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[EN] The presence of bisphenol A (BPA) in wastewater, its toxicity and the hardness for its complete degradation constitutes a serious environmental problem and a scientific challenge. The enzyme laccase is an oxidoreductase capable of oxidizing phenolic compounds, being one of the most efficient biocatalysts for BPA removal. The immobilization of laccases to lead solid biocatalysts is essential for their applications as it allows their stabilization and easy recovery from the liquid reaction media and subsequent reuse. This work compares the efficiency of siliceous-type supports, which have traditionally been used for this purpose, with the use of MOF-type supports. Thanks to the versatility in the synthesis methods offered by MOFs, MOF-type biocatalysts can be prepared in a one-pot using an in-situ procedure (Lac@NH-MIL-53(Al)) under conditions compatible with enzymatic activity (room temperature, aqueous solution and moderate pH values). The resulting biocatalyst displays a high enzyme loading of 100 mg.g which is also permanent: free of enzyme leaching. As opposed, post-synthesis in two steps: Lac#NH-MIL-53(Al) (with low enzyme loading) and Lac#Amorphous silica-NH (100 mg.g enzyme loading, but high rate of enzyme leaching) were also prepared and compared. In contrast to the elimination of 90% of BPA in 60 min obtained with Lac#Amorphous silica-NH, the one-pot immobilization of laccase on MOFs resulted much more efficient. The own enzyme-free MOF-type support (NH-MIL-53(Al)) removed more than 90% of BPA from aqueous solution in 30 min, but such performance was widely surpassed by the biocatalyst Lac@NH-MIL-53(Al) (100% in just 3 min). The significant improvement of BPA degradation was attributed to the mutual and simultaneous contribution of both support and enzyme. In addition, the biocatalyst Lac@NH-MIL-53(Al) was able to remove BPA in five successive cycles with efficiency higher than 85% at the end of the last batch., This work has been financed by CSIC: Consejo Superior de Investigaciones Científicas, Spain (2019AEP076, COOPB20377 and COOPA20271). M.A.M. acknowledges Spanish MINECO for PhD student contract BES-2017–082077 and UAM: Universidad Autónoma de Madrid, for PhD Program in Applied Chemistry. The authors acknowledge the CYTED-LIDA network and the SAB group of ITESM

The industrial use of enzymes generally necessitates their immobilization onto solid supports. The well-known high affinity of enzymes for metal-organic framework (MOF) materials, together with the great versatility of MOFs in terms of structure, composition, functionalization and synthetic approaches, has led the scientific community to develop very different strategies for the immobilization of enzymes in/on MOFs. This review focuses on one of these strategies, namely, the one-pot enzyme immobilization within sustainable MOFs, which is particularly enticing as the resultant biocomposite Enzyme@MOFs have the potential to be: (i) prepared in situ, that is, in just one step; (ii) may be synthesized under sustainable conditions: with water as the sole solvent at room temperature with moderate pHs, etc.; (iii) are able to retain high enzyme loading; (iv) have negligible protein leaching; and (v) give enzymatic activities approaching that given by the corresponding free enzymes. Moreover, this methodology seems to be near-universal, as success has been achieved with different MOFs, with different enzymes and for different applications. So far, the metal ions forming the MOF materials have been chosen according to their low price, low toxicity and, of course, their possibility for generating MOFs at room temperature in water, in order to close the cycle of economic, environmental and energy sustainability in the synthesis, application and disposal life cycle.

The survival rate in lung cancer remains stubbornly low and there is an urgent need for the identification of new therapeutic targets. In the last decade, several members of the SWI/SNF chromatin remodeling complexes have been described altered in different tumor types. Nevertheless, the precise mechanisms of their impact on cancer progression, as well as the application of this knowledge to cancer patient management are largely unknown. In this study, we performed targeted sequencing of a cohort of lung cancer patients on genes involved in chromatin structure. In addition, we studied at the protein level the expression of these genes in cancer samples and performed functional experiments to identify the molecular mechanisms linking alterations of chromatin remodeling genes and tumor development. Remarkably, we found that 20% of lung cancer patients show ARID2 protein loss, partially explained by the presence of ARID2 mutations. In addition, we showed that ARID2 deficiency provokes profound chromatin structural changes altering cell transcriptional programs, which bolsters the proliferative and metastatic potential of the cells both in vitro and in vivo. Moreover, we demonstrated that ARID2 deficiency impairs DNA repair, enhancing the sensitivity of the cells to DNA-damaging agents. Our findings support that ARID2 is a bona fide tumor suppressor gene in lung cancer that may be exploited therapeutically., IV is supported by SAF2012-31627 and SAF2016-76758-R grants from the Spanish Ministerio de Economía y Competitividad (MINECO), by a Fundación Ramón Areces grant and by ERC2014-StG637904 grant from the European Research Council. IV has been awardee of the Programa Ramón y Cajal (MINECO, Spain). TM has been awardee of the Ayudas para la contratación de investigadores predoctorales (MINECO, Spain). BM is awardee of the Ayudas para la formación de profesorado universitario (FPU, Ministerio de Educación y Formación Profesional, Spain). PC laboratory is supported by grant SAF-2015-63638R (MINECO/FEDER, UE); by Centro de Investigación Biomédica en Red de Cáncer (CIBERONC) and by Asociación Española Contra el Cáncer (AECC), grant GCB141423113. BC has been supported by a Retos Jóvenes Investigadores grant SAF-2015-73364-JIN (AEI/FEDER, UE) and a grant from Fundación Francisco Cobos. PS is supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001152) and the UK Medical Research Council (FC001152). HUCA/IUOPA is jointly financed by Servicio de Salud del Principado de Asturias, Instituto de Salud Carlos III, and Fundación Bancaria Cajastur. This research was funded in part by the Wellcome Trust [FC001152].

