Rosa M. Blanco

Immobilization-stabilization of enzymes; variables that control the intensity of the trypsin (amine)-agarose (aldehyde) multipoint attachment

Abstract We have developed a strategy for immobilization-stabilization of trypsin by multipoint covalent attachment to agarose (aldehyde) gels. We have studied the role of four main variables that control the intensity of the trypsin (amine)-agarose (aldehyde) multiinteraction processes: (a) surface density of aldehyde groups in the activated gels, (b) pH of the multiinteraction medium, (c) contact time between insolubilized enzyme and activated support prior to borohydride reduction of the derivatives, and (d) temperature. Different combinations of these four variables have been tested to prepare a number of trypsin-agarose derivatives. All these derivatives preserved 100% of catalytic activity but showed very different stability values. The less stable derivative had exactly the same stability of soluble trypsin in the absence of autolysis phenomena. On the other hand, the three-dimensional structure of the most stable derivative was 5000-fold more stable than the one corresponding to unmodified trypsin. Amino acid analysis of hydrolysates of this very stable derivative reveals that seven lysine residues per trypsin molecule have reacted with the activated support during the process of preparation of the derivative.

Stabilization of enzymes by multipoint covalent attachment to agarose-aldehyde gels. Borohydride reduction of trypsin-agarose derivatives

Abstract The process of borohydride reduction of one-point-attached and multipoint-attached trypsin (amine)-agarose (aldehyde) derivatives has been studied. We have tested the effect of different variables that control the intensity of this reduction process on the activity and stability of the resulting derivatives. In this way, we have been able to establish the optimal conditions for this process: 1 mg ml −1 borohydride, pH 10.0, 25°C, 30 min of reaction time, and presence of benzamidine (competitive inhibitor of trypsin). In these conditions, the reduction of Schiffs bases formed between the enzyme and the support is very intense, the remaining aldehyde groups on the support are reduced completely, and the deleterious effects of borohydride on trypsin structure are negligible. We have also studied these deleterious effects of borohydride when reductions were performed in more drastic experimental conditions. A broad range of experimental conditions seems to be useful to test the reduction of a broad spectrum of multipoint-attached enzyme (amine)-support (aldehyde) derivatives.

Immobilization-stabilization of penicillin G acylase from Escherichia coli

AbstractWe have developed a strategy for immobilization-stabilization of penicillin G acylase from E.coli, PGA, by multipoint covalent attachment to agarose (aldehyde) gels. We have studied the role of three main variables that control the intensity of these enzyme-support multiinteraction processes:1.surface density of aldehyde groups in the activated support;2.temperature; and3.contact-time between the immobilized enzyme and the activated support prior to borohydride reduction of the derivatives. Different combinations of these three variables have been tested to prepare a number of PGA-agarose derivatives. All these derivatives preserve 100% of catalytic activity corresponding to the soluble enzyme that has been immobilized but they show very different stability. The less stable derivative has exactly the same thermal stability of soluble penicillin G acylase and the most stable one is approximately 1,400 fold more stable. A similar increase in the stability of the enzyme against the deleterious effect of organic solvents was also observed. On the other hand, the agarose aldehyde gels present a very great capacity to immobilize enzymes through multipoint covalent attachment. In this way, we have been able to prepare very active and very stable PGA derivatives containing up to 200 International Units of catalytic activity per mL. of derivative with 100% yields in the overall immobilization procedure.

SKP2 Oncogene Is a Direct MYC Target Gene and MYC Down-regulates p27KIP1 through SKP2 in Human Leukemia Cells

