Montreal Children's Hospital

About: Montreal Children's Hospital is a healthcare organization based out in Montreal, Quebec, Canada. It is known for research contribution in the topics: Population & Poison control. The organization has 3842 authors who have published 4816 publications receiving 200198 citations.

Microvesicles: messengers and mediators of tumor progression.

Abstract: Cellular interactions play a crucial role in progression, angiogenesis and invasiveness of tumors, including glioma. The traditionally accepted view is that medium and long-range cellular communications occur primarily through gradients of soluble ligands, recognizable by the cell-associated receptors. Recent findings, however, suggest the existence of another mode of intercellular communication, where the ‘units’ of information are microvesicles containing a multitude of biologically active protein and RNA species, including oncogenic receptors, such as EGFRvIII. Moreover, microvesicles can be retrieved from the circulating blood of cancer patients, and reveal the presence of oncogenes in their tumors, thereby potentially serving as information-rich prognostic and predictive biomarkers.

A rapid procedure for extracting genomic DNA from leukocytes

Abstract: 1. Collect 5 ml of blood in a vacutainer tube (Becton Dickinson) containing EDTA and mix. 2. Make volume up to 10 ml with solution 1 (10 mM Tris pH 7.6; 10 mM KC1; 10 mM MgCy. 3. Add 120 /il Nonidet P40 (BDH) to lyse the cells. Mix well by inverting several times. 4. Spin down the nuclear pellet at 2000 rpm for 10 mins. 5. Pour off the supernatant without dislodging the pellet. The pellets can be stored frozen. 6. Gently resuspend pellet well in 800 /il of solution 2 (10 mM Tris pH 7.6; 10 mM KC1; 10 mM MgCl2; 0.5 M NaCl; 0.5% SDS; 2 mM EDTA). Solution 2 will lyse the nuclei so be careful not to shear the DNA. Transfer to a 1.5 ml microcentrifuge tube. 7. Add 400 /il of distilled phenol (saturated with 1 M Tris pH 8.0) and mix well. 8. Microfuge for 1 min at 12000 rpm. Transfer upper phase to a clean microfuge tube. Do not worry about transferring a little of the interface. 9. Add 200 /tl of phenol and 200 /il of chloroform :isoamyl alcohol (24:1). Mix well by inverting. 10. Spin for 1 min at 12000 rpm. Transfer upper phase to a clean microfuge tube. 11. Add 700 /tl of chloroform:isoamyl alcohol and extract as above. 12. Transfer upper aqueous phase to a small clean container. Avoid removing the interface. Add 2 volumes of ice cold ethanol and mix to precipitate the DNA*. 13. With the sealed tip of a pasture pipette, transfer the DNA fibres to a microcentrifuge tube containing 1 ml of 70% ethanol. Mix well to wash the DNA. 14. Spin for 5 mins at full speed. Discard the ethanol and dry pellet in a speed vac. Resuspend DNA in sterile water at 65°C. Do not over-dry genomic DNA or it will be difficult to resuspend. SIZE A / /Kb Hindll l

Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase

Abstract: Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of methylenetetrahydrofolate to methyltetrahydrofolate, the major methyl donor for the conversion of homocysteine to methionine. Two common polymorphisms of the human enzyme have been identified: 677C>T, which leads to the substitution of Ala-222 by valine, and 1298A>C, which leads to the replacement of Glu-429 by alanine; the former polymorphism is the most frequent genetic cause of mild hyperhomocysteinemia, a risk factor for cardiovascular disease. By using a baculovirus expression system, recombinant human MTHFR has been expressed at high levels and purified to homogeneity in quantities suitable for biochemical characterization. The Glu429Ala protein has biochemical properties that are indistinguishable from the wild-type enzyme. The Ala222Val MTHFR, however, has an enhanced propensity to dissociate into monomers and to lose its FAD cofactor on dilution; the resulting loss of activity is slowed in the presence of methyltetrahydrofolate or adenosylmethionine. This biochemical phenotype is in good agreement with predictions made on the basis of studies comparing wild-type Escherichia coli MTHFR with a mutant, Ala177Val, homologous to the Ala222Val mutant human enzyme [Guenther, B. D., et al. (1999) Nat. Struct. Biol. 6, 359–365].