© 2021 The Author(s)., Chronic myelogenous leukemia (CML) is a hematological malignancy that highly depends on the BCR-ABL1/STAT5 signaling pathway for cell survival. First-line treatments for CML consist of tyrosine kinase inhibitors that efficiently target BCR-ABL1 activity. However, drug resistance and intolerance are still therapeutic limitations in Ph+ cells. Therefore, the development of new anti-CML drugs that exhibit alternative mechanisms to overcome these limitations is a desirable goal. In this work, the antitumoral activity of JKST6, a naphthoquinone-pyrone hybrid, was assessed in imatinib-sensitive and imatinib-resistant human CML cells. Live-cell imaging analysis revealed JKST6 potent antiproliferative activity in 2D and 3D CML cultures. JKST6 provoked cell increase in the subG1 phase along with a reduction in the G0/G1 phase and altered the expression of key proteins involved in the control of mitosis and DNA damage. Rapid increases in Annexin V staining and activation/cleavage of caspases 8, 9 and 3 were observed after JKST6 treatment in CML cells. Of interest, JKST6 inhibited BCR-ABL1/STAT5 signaling through oncokinase downregulation that was preceded by rapid polyubiquitination. In addition, JKST6 caused a transient increase in JNK and AKT phosphorylation, whereas the phosphorylation of P38-MAPK and Src was reduced. Combinatory treatment unveiled synergistic effects between imatinib and JKST6. Notably, JKST6 maintained its antitumor efficacy in BCR-ABL1-T315I-positive cells and CML cells that overexpress BCR-ABL and even restored imatinib efficacy after a short exposure time. These findings, together with the observed low toxicity of JKST6, reveal a novel multikinase modulator that might overcome the limitations of BCR-ABL1 inhibitors in CML therapy., This research has been funded by Spanish Ministry of Economy and Competitiveness - MINECO - (SAF 2015–65113-C2–1-R and RTI2018–094356-B-C21 to AEB, SAF2015–65113-C2–2 to LFP, SAF2017–88026-R to JL) with the co-funding of European Regional Development Fund (EU-ERDF), Canary Islands Government (CEI2018–23/ACIISI to BG, CEI2019–08/ACIISI to BG and LFP, ProID2021010037 to AEB, LFP and BG) and "Juan de la Cierva Incorporacion" Grant Program from the Ministry of Science, Innovation and Universities (IJC2018-035193-I to CR). This project has been also supported by Alfredo Martin-Reyes Foundation (Arehucas)-Canary Islands Foundation for Cancer Research (FICIC). HAT is recipient of a predoctoral program grant from ULPGC (2016). JCM was funded by the Instituto de Salud Carlos III through a Miguel Servet program (CPII17/00015).

Metal-organic framework (MOF) materials possess the widest versatility in structure, composition, and synthesis procedures amongst the known families of materials. On the other hand, the extraordinary affinity between MOFs and enzymes has led to widely investigating these materials as platforms to support these catalytic proteins in recent years. In this work, the MOF material NH2-MIL-53(Al) has been tested as a support to immobilize by one-step methodology (in situ) the enzyme lipase CaLB from Candida antarctica by employing conditions that are compatible with its enzymatic activity (room temperature, aqueous solution, and moderate pH values). Once the nature of the linker deprotonating agent or the synthesis time were optimized, the MOF material resulted in quite efficient entrapping of the lipase CaLB through this in situ approach (&gt, 85% of the present enzyme in the synthesis media) while the supported enzyme retained acceptable activity (29% compared to the free enzyme) and had scarce enzyme leaching. The equivalent post-synthetic method led to biocatalysts with lower enzyme loading values. These results make clear that the formation of MOF support in the presence of the enzyme to be immobilized substantially improves the efficiency of the biocatalysts support for retaining the enzyme and limits their leaching.

BACKGROUND: The biocatalysis field demands ‘universal’ supports able to encapsulate enzymes with a straightforward methodology, and at the same time, capable of retaining their catalytic activity. The employment of siliceous materials for such a purpose is a big challenge because drastic synthesis conditions are required and improved functionalization is needed to increase affinities towards the targeted enzyme. In this work, a compromise between the development of a well‐formed mesostructured support and an acceptable enzymatic activity was attempted via the in‐situ immobilization approach. RESULTS: The immobilization of β‐glucosidase (EC 3.2.1.21) from Aspergillus niger was approached using different strategies. After trying to immobilize β‐glucosidase with a post‐synthesis approach, nonhigh loadings were achieved both with covalent linkage (using epoxy activated supports; 3.5 mgE g⁻¹) and with noncovalent bonding (using amine‐functionalized materials; 7.6 mgE g⁻¹). However, when the in‐situ approach was attempted, success in reaching the highest enzyme loading (close to 200 mgE g⁻¹) was achieved. CONCLUSION: In this work, the support cages around the in‐situ encapsulated enzyme fully prevented its release through the narrow windows connecting cages, achieving a less than 5% release of the initially desorbed protein, as well as a further total absence of leaching. This enabled the biocatalyst to be reused at least eight times more without any loss in activity. © 2018 Society of Chemical Industry