SKP2 is the ubiquitin ligase subunit that targets p27KIP1 (p27) for degradation. SKP2 is induced in the G1-S transit of the cell cycle, is frequently overexpressed in human cancer, and displays transformation activity in experimental models. Here we show that MYC induces SKP2 expression at the mRNA and protein levels in human myeloid leukemia K562 cells with conditional MYC expression. Importantly, in these systems, induction of MYC did not activate cell proliferation, ruling out SKP2 up-regulation as a consequence of cell cycle entry. MYC-dependent SKP2 expression was also detected in other cell types such as lymphoid, fibroblastic, and epithelial cell lines. MYC induced SKP2 mRNA expression in the absence of protein synthesis and activated the SKP2 promoter in luciferase reporter assays. With chromatin immunoprecipitation assays, MYC was detected bound to a region of human SKP2 gene promoter that includes E-boxes. The K562 cell line derives from human chronic myeloid leukemia. In a cohort of chronic myeloid leukemia bone marrow samples, we found a correlation between MYC and SKP2 mRNA levels. Analysis of cancer expression databases also indicated a correlation between MYC and SKP2 expression in lymphoma. Finally, MYC-induced SKP2 expression resulted in a decrease in p27 protein in K562 cells. Moreover, silencing of SKP2 abrogated the MYC-mediated down-regulation of p27. Our data show that SKP2 is a direct MYC target gene and that MYC-mediated SKP2 induction leads to reduced p27 levels. The results suggest the induction of SKP2 oncogene as a new mechanism for MYC-dependent transformation.

Protecting effect of competitive inhibitors during very intense insolubilized enzyme-activated support multipoint attachments: trypsin (amine)-agarose (aldehyde) system

Abstract The effect of the presence of benzamidine, a competitive inhibitor of trypsin, during two different enzyme inactivation processes was studied. The inactivation processes were: thermal inactivation of soluble and insolubilized trypsin and inactivation of trypsin by multipoint covalent attachment to highly activated agarose (aldehyde) gels. In both cases, the presence of benzamidine, strongly adsorbed on the active site of the enzyme, greatly reduces the inactivation of trypsin. In the insolubilization-stabilization of trypsin by multipoint covalent attachment, the presence of benzamidine preserved the enzyme fully active during the insolubilized enzyme-activated support multiinteraction processes. This result was observed even during the most intense process in which eight lysine residues per trypsin molecule remained attached to the support. As a consequence of this result, the insolubilized derivative becomes 10 000-fold more stable than soluble trypsin. On the other hand, the presence of the inhibitor reduces the overall possibilities of enzyme-support multiinteraction; as a result, derivatives prepared in the presence of benzamidine are more active but less stable than derivatives prepared in absence of the inhibitor.

Myc Inhibits p27-Induced Erythroid Differentiation of Leukemia Cells by Repressing Erythroid Master Genes without Reversing p27-Mediated Cell Cycle Arrest

ABSTRACT Inhibition of differentiation has been proposed as an important mechanism for Myc-induced tumorigenesis, but the mechanisms involved are unclear. We have established a genetically defined differentiation model in human leukemia K562 cells by conditional expression of the cyclin-dependent kinase (Cdk) inhibitor p27 (inducible by Zn2+) and Myc (activatable by 4-hydroxy-tamoxifen). Induction of p27 resulted in erythroid differentiation, accompanied by Cdk inhibition and G1 arrest. Interestingly, activation of Myc inhibited p27-mediated erythroid differentiation without affecting p27-mediated proliferation arrest. Microarray-based gene expression indicated that, in the presence of p27, Myc blocked the upregulation of several erythroid-cell-specific genes, including NFE2, JUNB, and GATA1 (transcription factors with a pivotal role in erythropoiesis). Moreover, Myc also blocked the upregulation of Mad1, a transcriptional antagonist of Myc that is able to induce erythroid differentiation. Cotransfection experiments demonstrated that Myc-mediated inhibition of differentiation is partly dependent on the repression of Mad1 and GATA1. In conclusion, this model demonstrates that Myc-mediated inhibition of differentiation depends on the regulation of a specific gene program, whereas it is independent of p27-mediated cell cycle arrest. Our results support the hypothesis that differentiation inhibition is an important Myc tumorigenic mechanism that is independent of cell proliferation.

Equilibrium controlled synthesis of cephalothin in water-cosolvent systems by stabilized penicillin G acylase

Synthesis of cephalothin from thienylacetic acid (TAA) and 7-aminocephalosporanic acid (7ACA) has been carried out in the presence of high concentrations of organic cosolvents (e.g., 50% N,N′dimethyl-formamide) and under a wide range of experimental conditions (pH, temperature, etc.) by using very active and highly stabilized derivatives of Penicillin G acylase. We have been able to find the compromising solutions under which: (a) synthetic yields were markedly increased compared to those obtained in fully aqueous medium, (b) derivatives preserved a good percentage of catalytic activity, (c) derivatives were quite stable, and (d) high concentrations of substrates could be used. Under optimal conditions, 50 mM solutions of 7ACA in the presence of a slight excess of TAA were converted to cephalothin with yields higher than 95% and final concentrations of product up to 20 g/L were obtained.

