Crystallization and preliminary X-ray diffraction studies of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Methanothermus fervidus.

Journal ArticleThe homotetrameric holo-D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Methanothermus fervidus has been crystallized in the presence of NADP+ using the hanging-drop vapour-diffusion method. Crystals grew from a solution containing 2-methyl-2,4-pentanediol and magnesium acetate. A native data set has been collected to 2.1 A using synchrotron radiation and cryocooling. Diffraction data have been processed in the orthorhombic system (space group P21212) with unit-cell dimensions a = 136.7, b = 153.3, c = 74.9 A and one tetramer per asymmetric unit

17O NMR and FT-IR study of the ionization state of peptides in aprotic solvents Application to Leu-enkephalin

AbstractThe ionization state of Leu-enkephalin in DMSO and MeCN/DMSO (4/1) solution was studied by the combined use of 17O NMR and FT-IR spectroscopy. After lyophilization or an aqueous solution at nearly neutral pH, Leu-enkephalin essentially exists in the uncharged state in MeCN/DMSO (4/1) solution. In pure DMSO, only 40% of the Leu-enkephalin molecules are in the zwitterionic state under the same conditions

A Reliable Method for the Selection of Exploitable Melanoma Archival Paraffin Embedded Tissues for Transcript Biomarker Profiling

The source tissue for biomarkers mRNA expression profiling of tumors has traditionally been fresh-frozen tissue. The adaptation of formalin-fixed, paraffin-embedded (FFPE) tissues for routine mRNA profiling would however be invaluable in view of their abundance and the clinical information related to them. However, their use in the clinic remains a challenge due to the poor quality of RNA extracted from such tissues. Here, we developed a method for the selection of melanoma archival paraffin-embedded tissues that can be reliably used for transcript biomarker profiling. For that, we used qRT-PCR to conduct a comparative study in matched pairs of frozen and FFPE melanoma tissues of the expression of 25 genes involved in angiogenesis/tumor invasion and 15 housekeeping genes. A classification method was developed that can select the samples with a good frozen/FFPE correlation and identify those that should be discarded on the basis of paraffin data for four reference genes only. We propose therefore a simple and inexpensive assay which improves reliability of mRNA profiling in FFPE samples by allowing the identification and analysis of “good” samples only. This assay which can be extended to other genes would however need validation at the clinical level and on independent tumor series

In Vitro Transformation of Primary Human CD34+ Cells by AML Fusion Oncogenes: Early Gene Expression Profiling Reveals Possible Drug Target in AML

Different fusion oncogenes in acute myeloid leukemia (AML) have distinct clinical and laboratory features suggesting different modes of malignant transformation. Here we compare the in vitro effects of representatives of 4 major groups of AML fusion oncogenes on primary human CD34+ cells. As expected from their clinical similarities, MLL-AF9 and NUP98-HOXA9 had very similar effects in vitro. They both caused erythroid hyperplasia and a clear block in erythroid and myeloid maturation. On the other hand, AML1-ETO and PML-RARA had only modest effects on myeloid and erythroid differentiation. All oncogenes except PML-RARA caused a dramatic increase in long-term proliferation and self-renewal. Gene expression profiling revealed two distinct temporal patterns of gene deregulation. Gene deregulation by MLL-AF9 and NUP98-HOXA9 peaked 3 days after transduction. In contrast, the vast majority of gene deregulation by AML1-ETO and PML-RARA occurred within 6 hours, followed by a dramatic drop in the numbers of deregulated genes. Interestingly, the p53 inhibitor MDM2 was upregulated by AML1-ETO at 6 hours. Nutlin-3, an inhibitor of the interaction between MDM2 and p53, specifically inhibited the proliferation and self-renewal of primary human CD34+ cells transduced with AML1-ETO, suggesting that MDM2 upregulation plays a role in cell transformation by AML1-ETO. These data show that differences among AML fusion oncogenes can be recapitulated in vitro using primary human CD34+ cells and that early gene expression profiling in these cells can reveal potential drug targets in AML

N-Méthyl peptides. VIII étude radiocristallographique du repliement ßVI des séquences homochirales

Les structures cristallines des deux dipeptides tBuCO-L-Pro-Me-L-X-NHMe avec X = Leu et Phe ont été résolues par diffraction des rayons X. Tous deux adoptent la forme repliée βVI caractérisée par une liaison hydrogène unissant les sites NH et CO extrêmes et par une conformation cis de la liaison amide médiane N-méthylée. Il apparaît que ce type de repliement est particulièrement favorisé dans le cas d'une séquence peptidique homochirale N-méthylée

N-methyl peptides

The influence of N-methylation on the preferential orientations of the peptide side-substituents has been studied on Pro-X and X-Pro sequences (X = Ala, Leu or Phe), protected on both ends by amide functions. We have considered the vicinal coupling constants in the CαH-CβH2 fragment and the upfield shifts of the Pro proton signals due to the Phe phenyl ring, on the basis of the conformations assumed by the peptide backbone. It appears that N-methylation does not affect the Cα-Cβ rotamer distribution for leucine, but greatly destabilizes the g- g+ conformation for phenylalanine In the latter case, the release of an attractive interaction between Phe-NH and π-orbitals, due to N-methylation. must also be considered

Mise au point Modulations conformationnelles du repliement β en serie peptidique et pseudopeptidique

L'unité structurale appelée repliement β dans les peptides et les protéines concerne une courte séquence de quatre résidus peptidiques qui est le siège d'une liaison hydrogène N — H...O = C fermant un cycle à dix atomes. Ce motif permet une inversion de la direction de propagation de la chaîne peptidique et constitue une zone exposée aux contacts intermoléculaires.L'une des voies de recherche en matière d’utilisation thérapeutique des peptides consiste à ralentir leur degradation enzymatique par modification chimique du squelette peptidique Cα—CO —NH. Toutefois, il peut aussi en résulter un changement des propriétés conformationnelles, propice ou néfaste à l’effet recherché. La présente revue examine l'influence de telles modifications sur la stabilité du repliement β. Certaines ont fait l'objet de nombreuses études (inversion de la configuration du carbone Cα, Cα-méthylation...), mais la plupart ont été peu examinées d'un point de vue conformationnel.L'examen de composés dérivés de la séquence minimale pour la réalisation du repliement β fournit des informations précises sur l'influence des diverses modifications introduites dans les analogues des peptides. Les résultats de ces études spectroscopiques (IR et RMN) en solution et radiocristallographiques sur l'état solide sont présentés et complétés par les observations rapportées dans la littérature

A sample chamber for in situ high-energy X-ray studies of crystal growth at deeply buried interfaces in harsh environments

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