Interactions between gangliosides and proteins in the exoplasmic leaflet of neuronal plasma membranes: A study performed with a tritium-labeled GM1 derivative containing a photoactivable group linked to the oligosaccharide chain

Interactions between gangliosides and proteins at the exoplasmic surface of the sphingolipid-enriched membrane domains can be studied by ganglioside photolabeling combined with cell surface biotin labeling. In the present paper, we report on the results obtained using a novel radioactive photoactivable derivative of GM1 ganglioside, carrying the photoactivable nitrophenylazide group at the external galactose.After cell photolabeling with the radioactive photoactivable derivative of GM1 and cell surface biotin labeling, sphingolipid-enriched domains were prepared from rat cerebellar neurons differentiated in culture and further purified by immunoprecipitation with streptavidin-coupled beads. Among proteins belonging to the sphingolipid-enriched domains that were biotin labeled, thus bearing an exoplasmic domain, a few were also cross-linked by the radioactive photoactivable ganglioside. In particular, two protein bands showing apparent molecular mass of 135 and 35 kDa were intensely photolabeled. The 135 kDa protein was immunologically identified as the GPI-anchored neural cell adhesion molecule TAG-1. These data suggest that hydrophilic interaction between the exoplasmic domains of the protein and the ganglioside sialooligosaccharide chain could exist. Published in 2004.     

Synthesis of radioactive and photoactivable ganglioside derivatives for the study of ganglioside-protein interactions

The procedures for the preparation of radioactive and photoactivable ganglioside derivatives have been continuously developed from 1989, when for the first time the synthesis of photoactivable tritium labeled GM1 ganglioside was presented. We described previously the synthesis of photoactivable derivatives of GM3 and GM1 gangliosides, tritium-labeled at acetyl group of sugar units, and of photoactivable GM1 and GD1b gangliosides, tritium-labeled at position 6 of the external galactose. These procedures are reviewed in detail in the present paper.The use of these ganglioside derivatives to study the ganglioside-protein interactions and to identify proteins that specifically interact with gangliosides (including GPI-anchored proteins of the outer membrane leaflet, proteins anchored to the cytoplasmic side of the plasma membrane through a fatty acyl chain, transmembrane proteins, and soluble cytoplasmic proteins) is discussed.     

Pharmacogenomics in colorectal carcinomas: future perspectives in personalized therapy

The recent introduction of new drugs such as capecitabine, irinotecan, and oxaliplatinum has greatly improved the clinical outcome of patients with advanced/metastatic colorectal cancer. Nevertheless, some patients may suffer from the adverse drug reactions which will probably be the main cause of chemotherapy failure. The goal of pharmacogenomics is to find correlations between therapeutic responses to drugs and the genetic profiles of patients; the different responses to a particular drug are due, in fact, not only to the specific clinico-pathological features of the patient or to environmental factors, but also to the ethnic origins and the particular individual's genetic profile. Genes which codify for the metabolism enzymes, receptor proteins, or protein targets of chemotherapy agents often present various genetic polymorphisms. The main aim of this review is to provide an overview of the known polymorphisms present in the genes which codify for factors (thymidylate synthase dihydropyrimidine dehydrogenase, uridine diphosphate (UDP)-glucuronosyl-transferase 1A1, enzymes implicated in DNA repair) involved in the action mechanisms of the drugs now utilized in chemotherapeutic treatment of colorectal carcinoma, such as fluoropyrimidines, irinotecan, and platinum agents.     

Putrescine Accumulation in Wine: Role of Oenococcus oeni

Putrescine, the most abundant biogenic amine in wine, was proved to be produced by Oenococcus oeni strains in wine not only from ornithine but also from arginine. In this case, putrescine may originate from strains possessing the complete enzyme system to convert arginine to putrescine or by a metabiotic association, with an exchange of ornithine, between strains capable of metabolizing arginine to ornithine but unable to produce putrescine and strains capable of producing putrescine from ornithine but unable to degrade arginine. Putrescine production by this metabiotic association occurred once the malolactic fermentation was completed, whereas conversion of ornithine to putrescine by a single culture of the ornithine decarboxylating strain concurred with the degradation of malic acid. Moreover, in the former case, putrescine formation proceeded more slowly than in the latter. Metabiosis may play an important role in the accumulation of putrescine in wine, arginine being one of the major amino acids found in wine.     

Transcript profiling reveals novel marker genes involved in fruiting body formation in Tuber borchii

cDNA arrays were used to explore mechanisms controlling fruiting body development in the truffle Tuber borchii. Differences in gene expression were higher between reproductive and vegetative stage than between two stages of fruiting body maturation. We suggest hypotheses about the importance of various physiological processes during the development of fruiting bodies.     

Cryomacroscopy of vitrification, Part II: Experimental observations and analysis of fracture formation in vitrified VS55 and DP6

A new imaging device, termed a "cryomacroscope", was used to observe macrofractures in the cryoprotectant cocktails DP6 and VS55. Details of the design and construction of the cryomacroscope were presented in Part I of this report, which focused on describing the apparatus and observations of crystallization. Part I and the current paper (Part II) describe events that occur as 1 m? of cryoprotectant contained in a glass vial is cooled from room temperature down to cryogenic temperatures (～ -135°C). The presence of cracking, as well as patterns in their position and orientation, are found to be dependent on the cooling rate and on the specific cryoprotectant cocktail. Cracks, if present, disappear upon rewarming, although they appear to be sites for later preferential crystallization. Computations which predict temperatures and mechanical stresses are used to explain observations of cracking. In conjunction with these reports, additional photos of cryomacroscopy of vitrification, crystallization, and fracture formation are available at http://www.me.cmu.edu/faculty1/rabin/CryomacroscopyImages01.htm.     

Discovery of (phenylureido)piperidinyl benzamides as prospective inhibitors of bacterial autolysin E from Staphylococcus aureus

Autolysin E (AtlE) is a cell wall degrading enzyme that catalyzes the hydrolysis of the β-1,4-glycosidic bond between the N-acetylglucosamine and N-acetylmuramic acid units of the bacterial peptidoglycan. Using our recently determined crystal structure of AtlE from Staphylococcus aureus and a combination of pharmacophore modeling, similarity search, and molecular docking, a series of (Phenylureido)piperidinyl benzamides were identified as potential binders and surface plasmon resonance (SPR) and saturation-transfer difference (STD) NMR experiments revealed that discovered compounds bind to AtlE in a lower micromolar range. (phenylureido)piperidinyl benzamides are the first reported non-substrate-like compounds that interact with this enzyme and enable further study of the interaction of small molecules with bacterial AtlE as potential inhibitors of this target.