Metal-organic frameworks (MOFs) have revolutionized the potential applications of nanoporous materials. One of the most recent and promising applications of these materials is their use as supports for enzyme immobilization. In this context, the in-situ (one-step) methodologies, which do not require the use of MOFs with pores larger than the enzyme to be immobilized, seem to be particularly encouraging. This work presents a systematic study of the semi-crystalline Fe-BTC MOF material (commercialized as Basolite F300) employed as support of the enzymes laccase and lipase through either in-situ or post-synthesis methodology. The presence of the enzyme in the resultant solid biocatalysts was proved by CHNS chemical analysis, thermogravimetric analysis, Bradford assays and by SDS-PAGE electrophoresis. The enzymatic activity of the resultant Fe-BTC-based biocatalysts was also tested. The in-situ approach is particularly relevant due to various reasons: (i) the enzyme immobilization is given in one step; (ii) it is rapid (10 min); (iii) it is very efficient in terms of encapsulation capacity (≥98% for laccase and ≥87% for lipase); (iv) the enzymes are fully retained and no leaching is observed after an initial release of only around 10% of the enzyme molecules weakly immobilized; and (v) the activity of the retained enzyme can be substantially maintained (97% with respect to the free enzyme in the case of lipase). Any of these parameters systematically improves these given by the post-synthesis (two-step) approach. Moreover, Fe-BTC widely surpasses the benefits given by other MOF-based supports either by in-situ or post-synthesis approaches., This work has been partially financed by the Spanish State Research Agency (Agencia Española de Investigación, AEI) and the European Regional Development Fund (Fondo Europeo de Desarrollo Regional, FEDER) through the Project MAT2016-77496-R (AEI/FEDER, UE).

The presence of two different additives during non-covalent immobilization of lipase was studied. Lipase was immobilized via hydrophobic interactions on an amorphous silica with large pore size bea...

This work has been partially financed by the Spanish State Research Agency (Agencia Espanola de Investigacion, AEI) and the European Regional Development Fund (Fondo Europeo de Desarrollo Regional, FEDER) through the Project MAT2016‐77496‐R (AEI/FEDER, UE).

The use of a metal–organic framework (MOF) as a support for the in situ immobilization of enzymes was explored. The MOF support, a Basolite F300‐like material, was prepared from FeCl3 and the tridentate linker trimesic acid. Immobilization of alcohol dehydrogenase, lipase, and glucose oxidase was performed in situ under mild conditions (aqueous solution, neutral pH, and at room temperature) in a rapid and facile manner with retention of activity for at least 1 week. The catalytic activities of lipase and glucose oxidase were similar to the activities of the free enzymes; with alcohol dehydrogenase, there was a substantial decrease in activity on immobilization that may arise from diffusion limitations. The approach demonstrates that a MOF material, prepared from cheap and commercially available materials, can be successively utilized to prepare stable and catalytically active biocatalysts in a rapid and facile manner., Support from the Science Foundation Ireland (12/RC/2275), the Programme for Third Level Institutions, the Irish Research Council (postdoctoral fellowship to V.G. GOIPD/2015/287), and the Spanish Government (MAT‐2012‐31127) is gratefully acknowledged. This work was also partially financed by the Spanish State Research Agency (Agencia Española de Investigación, AEI) and the European Regional Development Fund (Fondo Europeo de Desarrollo Regional, FEDER) through the Project MAT2016‐77496‐R (AEI/FEDER, UE).

MXD1 is a protein that interacts with MAX, to form a repressive transcription factor. MXD1-MAX binds E-boxes. MXD1-MAX antagonizes the transcriptional activity of the MYC oncoprotein in most models. It has been reported that MYC overexpression leads to augmented RNA synthesis and ribosome biogenesis, which is a relevant activity in MYC-mediated tumorigenesis. Here we describe that MXD1, but not MYC or MNT, localizes to the nucleolus in a wide array of cell lines derived from different tissues (carcinoma, leukemia) as well as in embryonic stem cells. MXD1 also localizes in the nucleolus of primary tissue cells as neurons and Sertoli cells. The nucleolar localization of MXD1 was confirmed by co-localization with UBF. Co-immunoprecipitation experiments showed that MXD1 interacted with UBF and proximity ligase assays revealed that this interaction takes place in the nucleolus. Furthermore, chromatin immunoprecipitation assays showed that MXD1 was bound in the transcribed rDNA chromatin, where it co-localizes with UBF, but also in the ribosomal intergenic regions. The MXD1 involvement in rRNA synthesis was also suggested by the nucleolar segregation upon rRNA synthesis inhibition by actinomycin D. Silencing of MXD1 with siRNAs resulted in increased synthesis of pre-rRNA while enforced MXD1 expression reduces it. The results suggest a new role for MXD1, which is the control of ribosome biogenesis. This new MXD1 function would be important to curb MYC activity in tumor cells., The work was supported by grants SAF2014-53526 (to JL), BFU2014-54754P (to ML and MTB) from Spanish Economy and Competitiveness Ministry (MINECO), and grants RETIC-RD012-036-033 (to JL) and CIBERNED CB06/05/0037 (to ML) from Instituto Carlos III to JL. These funding were co-sponsored by the FEDER program. MCL was recipient of a fellowship from the FPU program from MINECO.

