myc proto-oncogene family promote cell division, but how they do this is poorly understood. Here we address the functions of Drosophila Myc (dMyc) during development. Using mosaic analysis in the fly wing, we show that loss of dMyc retards cellular growth (accumulation of cell mass) and reduces cell size, whereas dMyc overproduction increases growth rates and cell size. dMyc-induced growth promotes G1/S progression but fails to accelerate cell division because G2/M progression is independently controlled by Cdc25/String. We also show that the secreted signal Wingless patterns growth in the wing primordium by modulating dMyc expression. Our results indicate that dMyc links patterning signals to cell division by regulating primary targets involved in cellular growth and metabolism.

Alleles↗
PubMed1999

Local intramural heparin delivery during primary angioplasty for acute myocardial infarction: results of the Local PAMI Pilot Study.

The feasibility and safety of local heparin delivery during acute infarct angioplasty was evaluated in a prospective, multicenter, 120-patient series. Angioplasty was performed using standard techniques, after which heparin (4,000 U) was delivered locally; 25% of patients received stents. Procedural success was reported in 98% of patients; 6.7% of patients suffered death, reinfarction, recurrent ischemia, or stroke during the index hospitalization. The 6-month target vessel revascularization rate was 12.5%. Local heparin therapy with provisional stenting in acute myocardial infarction patients is safe, feasible, associated with a low rate of infarct artery revascularization at 6 months, and may potentially eliminate the need for systemic heparin following the procedure.

Aged↗
PubMed1998

A Drosophila RNA helicase gene, pitchoune, is required for cell growth and proliferation and is a potential target of d-Myc.

This article describes the characterization of a new Drosophila gene that we have called pitchoune (pit) (meaning small in Provence) because mutations in this gene produce larvae that cannot grow beyond the first instar larval stage although they can live as long as 7-10 days. All the tissues are equally affected and the perfectly shaped larvae are indistinguishable from first instar wild-type animals. Analysis of mutant somatic clones suggests a function in cell growth and proliferation, which is supported by the fact that cell proliferation is promoted by pit overexpression. Tagged-Pit, when transfected in S2 cells, localizes mainly to the nucleolus, pointing towards a possible role in ribosome biogenesis and, consequently, in protein biosynthesis. pit encodes a DEAD-box RNA helicase, a family of proteins involved in the control of RNA structure in many cellular processes and its closest homologue is a human DEAD-box RNA helicase, MrDb, whose corresponding gene transcription is directly activated by Myc-Max heterodimers (Grandori, C., Mac, J., Siëbelt, F., Ayer, D. E. and Eisenman, R. N. (1996) EMBO J. 15, 4344-4357). The patterns of expression of d-myc and pit are superimposable. Ectopic expression of myc in the nervous system drives an ectopic expression of pit in this tissue indicating that in Drosophila as well, pit is a potential target of d-Myc. These results suggest that myc might promote cell proliferation by activating genes that are required in protein biosynthesis, thus linking cell growth and cell proliferation.

Amino Acid Sequence↗
PubMed1996

Myc and Max homologs in Drosophila.

The proteins encoded by the myc proto-oncogene family are involved in cell proliferation, apoptosis, differentiation, and neoplasia. Myc acts through dimerization with Max to bind DNA and activate transcription. Homologs of the myc and max genes were cloned from the fruit fly Drosophila melanogaster and their protein products (dMyc and dMax) were shown to heterodimerize, recognize the same DNA sequence as their vertebrate homologs, and activate transcription. The dMyc protein is likely encoded by the Drosophila gene diminutive (dm), a mutation in which results in small body size and female sterility caused by degeneration of the ovaries. These findings indicate a potential role for Myc in germ cell development and set the stage for genetic analysis of Myc and Max.

Amino Acid Sequence↗
PubMed1996

A eubacterial Mycobacterium tuberculosis tRNA synthetase is eukaryote-like and resistant to a eubacterial-specific antisynthetase drug.

We report here the cloning and primary structure of Mycobacterium tuberculosis isoleucyl-tRNA synthetase. The predicted 1035-amino acid protein is significantly more similar in sequence to eukaryote cytoplasmic than to other eubacterial isoleucyl-tRNA synthetases. This similarity correlates with the enzyme being resistant to pseudomonic acid A, a potent inhibitor of Escherichia coli and other eubacterial isoleucyl-tRNA synthetases, but not of eukaryote cytoplasmic enzymes. Consistent with its eukaryote-like features, and unlike E. coli isoleucyl-tRNA synthetase, the M. tuberculosis enzyme charged yeast isoleucine tRNA. In spite of these eukaryote-like features, M. tuberculosis isoleucyl-tRNA synthetase exhibited highly specific cross-species aminoacylation, as demonstrated by its ability to complement isoleucyl-tRNA synthetase-deficient mutants of E. coli. When introduced into a pseudomonic acid-sensitive wild-type strain of E. coli, the M. tuberculosis enzyme conferred trans-dominant resistance to the drug. The results demonstrate that the sequence of a tRNA synthetase could have predictive value with respect to the interaction of that synthetase with a specific inhibitor. The results also demonstrate that mobilization of a pathogen's gene for a drug-resistant protein target can spread resistance to other, normally drug-sensitive pathogens infecting the same host.

Amino Acid Sequence↗
PubMed1996

Expression analysis of an Arabidopsis C2H2 zinc finger protein gene.

