Improvement of stability and enzymatic activity by site-directed mutagenesis of E. coli asparaginase II

Bacterial asparaginases (EC 3.5.1.1) have attracted considerable attention because enzymes of this group are used in the therapy of certain forms of leukemia. Class II asparaginase from Escherichia coli (EcA), a homotetramer with a mass of 138 kDa, is especially effective in cancer therapy. However, the therapeutic potential of EcA is impaired by the limited stability of the enzyme in vivo and by the induction of antibodies in the patients. In an attempt to modify the properties of EcA, several variants with amino acid replacements at subunit interfaces were constructed and characterized. Chemical and thermal denaturation analysis monitored by activity, fluorescence, circular dichroism, and differential scanning calorimetry showed that certain variants with exchanges that weaken dimer–dimer interactions exhibited complex denaturation profiles with active dimeric and/or inactive monomeric intermediates appearing at low denaturant concentrations. By contrast, other EcA variants showed considerably enhanced activity and stability as compared to the wild-type enzyme. Thus, even small changes at a subunit interface may markedly affect EcA stability without impairing its catalytic properties. Variants of this type may have a potential for use in the asparaginase therapy of leukemia.     

Relationship between the concanavalin A-agglutinability and deformability of human erythrocytes

Cellular deformability has been proposed in the past as a major determinant of lectin-mediated agglutination of cells. In this paper we have evaluated the correlation between deformability and Con A-agglutinability of human erythrocytes by subjecting them to agents that alter either one of the properties and evaluating the effect on the other property. The following results have been obtained: (i) Treatment with pronase or trypsin, which makes the Con A-nonagglutinable normal red cells highly agglutinable, has practically no effect on deformability; while neuraminidase treatment, with a similar effect on agglutinability, produces a small but statistically significant reduction in deformability. (ii) Diamide treatment, on the other hand, produces a drastic reduction in the deformability of pronase-treated erythrocytes but has no effect on the Con A-agglutinability of the cells. Dinitrophenol also reduces deformability but without altering the agglutinability, (iii) Chlorpromazine, at 2 x 10(-5) M, does not have any effect on the deformability of trypsinized cells, but increases the agglutinability substantially. When the Con A-agglutinability of the cells and their deformability after these treatments are compared, a correlation coefficient r = -0.353 (P greater than 0.1) is obtained. This indicates the lack of any direct correlation between the two parameters, and rules out any significant role of deformability in the determination of Con A-agglutinability of erythrocytes. The agglutination with the lectin is completely reversed by methyl alpha-D-mannoside, the specific inhibitory sugar for Con A, also ruling out any secondary role for deformability in the non-lectin-mediated stabilization of clumps. Upon incubation of normal erythrocytes with Con A. a dose-dependent decrease in deformability is observed, with the deformability index falling to almost 25% of the normal value with 500 microgram/ml Con A. This indicates that Con A binding to its receptor produces changes in the membrane probably by altering properties of the membrane skeleton.     

Oxidized LDL Triggers Pro-Oncogenic Signaling in Human Breast Mammary Epithelial Cells Partly via Stimulation of MiR-21

Dyslipidemia and obesity are primary risk factors for the development of atherosclerosis and are also epidemiologically linked to increased susceptibility to a variety of cancers including breast cancer. One of the prominent features of dyslipidemia is enhanced production of oxidized LDL (ox-LDL), which has been shown to be implicated in key steps of atherogenesis including inflammatory signaling and proliferation of vascular cells. In this study we analyzed the effects of ox-LDL in human mammary epithelial cells (MCF10A). MCF10A cells avidly internalized dil-ox-LDL and exhibited increased proliferative response to ox-LDL within the range of 1–50 µg/ml in a dose-dependent manner. Treatment of cells with 20 µg/ml ox-LDL for 2 and 12 hours was associated with upregulation of LOX-1 and CD36 scavenger receptors while MSR1 and CXLC16 receptors did not change. Ox-LDL-treated cells displayed significant upregulation of NADPH oxidases (subunits P22^phox and P47^phox), lipoxygenases-12 and -15, and cytoplasmic, but not mitochondrial, SOD. Ox-LDL also triggered phosphorylation of IκBα coupled with nuclear translocation of NF-κB and stimulated p44/42 MAPK, PI3K and Akt while intracellular PTEN (PI3K/Akt pathway inhibitor and target of miR-21) declined. Quantitative PCR revealed increased expression of hsa-miR-21 in ox-LDL treated cells coupled with inhibition of miR-21 target genes. Further, transfection of MCF10A cells with miR-21 inhibitor prevented ox-LDL mediated stimulation of PI3K and Akt. We conclude that, similarly to vascular cells, mammary epithelial cells respond to ox-LDL by upregulation of proliferative and pro-inflammatory signaling. We also report for the first time that part of ox-LDL triggered reactions in MCF10A cells is mediated by oncogenic hsa-miR-21 through inhibition of its target gene PTEN and consequent activation of PI3K/Akt pathway.