[EN] Ordered Mesoporous Materials offer wide application areas in chemistry and biotechnology. SBA-15 displays hexagonal arrangement of parallel unidirectional channels, where pore size and particle morphology are the controlling parameters when used as enzyme support to favour the diffusion of the protein molecules. The synthesis conditions can be controlled to yield the desired structural and morphological features of the material, as well as the functionalization of its surface to improve chemical affinity with the enzyme. Functionalization with amine groups has been performed to immobilize laccase on SBA-15. Particles of SBA-15 with short channels (300–500 nm length) and large pore diameter (10–14 nm) have been obtained. The experimental parameters varied to control the structural features were surfactant, micelle expander and silica source. Upon functionalization with amine groups, laccase was immobilized at a pH in the range between the isoelectric point of the enzyme (pI = 4.2) and the pKa value of the amino groups (~10) via non-covalent (electrostatic) interaction between carboxylate groups of the enzyme and the protonated amino groups. The enzyme was successfully immobilized on all the supports tested (loading from 18 to 57 mg/g) with fair catalytic activity and observing enzyme desorption (leaching) below 9% after 48 h in all cases., This work has been funded by the Spanish Research Agency (AEI) and the European Regional Development Fund (FEDER) through the Project MAT2016-77496-R. M.A.M. acknowledges MINECO for graduate student contract BES-2017-082077. The support of CYTED (Ibero-American Program on Science and Technology for Development) under the LIDA Thematic Network is also acknowledged.

Cell cycle stimulation is a major transforming mechanism of Myc oncoprotein. This is achieved through at least three concomitant mechanisms: upregulation of cyclins and Cdks, downregulation of Cdk inhibitors p15 and p21 and the degradation of p27. The Myc-p27 antagonism has been shown to be relevant in human cancer. To be degraded, p27 must be phosphorylated at Thr-187 to be recognized by Skp2, a component of the ubiquitination complex. We previously described that Myc induces Skp2 expression. Here we show that not only Cdk2 but Cdk1 phosphorylates p27 at the Thr187, which was previously unreported. Moreover, Myc induced p27 degradation in murine fibroblasts through Cdk1 activation, which was achieved by Myc-dependent cyclin A and B induction. In the absence of Cdk2, p27 phosphorylation at Thr-187 was mainly carried out by cyclin A2-Cdk1 and cyclin B1-Cdk1. We also show that Cdk1 inhibition was sufficient for the synthetic lethal interaction with Myc. This result is relevant because Cdk1 is the only Cdk strictly required for cell cycle and the reported synthetic lethal interaction between Cdk1 and Myc.

Human Chronic Myelogenous Leukemia (CML) is a hematological stem cell disorder which is associated with activation of Bcr-Abl-Stat5 oncogenic pathway. Direct Bcr-Abl inhibitors are initially successful for the treatment of CML but over time many patients develop drug resistance. In the present study, the effects of CM363, a novel naphthoquinone (NPQ) derivative, were evaluated on human CML-derived K562 cells. CM363 revealed an effective cell growth inhibition (IC50 = 0.7 ± 0.5 μM) by inducing cancer cells to undergo cell cycle arrest and apoptosis. CM363 caused a dose- and time-dependent reduction of cells in G0/G1 and G2/M phases. This cell cycle arrest was associated with increased levels of cyclin E, pChk1 and pChk2 whereas CM363 downregulated cyclin B, cyclin D3, p27, pRB, Wee1, and BUBR1. CM363 increased the double-strand DNA break marker γH2AX. CM363 caused a time-dependent increase of annexin V-positive cells, DNA fragmentation and increased number of apoptotic nuclei. CM363 triggered the mitochondrial apoptotic pathway as reflected by a release of cytochrome C from mitochondria and induction of the cleavage of caspase-3 and -9, and PARP. CM363 showed multikinase modulatory effects through an early increased JNK phosphorylation followed by inhibition of pY-Bcrl-Abl and pY-Stat5. CM363 worked synergistically with imatinib to inhibit cell viability and maintained its activity in imatinib-resistant cells. Finally, CM363 (10 mg/Kg) suppressed the growth of K562 xenograft tumors in athymic mice. In summary, CM363 is a novel multikinase modulator that offers advantages to circumvent imanitib resistance and might be therapeutically effective in Bcrl-Abl-Stat5 related malignancies.

