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A study was conducted to examine the binding of Ca+ and Mg2+ with SynCam which was a synthetic hybrid of CaM that could activate all the CaM enzymes. The thermodynamic binding parameters of Ca2+-CaM and Mg2+-CaM complexes were characterized through the use of flow dialysis and microcalorimetry under varying conditions. Results revealed that Ca2+-induced change in CaM dynamics contributed to the latter's capabilities while Mg2+ functioned as allosteric effector.

A novel chromosome mapping technique was developed based on a low coverage shotgun approach and directed sequencing steps. The method was utilized for nucleotide sequencing of the 115 kb chromosomal fragment of the Bacillus subtilis genome. Low coverage shotgun approach for the mapping of the 115 kb chromosomal fragment indicated the presence of 21 open reading frames that belong to seven transcription units. The G+C content of the segment is greater than 47% and contains genes that regulate the synthesis of exopolysaccharide.

Electrospray ionization mass spectrometry (ESI-MS) studies on the binding of Ca and Mg ions to four calmodulins, (CaM) SynCaM 1, SynCaM 8, and SynCaM 12A and SynCaM 18A, indicate that CaMs can bind four to six Ca ions and two Mg ions on auxiliary sites. This binding takes place between pH 6 and 8. There are three kinds of sites, the first one being of high affinity and the other two of low affinity. The auxilarry sites probably have a regulatory role since both Mg2+ ions and Ca2+ ions do not bind to the same site.

The lacA gene encoding beta-galactosidase in Bacillus subtilis and a regulator gene, lacR were analyzed to determine its characterization. The experiment utilized a transposon mutaganesis method. Results revealed that putative operon containing lacA is a negative regulator. On the other hand, lacR effectively encodes a product related to a family of regulators and includes the lactose repressor of Escherichia coli.

Abstract Background Tumor initiating cells (TICs) provide a new paradigm for developing original therapeutic strategies. Methods We screened for TICs in 47 human adult brain malignant tumors. Cells forming floating spheres in culture, and endowed with all of the features expected from tumor cells with stem-like properties were obtained from glioblastomas, medulloblastoma but not oligodendrogliomas. Results A long-term self-renewal capacity was particularly observed for cells of malignant glio-neuronal tumors (MGNTs). Cell sorting, karyotyping and proteomic analysis demonstrated cell stability throughout prolonged passages. Xenografts of fewer than 500 cells in Nude mouse brains induced a progressively growing tumor. CD133, CD15/LeX/Ssea-1, CD34 expressions, or exclusion of Hoechst dye occurred in subsets of cells forming spheres, but was not predictive of their capacity to form secondary spheres or tumors, or to resist high doses of temozolomide. Conclusions Our results further highlight the specificity of a subset of high-grade gliomas, MGNT. TICs derived from these tumors represent a new tool to screen for innovative therapies.

Abstract Background Pharmacological high-throughput screening (HTS) represents a powerful strategy for drug discovery in genetic diseases, particularly when the full spectrum of pathological dysfunctions remains unclear, such as in Friedreich ataxia (FRDA). FRDA, the most common recessive ataxia, results from a generalized deficiency of mitochondrial and cytosolic iron-sulfur cluster (ISC) proteins activity, due to a partial loss of frataxin function, a mitochondrial protein proposed to function as an iron-chaperone for ISC biosynthesis. In the absence of measurable catalytic function for frataxin, a cell-based assay is required for HTS assay. Methods Using a targeted ribozyme strategy in murine fibroblasts, we have developed a cellular model with strongly reduced levels of frataxin. We have used this model to screen the Prestwick Chemical Library, a collection of one thousand off-patent drugs, for potential molecules for FRDA. Results The frataxin deficient cell lines exhibit a proliferation defect, associated with an ISC enzyme deficit. Using the growth defect as end-point criteria, we screened the Prestwick Chemical Library. However no molecule presented a significant and reproducible effect on the proliferation rate of frataxin deficient cells. Moreover over numerous passages, the antisense ribozyme fibroblast cell lines revealed an increase in frataxin residual level associated with the normalization of ISC enzyme activities. However, the ribozyme cell lines and FRDA patient cells presented an increase in Mthfd2 transcript, a mitochondrial enzyme that was previously shown to be upregulated at very early stages of the pathogenesis in the cardiac mouse model. Conclusion Although no active hit has been identified, the present study demonstrates the feasibility of using a cell-based approach to HTS for FRDA. Furthermore, it highlights the difficulty in the development of a stable frataxin-deficient cell model, an essential condition for productive HTS in the future.

Background: Pharmacological high-throughput screening (HTS) represents a powerful strategy for drug discovery in genetic diseases, particularly when the full spectrum of pathological dysfunctions remains unclear, such as in Friedreich ataxia (FRDA). FRDA, the most common recessive ataxia, results from a generalized deficiency of mitochondrial and cytosolic iron-sulfur cluster (ISC) proteins activity, due to a partial loss of frataxin function, a mitochondrial protein proposed to function as an iron-chaperone for ISC biosynthesis. In the absence of measurable catalytic function for frataxin, a cell-based assay is required for HTS assay.Methods: Using a targeted ribozyme strategy in murine fibroblasts, we have developed a cellular model with strongly reduced levels of frataxin. We have used this model to screen the Prestwick Chemical Library, a collection of one thousand off-patent drugs, for potential molecules for FRDA.Results: The frataxin deficient cell lines exhibit a proliferation defect, associated with an ISC enzyme deficit. Using the growth defect as end-point criteria, we screened the Prestwick Chemical Library. However no molecule presented a significant and reproducible effect on the proliferation rate of frataxin deficient cells. Moreover over numerous passages, the antisense ribozyme fibroblast cell lines revealed an increase in frataxin residual level associated with the normalization of ISC enzyme activities. However, the ribozyme cell lines and FRDA patient cells presented an increase in Mthfd2 transcript, a mitochondrial enzyme that was previously shown to be upregulated at very early stages of the pathogenesis in the cardiac mouse model.Conclusion: Although no active hit has been identified, the present study demonstrates the feasibility of using a cell-based approach to HTS for FRDA. Furthermore, it highlights the difficulty in the development of a stable frataxin-deficient cell model, an essential condition for productive HTS in the future. [ABSTRACT FROM AUTHOR]