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B Garat

Publications and source records attributed to B Garat.

8 recordsLinked to original sources
PubMed2000

Characterisation of the proximal regulatory domain of the Echinococcus granulosus homeodomain-containing gene EgHbx1.

In an attempt to understand the molecular basis of the development of Echinococcus granulosus, we have previously isolated several homeobox-containing genes (EgHbx1-5). Here we report the characterisation of the EgHbx1 proximal regulatory domain. EgHbx1 codes for an amino acid sequence presenting 90% identity at the homeodomain level with the Drosophila melanogaster NK1-S59 transcription factor. The proximal regulatory domain sequence and the transcription start site were determined. Electrophoretic mobility shift assays using nuclear extracts from protoscoleces revealed the presence of specific DNA-protein complexes. These results constitute a hint and provide new tools to decipher regulatory cascades during E. granulosus development.

Animals↗
PubMed1998

Echinococcus granulosus: preparation of protein extracts from protoscolex nuclei for mobility-shift assays.

Nuclei from Echinococcus granulosus protoscolices were isolated from infected sheep. Protein extracts were prepared for analysis of DNA-protein interactions involving specific transcriptional regulatory factors. Gel mobility-shift assays were done using a heterologous probe containing binding sites for widespread transcription factors. A fragment of the promoter of GATA-1 transcription factor from the chicken was selected. When nuclear extracts from E. granulosus protoscolices were assayed a specific band shift was observed. The methodologies developed in this study could provide an important contribution for the characterization of the DNA-protein interactions involved in transcriptional regulation within the context of recent developments in the molecular biology of this parasite.

Animals↗
PubMed1997

Presence of a conserved domain of GATA transcription factors in Echinococcus granulosus.

In order to characterize GATA transcription factors in Echinococcus granulosus, a PCR-based cloning strategy was developed. Degenerate oligonucleotides were designed for the most conserved sequence in GATA proteins that include 20 amino acids of the zinc domain. A 60 bp fragment was isolated that had high homology among this sequence and those reported in other species. An analogous sequence was obtained by performing the same procedure with DNA from the free living platyhelminth Dugesia tigrina. High stringency Southern blotting experiments confirmed the presence of this sequence in the parasite genome.

Amino Acid Sequence↗
PubMed1987

Antibody heterogeneity: theoretical and experimental evaluation of a simple procedure to describe differing affinities in hapten binding reactions.

Heterogeneity in antibody binding affinity for hapten is easily and comprehensibly described by plotting the histogram of delta r, the change in the fraction of occupied active sites, vs the logarithm of the concn of free ligand. Computer simulations of binding reactions revealed that multimodal histograms occur only if differences in affinity are greater than 10-fold. Otherwise, the delta r histogram closely approximates a normal distribution, the variance of which increases with increasing heterogeneity. In biochemical experiments delta r histograms successfully detected mixtures of anti-DNP antibodies of varying affinities as well as changes between early and late anti-DNP immune responses in rabbits immunized with or without Freund's complete adjuvant.

Animals↗
PubMed1985

Presence of a membrane bound pyroglutamyl amino peptidase degrading thyrotropin releasing hormone in rat brain.

In the present work we studied the pattern of degradation of [3H-Pro]-TRH by soluble and membrane fractions from rat brain. Demonstration of the membrane bound or soluble nature of the activities was obtained by comparing their distribution to that of lactate dehydrogenase and by looking at the effect of NaCl washes on the membrane fractions. We observed that the pyroglutamyl amino peptidase activity detected in brain homogenates is a result of two different enzymes. One of them is a soluble enzyme previously characterized, that needs DTT and EDTA for its expression, is inhibited by SH-blocking agents such as iodoacetamide and utilizes p-glu-beta-naphtylamide as a substrate. The other one, a membrane enzyme, is inhibited by chelating agents such as EDTA and DTT, is not affected by iodoacetamide and does not degrade p-glu-beta-naphtylamide. The later presents some specificity towards TRH as shown by competition experiments with TRH analogs. We were able to corroborate that the post proline cleaving enzyme acting on TRH is a soluble enzyme. In membranes we demonstrated also the presence of a post-proline dipeptidyl aminopeptidase. The membrane bound pyroglutamidase activity is a potential new source of L-his-L-pro-diketopiperazine in brain. The presence of a TRH degrading enzyme in membrane fractions is of particular importance in searching an inactivation mechanism of this peptide once it is released into the synaptic cleft.

Aminopeptidases↗