It is thought that during immobilization enzymes, as dynamic biomolecules, may become distorted and this may alter their catalytic properties. However, the effects of different immobilization strategies on enzyme rigidity or flexibility and their consequences in specificity and stereochemistry at large scale has not been yet clearly evaluated and understood. This was here investigated by using as model an ester hydrolase, isolated from a bacterium inhabiting a karstic lake, with broad substrate spectrum (72 esters being converted; 61.5 U mg−1 for glyceryl tripropionate) but initially non-enantiospecific. We found that the enzyme (7 nm × 4.4 nm × 4.2 nm) could be efficiently ionic exchanged inside the pores (9.3 nm under dry conditions) of amino-functionalized ordered mesoporous material (NH2-SBA-15), achieving a protein load of 48 mg g−1, and a specific activity of 4.5 ± 0.1 U mg−1. When the enzyme was site-directed immobilized through His interaction with an immobilized cationon the surface of two types of magnetic micro-particles through hexahistidine-tags, protein loads up to 10.2 μg g−1 and specific activities of up to 29.9 ± 0.3 U mg−1, were obtained. We found that ionically exchanged enzyme inside pores of NH2-SBA-15 drastically narrowed the substrate range (17 esters), to an extent much higher than ionically exchanged enzyme on the surface of magnetic micro-particles (up to 61 esters). This is attributed to differences in surface chemistry, particle size, and substrate accessibility to the active site tunnel. Our results also suggested, for the first time, that immobilization of enzymes in pores of similar size may alter the enzyme structures and produce enzyme active centers with different configuration which promote stereochemical conversions in a manner different to those arising from surface immobilization, where the strength of the ionic exchange also has an influence. This was shown by demonstrating that when the enzyme was introduced inside pores with a diameter (under dry conditions) slightly higher than that of the enzyme crystal structure a biocatalyst enantiospecific for ethyl (R)-4-chloro-3-hydroxybutyrate was produced, a feature not found when using wider pores. By contrast, immobilization on the surface of ferromagnetic microparticles produced selective biocatalysts for methyl (S)-(+)-mandelate or methyl (S)-lactate depending on the functionalization. This study illustrates the benefits of extensive analysis of the substrate spectra to better understand the effects of different immobilization strategies on enzyme flexibility/rigidity, as well as substrate specificity and stereochemistry. Our results will help to design tunable materials and interfaces for a controlled manipulation of specificity and to transform non-enantiospecific enzymes into stereo-chemically substrate promiscuous biocatalysts capable of converting multiple chiral molecules. This project received funding from the European Union’s Horizon 2020 research and innovation program Blue Growth: Unlockingthe potential of Seas and Oceans under grant agreement no. 634486 (project acronym INMARE). This research was also supported by the grants PCIN-2014-107 (within ERA NET IB2 grant nr. ERA-IB-14-030 - MetaCat), PCIN-2017-078 (within the ERA-MarineBiotech grant ProBone), BIO2014-54494-R, MAT2016-77496-R, BIO2017-85522-R, and CTQ2016-79138-R from the Spanish Ministry of Economy, Industry and Competitiveness. A.B. acknowledges the support from the Spanish Ministry of Economy, Industry and Competitiveness (MAT2017-88808-R grant), María de Maeztu Units of Excellence Programme (MDM-2016-0618), and the Diputación de Guipúzcoa for current funding in the frame of Gipuzkoa Fellows program. G.D. thanks the German Federal Ministry of Education and Research (BMBF, Grant No. 031A095C) for funding in the frame of the Molecular Interaction Engineering program (Biotechnologie 2020+). The authors gratefully acknowledge financial support provided by the European Regional Development Fund (ERDF). C.C. thanks the Spanish Ministry of Economy, Industry and Competitiveness for a PhD fellowship (Grant BES-2015-073829).

MNT is a transcription factor of the MXD family. MNT-MAX dimers down-regulate genes by binding to E-box sequences, which can also be bound by MYC-MAX to activate transcription. MNT has been described as a modulator of MYC activity but little is known about MNT regulation and whether MNT has MAX-independent functions. Using a MAX deficient cell line and siRNA-mediated silencing of MAX, we show that in the absence of MAX, the total MNT levels are elevated and that MNT localizes both in the cytoplasm and the nucleus. In contrast, MNT is predominantly nuclear when MAX is expressed. MNT is required for optimal cell proliferation even in the absence of MAX, being the first report of a MAX-independent function of MNT. Interestingly, MNT forms homodimers and autoregulates its expression by repressing its own promoter. The tight MNT regulation and its activity in absence of MAX suggest its importance on cell homeostasis.

Mesoporous silica materials are promising carriers for enzyme immobilization in heterogeneous biocatalysis applications. By tailoring their pore structural framework, these materials are designable for appropriate enzyme binding capacity and internal diffusivity. To supply O2 efficiently to solid-supported immobilized enzymes represents a core problem of heterogeneously catalyzed oxidative biotransformations. In this study, therefore, we synthesized and compared three internally well-ordered and two amorphous silica materials as enzyme carriers, each of those with pore sizes of ≥10 nm, to enable the coimmobilization of d-amino-acid oxidase (79 kDa) and catalase (217 kDa). Both enzymes were fused to the silica-binding module Zbasic2 to facilitate their selective and oriented immobilization directly from crude protein mixtures on native silica materials. Analyzing the effects of varied pore architecture and internal surface area on the performance of the immobilized bienzymatic system, we showed that a uniform pore structural framework was beneficial for enzyme loading (≥70 mg protein/g carrier), immobilization yield (≥90%), surface and pore volume filling without hindered adsorption, and catalytic effectiveness (≥60%) of the coimmobilizate. Using the best carrier LP-SBA-15, we obtained a solid oxidase-catalase preparation with an activity of 2000 μmol/(min g_material) that was recyclable and stable during oxidation of d-methionine. These results affirm a strategy of optimizing immobilized O2-dependent enzymes via tunable internal structuring of the silica material used as carrier. They thus make a significant advance toward the molecular design of heterogeneous oxidation biocatalysts on mesoporous silica supports., V.G.P., C.M.-A., and R.M.B. acknowledge Spanish Government for financial support (MAT-2012-31127). This work has been partially financed by the Spanish State Research Agency (Agencia Española de Investigación, AEI) and the European Regional Development Fund (Fondo Europeo de Desarrollo Regional, FEDER) through the Projects MAT2016–77496-R (AEI/FEDER, UE).