C2H2 zinc finger protein genes encode nucleic acid-binding proteins involved in the regulation of gene activity. AtZFP1 (Arabidopsis thaliana zinc finger protein 1) is one member of a small family of C2H2 zinc finger-encoding sequences previously characterized from Arabidopsis. The genomic sequence corresponding to the AtZFP1 cDNA has been determined. Molecular analysis demonstrates that AtZFP1 is a unique, intronless gene which encodes a 1100 nucleotides mRNA highly expressed in roots and stems. A construct in which 2.5 kb of AtZFP1 upstream sequences is linked to the beta-glucuronidase gene was introduced into Arabidopsis by Agrobacterium-mediated transformation of roots. Histochemical analysis of transgenic Arabidopsis carrying the AtZFP1 promoter: beta-glucuronidase fusion shows good correlation with RNA blot hybridization analysis. This transgenic line will be a useful tool for analyzing the regulation of AtZFP1 to further our understanding of its function.

Amino Acid Sequence↗
PubMed1995

Protein kinases in the control of mitosis: focus on nucleocytoplasmic trafficking.

The eukaryotic cell nucleus is a highly dynamic organelle. This is illustrated most dramatically during mitosis, when the nuclear envelope breaks down, the nuclear lamina disassembles, chromosomes condense, and a microtubule-based spindle apparatus distributes sister chromatids to the dividing daughter cells. Many of these dramatic changes in nuclear architecture and microtubule organization are controlled by phosphorylation and dephosphorylation events. Whereas the cardinal role of cyclin-dependent kinases (CDKs) in the regulation of mitosis is well established, there is now clear evidence for the requirement of additional mitotic protein kinases. Studies into the regulation of CDKs and other mitotic kinases have revealed that these enzymes undergo cell cycle dependent changes in subcellular distribution, suggesting that localization may contribute to regulating their activities. This article describes some recent findings relating to the nucleocytoplasmic translocation of CDK/cyclin complexes at the onset of mitosis. In addition, it summarizes recent information on two novel human protein kinases which have been implicated in the control of mitotic progression.

Amino Acid Sequence↗
PubMed1994

Identification of a novel vertebrate cyclin: cyclin B3 shares properties with both A- and B-type cyclins.

Cyclins play a key role in controlling progression through the cell cycle. They act as regulatory subunits of p34cdc2/CDC28 and related cyclin-dependent protein kinases (cdks). In vertebrates, cyclins B1 and B2 function during M phase, whereas cyclin A is required for S phase as well as the G2 to M phase transition. Here, we describe the identification and characterization of a novel vertebrate cyclin, termed cyclin B3. The assignment of this cyclin to the B-type subfamily is based on its cDNA-derived sequence and its pattern of expression in synchronized cells, both suggesting a distant relationship to other B-type cyclins. Interestingly, however, cyclin B3 also displays properties that resemble those of A- rather than B-type cyclins. Specifically, cyclin B3 localizes to the cell nucleus throughout the cell cycle, and is able to associate in vivo with at least two kinase subunits, p34cdc2 and p33cdk2. Furthermore, deletion of 26 amino acids from the C-terminus of cyclin B3 impairs both its interaction with kinase catalytic subunits and its nuclear localization, reminiscent of recent results obtained with cyclin A. Based on these observations, we conclude that cyclin B3 may share functional properties with both A- and B-type cyclins.

Amino Acid Sequence↗
PubMed1993

Nuclear localization of vertebrate cyclin A correlates with its ability to form complexes with cdk catalytic subunits.

Cyclins control the activities of cyclin-dependent protein kinases (cdks) and hence play a key role in cell cycle regulation. While B-type cyclins associate with p34cdc2 to trigger entry into mitosis, progression through S phase requires cyclin A, presumably in association with p33cdk2. Vertebrate A- and B-type cyclins display strikingly distinct subcellular localizations, but the mechanisms underlying these differential distributions are unknown. Here, we have begun to study the requirements for nuclear localization of cyclin A. We have isolated a cDNA coding for chicken cyclin A and constructed a series of deletion mutants. These were then transfected into HeLa cells, and the subcellular distribution of the mutant cyclin A proteins was determined by indirect immunofluorescence microscopy. In parallel, the cyclin A mutants were assayed for their ability to form complexes with cdk subunits. We found that deletion of more than 100 residues from the N terminus of cyclin A did not impair nuclear localization or cdk subunit binding and kinase activation. In contrast, removal of as few as 15 residues from the C terminus, or deletion of part of the internal cyclin box domain, abolished nuclear localization of cyclin A as well as its ability to bind to and activate cdk subunits. These results suggest that nuclear transport of cyclin A may depend on the formation of multiprotein complexes comprising cdk catalytic subunits.

Amino Acid Sequence↗
PubMed1992

Regulation of p34cdc2 protein kinase activity by phosphorylation and cyclin binding.

Activation of the protein kinase p34cdc2 is required for entry into meiotic or mitotic M phase in all eukaryotic cells. One important mechanism regulating the activity of p34cdc2 during the cell cycle is based on phosphorylation/dephosphorylation. Avian p34cdc2 is phosphorylated on threonine 14 (Thr14), tyrosine 15 (Tyr15), threonine 161 (Thr161) and serine 277 (Ser277). Dephosphorylation of both Thr14 and Tyr15 is required for activation of p34cdc2 at the G2/M transition, indicating that phosphorylation of these residues negatively regulates p34cdc2 activity. Conversely, phosphorylation of Thr161 is required for kinase activity. Whether modification of this residue is due to intramolecular autophosphorylation or to the action of an as yet unidentified kinase remains unresolved. Likewise, the role of phosphorylation of p34cdc2 on Ser277 during G1 phase of the cell cycle remains to be determined. The function of p34cdc2 is regulated also by cell cycle-dependent complex formation with cyclin proteins. We found that chicken cyclin B2 undergoes a striking redistribution from the cytoplasm to the nucleus just prior to the onset of mitosis. Expression of a non-destructible cyclin B2 mutant causes HeLa cells to arrest in mitosis. Frequently, arrested cells displayed multiple mitotic spindles.

Amino Acid Sequence↗