The 2-phenylaminopyrimidine derivative STI571 has been shown to selectively inhibit the tyrosine kinase domain of the oncogenicbcr/abl fusion protein. The activity of this inhibitor has been demonstrated so far both in vitro with bcr/abl expressing cells derived from leukemic patients, and in vivo on nude mice inoculated with bcr/abl positive cells. Yet, no information is available on whether leukemic cells can develop resistance to bcr/ablinhibition. The human bcr/abl expressing cell line LAMA84 was cultured with increasing concentrations of STI571. After approximately 6 months of culture, a new cell line was obtained and named LAMA84R. This newly selected cell line showed an IC50 for the STI571 (1.0 μM) 10-fold higher than the IC50 (0.1 μM) of the parental sensitive cell line. Treatment with STI571 was shown to increase both the early and late apoptotic fraction in LAMA84 but not in LAMA84R. The induction of apoptosis in LAMA84 was associated with the activation of caspase 3–like activity, which did not develop in the resistant LAMA84R cell line. LAMA84R cells showed increased levels of bcr/abl protein and mRNA when compared to LAMA84 cells. FISH analysis with BCR- and ABL-specific probes in LAMA84R cells revealed the presence of a marker chromosome containing approximately 13 to 14 copies of the BCR/ABL gene. Thus, overexpression of the Bcr/Abl protein mediated through gene amplification is associated with and probably determines resistance of human leukemic cells to STI571 in vitro.

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Trehalose has been widely used to stabilize cellular structures such as membranes and proteins. The effect of trehalose on the stability of the enzyme cutinase was studied. Thermal unfolding of cutinase reveals that trehalose delays thermal unfolding, thus increasing the temperature at the midpoint of unfolding by 7.2 degrees . Despite this stabilizing effect, trehalose also favors pathways that lead to irreversible denaturation. Stopped-flow kinetics of cutinase folding and unfolding was measured and temperature was introduced as experimental variable to assess the mechanism and thermodynamics of protein stabilization by trehalose. The main stabilizing effect of trehalose was to delay the rate constant of the unfolding of an intermediate. A full thermodynamic analysis of this step has revealed that trehalose induces the phenomenon of entropy-enthalpy compensation, but the enthalpic contribution increases more significantly leading to a net stabilizing effect that slows down unfolding of the intermediate. Regarding the molecular mechanism of stabilization, trehalose increases the compactness of the unfolded state. The conformational space accessible to the unfolded state decreases in the presence of trehalose when the unfolded state acquires residual native interactions that channel the folding of the protein. This residual structure results into less hydrophobic groups being newly exposed upon unfolding, as less water molecules are immobilized upon unfolding.

The adenovirus protease requires activation by an 11-residue peptide, GVQSLKRRRCF, to achieve maximum proteolytic activity. Derived from the C terminus of the viral protein pVI, the activating peptide (pVI-CT) forms a disulfide bond with cysteine 104 of the protease and causes a conformational change that accompanies the development of proteolytic activity. Results presented here show that the interaction of pVI-CT with the protease is dependent not only on the cysteine 10 but also on glycine 1 and valine 2. Removal of these residues, acetylation of the N-terminal glycine, or mutation of the valine to alanine or threonine significantly reduces or abolishes activation. Peptides lacking Gly-1 and Val-2 still form a disulfide bond with the protease but do not cause a conformational change in the protease also they are not effective inhibitors of activation as the interaction is readily reversed by full-length pVI-CT. These results suggest that pVI-CT causes activation by binding to two distinct regions of the protease and in doing so stabilizes the catalytic site. The reversible nature of the activation, suggested by the results presented here, may well reflect an in vivo regulatory mechanism.The protease coded by adenovirus plays an essential role in the replication cycle of the virus (1) and has distinctive properties that make it of intrinsic scientific interest and an attractive target for antiviral therapy. It is known to cleave several capsid proteins (2) suggesting that it has a role in virion maturation; it cleaves the preterminal protein (pTP), the protein primer for DNA replication, thereby altering the affinity of that protein for the viral polymerase (3). It has also been reported to cleave the cellular protein cytokeratin 18 (4) raising the intriguing possibility that it has a role in the escape of the mature virus from the cell.Its properties are distinctive in several ways. It has an unusual substrate specificity (5, 6) that depends primarily on recognizing a hydrophobic residue (M, L, or I) in the P 4 position (7) and accepting only a glycine in P 2 . Although it appears to be a cysteine protease (8), the catalytic histidine and cysteine (His-54 and Cys-122) are in the reverse order of that found in the archetypal cysteine protease, papain (Cys-25 and His-159), which has led to them being classified in separate families within the category of cysteine protease (9). Perhaps the most interesting facet of its mode of action, however, is that in contrast to most other proteases it does not require proteolytic activation (10). The development of significant proteolytic activity depends on the participation of an 11-residue peptide (GVQSLKRRRCF), which is derived from the C terminus of the viral capsid protein pVI (11, 12). There have also been reports that viral DNA is involved in the catalytic mechanism (12, 13), but other reports (3) suggest that DNA is not necessary for catalysis but may help to stabilize the protease in vitro and could enhance the interaction of protease and substrates in vivo.Previo...

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The cohesion of cork agglomerates is determined by the strength of the adhesive joint established between the polymeric adhesive and the cork particles. The ability of adhesives to form good joints depends, among other factors, on the wetting of cork by the adhesives. The main objective of this research was to study the behaviour of adhesive drops deposited on cork substrates through measurements of contact angles and their time dependence. Several polyurethane prepolymers were tested to establish a correlation between the wetting characteristics and the chemical structure and physical properties of the adhesives. The effect of the morphology of cork on the interfacial properties was also investigated. The initial contact angles were related to the chemical nature of the adhesive. The kinetics of the wetting process were found to depend mainly on the viscosity of the adhesive.

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