Siliceous ordered mesoporous materials (OMM) are gaining interest as supports for enzyme immobilization due to their uniform pore size, large surface area, tunable pore network and the introduction of organic components to mesoporous structure. We used SBA-15 type silica materials, which exhibit a regular 2D hexagonal packing of cylindrical mesopores of uniform size, for non-covalent immobilization of laccase. Synthesis conditions were adjusted in order to obtain supports with different particle shape, where those with shorter channels had higher loading capacity. Despite the similar isoelectric points of silica and laccase and the close match between the size of laccase and the pore dimensions of these SBA-15 materials, immobilization was achieved with very low leaching. Surface modification of macro-/mesoporous amorphous silica by grafting of amine moieties was proved to significantly increase the isoelectric point of this support and improve the immobilization yield. © 2014 by the authors., The authors thank the Spanish Government for financial support through the project MAT 2012-31127, and Ramiro Martinez (Novozymes, Spain) for his kind help with the supply of laccase. V. G. acknowledges Ministerio de Educación, Cultura y Deporte for a FPU PhD fellowship. We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

This study was designed to assess satisfaction with methadone treatment (MT) in a multidimensional manner among prison inmates and to identify significant independent predictors of inmates’ satisfaction with MT. A total of 158 prison inmates, who were currently on MT for at least the past 3 months, were assessed with the Verona Service Satisfaction Scale for MT. Inmate participants reported slight satisfaction with MT (3.1 on a 1-5 scale), with 51.3% of them feeling dissatisfied. Three factors were found to be independently associated with satisfaction with MT: HIV infection, odds ratio (OR) = 3.72, 95% confidence interval (CI) = [1.79, 7.73]; number of MT episodes, OR = 0.68, 95% CI = [0.53, 0.88]; and perceived influence on methadone dose changes, OR = 1.69, 95% CI = [1.25, 2.28]. Given that perceived involvement in treatment decisions is the only variable independently associated with MT satisfaction that can be modified by changing how MT is implemented, patient participation should be promoted and supported.

The synthesis of hydrophobic peptide as Leu-enkephalin derivatives (N-acetyl phenyl-l leucinamide) from hydrophobic esters (N-acetyl phenylalanine methyl ester) plus a high excess hydrophilic nucleophile (l-leucinamide) catalyzed by immobilized chymotrypsin (α-CT) was studied. By using biphasic systems, the biocatalyst and the necessary high excess of nucleophile remain in the aqueous phase. The hydrophobic acyl donor in the organic phase is partially partitioned into the aqueous phase allowing the synthesis of the peptide. Then the highly hydrophobic reaction product is completely partitioned to the organic phase and it cannot be hydrolyzed by the biocatalyst. Under these conditions, synthetic yields of 95% with respect to the acyl donor were obtained. The excess of nucleophile does not need to be recovered at the end of the synthesis because it remains “immobilized” in the aqueous phase and it can be re-used as well as the immobilized biocatalyst. In such biphasic systems, in each reaction cycle, the synthesis proceeded according to this interesting mass balance: 50 mM N-acetyl phenylalanine methyl ester plus 47.5 mM l-leucinamide were converted into 47.5 mM peptide derivative, that precipitated in the organic phase as a fluffy solid, and 2.5 mM N-acetyl phenyl acid as side product in the aqueous phase. The immobilized biocatalyst (inside a porous structure) is not in contact with the organic phase. Three consecutive reaction cycles were performed, and 95% of peptide was always obtained.

Matrix morphology and surface polarity effects were investigated for Candida antarctica lipase B immobilization. Measurements were made of the amount of lipase immobilized and the catalyst's tributyrin hydrolysis activity, along with a determination of the lipase's functional fraction by active-site titration. Soluble, purified lipase had an active fraction of 84%. Immobilization on the hydrophobic surface of macroporous poly(methylmethacrylate) resin resulted in the full retention the lipase active fraction, while immobilization on the hydrophobic surface of mesoporous, amorphous, octyl-modified silica allowed retention of just half of the lipase active fraction. The polar surface of unmodified mesoporous, amorphous silica bound the lipase in such a manner that all of the immobilized enzyme was active. Mesoporous, crystalline SBA-15 silica also bound lipase so that it all was active. The polar, non-porous surface of fumed silica retained only a small fraction (28%) of active lipase. Substantial differences were found among the various supports in their ability to preserve catalytic activity upon vacuum drying. These findings demonstrate that surface polarity alone is not the only determinant for immobilization, as hydrophobic poly(methylmethacrylate) and hydrophilic SBA-15 were equally competent as lipase supports. The ordered-channel mesostructure of SBA-15 may provide a critical balance of interactions with the enzyme to preserve its native conformation.

Lipase from Candida antarctica B, horseradish peroxidase and laccase have been entrapped in silica cages rising mesoporous structures. Lipase and laccase yielded the highest structured mesoporous material whereas horseradish peroxidase may have altered the symmetry giving as a result mesocelullar foam (MCF) type of cages. The possible effect in the final structure of the material of the nature, size and surface structure of the proteins as well as the presence of various additives in the enzyme extracts is currently under investigations.

23 páginas, 9 figuras.-- et al., 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 upregulation 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 activated the SKP2 promoter in luciferase reporter assays. By 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 (CML). In a cohort of CML 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. MYC-induced SKP2 expression resulted in a decrease in p27 protein in K562 cells. Moreover, silencing of SKP2 abrogated the MYC-mediated downregulation 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.