MYC antagonizes imatinib-induced differentiation in chronic myeloid leukemia cells through downregulation of p27KIP1

Chronic myeloid leukemia (CML) progresses from a chronic to a blastic phase where the leukemic cells are proliferative and undifferentiated. The CML is nowadays successfully treated with BCR-ABL kinase inhibitors as imatinib and dasatinib. In the CML-derived K562 cell line, low concentrations of imatinib induce proliferative arrest and erythroid differentiation. We found that imatinib upregulated the cell cycle inhibitor p27KIP1 (p27) in a time- and -concentration dependent manner, and that the extent of imatinib-mediated differentiation was severely decreased in cells with depleted p27. MYC (c-Myc) is a transcription factor frequently deregulated in human cancer. MYC is overexpressed in untreated CML and is associated to poor response to imatinib. Using K562 sublines with conditional MYC expression (induced by Zn2+ or activated by 4-hydroxy-tamoxifen) we show that MYC prevented the erythroid differentiation induced by imatinib and dasatinib. The differentiation inhibition is not due to increased proliferation of MYC-expressing clones or enhanced apoptosis of differentiated cells. As p27 overexpression is reported to induce erythroid differentiation in K562, we explored the effect of MYC on imatinib-dependent induction of p27. We show that MYC abrogated the imatinib-induced upregulation of p27 concomitantly with the differentiation inhibition, suggesting that MYC inhibits differentiation by antagonizing the imatinib-mediated upregulation of p27. This effect occurs mainly by p27 protein destabilization. This was in part due to MYC-dependent induction of SKP2, a component of the ubiquitin ligase complex that targets p27 for degradation. The results suggest that, although MYC deregulation does not directly confer resistance to imatinib, it might be a factor that contributes to progression of CML through the inhibition of differentiation.

Designing Functionalized Mesoporous Materials for Enzyme Immobilization: Locating Enzymes by Using Advanced TEM Techniques

One of the widely accepted uses of ordered mesoporous materials is as supports of enzymes for biotechnological applications. Enzymes have been trapped, anchored, or encapsulated in organized porous networks of the mesoporous range (2–50 nm). The reactivity of the surface of mesoporous materials has enabled the synthesis of various supports by using different forces for the immobilization process. To design catalysts for specific applications, we have developed functionalized mesoporous materials with tunable hydrophobicity for the immobilization of lipase. More recently, we moved to the immobilization of laccase with amino‐functionalized ordered mesoporous materials. In this case, it is required to use pore expanders along with optimized functionalization techniques. Advanced TEM techniques have been applied to locate not only the functional groups but also the macromolecules inside the silica matrix.

P21 as a transcriptional co-repressor of S-phase and mitotic control genes

It has been previously described that p21 functions not only as a CDK inhibitor but also as a transcriptional co-repressor in some systems. To investigate the roles of p21 in transcriptional control, we studied the gene expression changes in two human cell systems. Using a human leukemia cell line (K562) with inducible p21 expression and human primary keratinocytes with adenoviral-mediated p21 expression, we carried out microarray-based gene expression profiling. We found that p21 rapidly and strongly repressed the mRNA levels of a number of genes involved in cell cycle and mitosis. One of the most strongly down-regulated genes was CCNE2 (cyclin E2 gene). Mutational analysis in K562 cells showed that the N-terminal region of p21 is required for repression of gene expression of CCNE2 and other genes. Chromatin immunoprecipitation assays indicated that p21 was bound to human CCNE2 and other p21-repressed genes gene in the vicinity of the transcription start site. Moreover, p21 repressed human CCNE2 promoter-luciferase constructs in K562 cells. Bioinformatic analysis revealed that the CDE motif is present in most of the promoters of the p21-regulated genes. Altogether, the results suggest that p21 exerts a repressive effect on a relevant number of genes controlling S phase and mitosis. Thus, p21 activity as inhibitor of cell cycle progression would be mediated not only by the inhibition of CDKs but also by the transcriptional down-regulation of key genes.