This study compares the use of different mesoporous silica as lipase supports. Amorphous silica, ordered mesoporous materials SBA-15 and periodic mesoporous organosilica ethane-PMO have been prepared and modified for this study. A slight hydrophobic degree (10% of methyltriethoxysilane) has been introduced in SBA-15 (Me-SBA-15), and a more efficient octyltriethoxysilane modification has been introduced in the amorphous silica (Oct-AS), and compared with the PMO. An analysis of the influence of the hydrophobic nature of the supports along with the porous topology is presented. Enzyme loading, leaching and final activities of the biocatalysts have been tested, pointing to very promising results for the use of PMO as a support. This study is in agreement with previous studies that devoted efforts in designing heterogeneous biocatalysts in a very simple manner based on ordered mesoporous materials.

Lipase has been encapsulated in ordered mesoporous materials in a one-step process. The procedure is simple and leads to well structured materials with enzyme loadings higher than those obtained through the post-synthesis approach. In the final bottle-around-the-ship material the enzyme would be entrapped in silica cages whose entrances are smaller than the enzyme itself. Therefore, reagents and products may pass through, while maintaining the active site entrapped and ready for reuse. (C) 2009 Elsevier Inc. All rights reserved.

BACKGROUND: Enzymatic esterification of phytosterols with fatty acids from butterfat in equimolecular conditions to produce phytosteryl esters was performed in solvent-free medium. Commercial and immobilized Candida rugosa lipases were used as biocatalysts for the reaction. RESULTS: By this methodology, under simple and mild reaction conditions (without solvents, 50 °C and short reaction times), 94% and 99% (w/w) of phystosteroyl esters were obtained in 48 h and 9 h with the commercial and the immobilized lipase, respectively. The effects of temperature, fatty acid specificity, enzyme amount and residual activity of each lipase were also evaluated. CONCLUSIONS: The phytosteryl esters from butterfat produced in this study are expected to have lower melting point, improved oil and fat solubility and bioavailability compared to that of their corresponding free phytosterols. Copyright © 2008 Society of Chemical Industry

El pdf del artículo es la versión de autor.-- et al., p21Waf1 (p21) was described as a cyclin-dependent kinase inhibitor, but other p21 activities have subsequently been described, including its ability to inhibit apoptosis in some models. Comparative work on the human colon cancer isogenic cell lines HCT116 and HCT116p21-/- led to the proposal that p21 protects colon cancer cells against apoptosis by genotoxic drugs. We asked whether p21 also protected from cell death induced by non-genotoxic drugs, such as tyrosine kinase inhibitors. We found that p21-deficient cells were dramatically more sensitive towards imatinib and gefitinib than parental cells. Interestingly, HCT116p21-/- also showed higher basal activity of protein kinases as c-Abl, c-Src, and Akt. We generated HCT116p21-/- sublines with inducible p21 expression and found that p21 did not rescue the hypersensitivity to imatinib. Moreover, down-regulation of p21 by enforced c-Myc expression or by p21 siRNA did not sensitize parental HCT116 cells. We found that, in HCT116p21-/- cells, p53 showed higher stability, higher transcriptional activity and phosphorylation in serines associated with p53 activity. Furthermore, silencing of p53 with siRNA and inactivation of p53 with a dominant negative mutant rescued the hypersensitive response to kinases inhibitors, 5-fluorouracil and adriamycin in HCT116p21-/- cells. Consistently, HCT116p53-/- cells are more resistant to imatinib than parental cells, suggesting that imatinib activity is partly dependent on p53 in colon cancer cells. We conclude that high p53 activity, rather than p21 deficiency, is the mechanism responsible for hypersensitivity to drugs of HCT116p21-/- cells. Therefore the role of p21 on apoptosis of HCT116 colon cancer cells should be re-evaluated. © 2008 Elsevier B.V. All rights reserved., N.F. is funded by a predoctoral fellowship from the Spanish Ministry of Education and Science (MEC) and from the University of Cantabria. Work at the laboratory of J.L. is funded by MEC grants SAF2005-00461 and Spanish Ministry of Health and Consume (MSC) grant ISCIII-RETIC-RD06/0020. M.D.D. is funded by MSC grant FIS04-1083, and J.M.P. is funded by grants from Fundación de Investigación Médica Mutua Madrileña and MEC grant SAF2006-00371.

A systematic study dealing with the influence of several parameters on the immobilization of lipase in ordered mesoporous materials (OMM) is presented here. In a first step, a series of OMM have been synthesized trying to cover the most relevant structures. The aim is to get variation in the key properties susceptible of influencing their behavior as lipase supports, such as the structure (cubic or hexagonal), the nature of the pores (channel-like or cage-like), the connectivity of the porous network and the pore size. Also, by following the co-condensation technique, 5–10%-methylated analogues of the pure-silica materials have been prepared. All the samples have been fully characterized with XRD, TEM (including 3D reconstruction), SEM, TGA and N2 isotherms, and the incorporation of the organic function has been demonstrated by 29Si NMR. All of them have been tested as supports in the immobilization of Candida antarctica Lipase B (CaLB) and the leaching of the enzyme in aqueous media evaluated. With such a systematic approach, valuable information on the influence of the textural properties and the nature of the porous network on the yields of immobilization and enzyme desorption have been stated. Very interestingly, leaching of the enzyme can be diminished until it practically disappears without being covalently bonded to the wall, which places the ordered mesoporous materials at the starting point of a new scenario in enzyme immobilization on preexisting supports.

A mesoporous silica functionalized with octyl groups had been used as the support for lipase (from Candida antarctica ) immobilization. The hydrophobicity provided by the hydrocarbon chains together with the excellent morphologic characteristics of the solid leads to high enzyme loadings in monolayer fashion. However, in this kind of systems the aqueous enzyme solution cannot easily access all the inner surface of the pores because of the highly hydrophobic nature of these surfaces. Thus, the presence of low ethanol concentration decreases the hydrophobicity of the channels and the access of enzyme seems to be significantly improved. The monolayer capacity (400–500 mg protein/g of octyl silica) increases twice compared to the corresponding immobilization in the absence of ethanol (200 mg protein/g of octyl silica). The activity of the derivatives prepared in the presence of ethanol was also significantly improved: 33,000 tributyrin units/g of catalyst at the monolayer limit, which is four- to five-fold higher than the activities of the corresponding derivatives prepared in the absence of ethanol.

A support for enzyme immobilization was prepared by functionalization of mesoporous silica with octyltriethoxysilane. The features of the surface enables the adsorption of lipase from Candida antarctica B via strong hydrophobic interactions, enhancing the stability of the adsorbed enzyme molecules. Derivatives with a high enzyme loading (200 mg protein/g of silica) can be obtained due to the high porosity and surface properties of the support while the immobilization occurs in a monolayer fashion. The lack of inactive enzyme aggregates, together with the high enzyme loading, are responsible for the high catalytic activity achieved by these species. Derivatives were prepared with different lipase loading, and the activities were tested and compared to the commercial derivative Novozym 435. The stability of the catalyst and hence its industrial applicability were tested by performing subsequent reaction cycles of acylation of ethanolamine with lauric acid in acetonitrile. Conversion was quantitative even after 15 reaction cycles.

Thermomyces lanuginosa lipase was employed to catalyze the interesterification reaction between corn oil and tristearin at 45°C in a solvent-free system. HPLC and GC analyses were used to monitor both the distribution of TAG and the concentrations of MAG, DAG, and TAG as the reaction progressed. The positional distribution of the FA residues in the interesterified product was also determined for comparison to that of the original corn oil. Two different weight ratios of corn oil to tristearin were studied. Addition of molecular sieves to the reaction medium reduced the percentage of hydrolysis from 15 to 7. In order to improve the results obtained with Lipozyme TL IM, an immobilization of T. lanuginosa lipase was carried out. At a solids loading of 10% (w/w), the new immobilized lipase reduced the concentration of tristearin from 6 to 0.5% in only 30 min.

The present work aims to be a step forward in the synthesis of siliceous ordered mesoporous materials (OMM) as tailor made matrices to optimize the immobilization and stabilization of enzymes. Based on a classic non-covalent adsorption by electrostatic interactions we have developed the syntheses of materials especially designed for this enzyme, in order to optimize the properties of the final biocatalyst. Siliceous materials with a hexagonal arrangement of parallel mesoporous channels (SBA-15 type of structure) have been synthesized, whose pore diameter has been tuned according to the molecular dimensions of laccase. The synthesis conditions used allowed to obtain pore sizes large enough to permit laccase entrance and diffusion through the pore channels. Diffusion of the enzyme is crucial to obtain high immobilization yield since most of the surface area of the particles is the internal surface of the pores. A poor diffusion would involve retention of enzyme molecules in the pore mouths preventing new ones to access the channel and leading to a low enzyme loading of the catalyst. A micelle swelling agent has been used to expand the supramolecular aggregates that generate the pore architecture of SBA-15 silica. The surfaces of the supports were functionalized with amino groups aiming to strengthen electrostatic interactions between support and enzyme at a suitable pH. Two strategies of surface functionalization of the large-pore ordered mesoporous silica materials were followed: (1) anchoring of an amino-functional alkoxysilane on mesoporous silica and (2) direct co-condensation of a silicon alkoxide and an amino-functional alkoxysilane to obtain the functionalized material in one step. The possibility to prepare carriers where each characteristic has been separately studied and optimized has allowed to obtain biocatalysts with optimal properties. Enzyme loading up to 187 mg/g of catalyst and high activities were achieved with the amino-functionalized large-pore supports. Furthermore, immobilization improved enzyme stability in ethanol. Strong binding forces were capable of housing and retaining the enzyme irreversibly, fully preventing leaching in aqueous medium. Through the careful design of the support material, the biocatalysts obtained share the advantages of enzyme-support covalent attachment regarding absence of leaching and stability, while avoiding drawbacks like loss of activity and enabling the reuse of the support., The authors thank the Spanish Government for financial support through the project MAT 2012-31127, and Mr. Ramiro Martinez (Novozymes, Spain) for his kind help with the supply of Suberase. V. G. acknowledges Spanish Ministerio de Educación, Cultura y Deporte for a FPU PhD fellowship (AP2010-2145).

β- d -glucopyranosidase (βG, EC 3.2.1.21) is an enzyme of considerable importance in food technology for increasing the aroma of wines, musts, fruit juices and alcoholic beverages. In this research we have studied the stabilization of a commercial βG preparation, by covalent immobilization of its carbohydrate moiety to an amine agarose gel. The findings showed total adsorption of the enzyme, previously purified [G. Spagna, D. Romagnoli, A. Martion, G. Bianchi, P.G. Pifferi, Enzyme Microb. Technol., 22 (1998) 298], on the matrix, its low reduction in activity and finally a high stabilization over time.

Two types of highly ordered periodic mesoporous organosilicas have been synthesized as tailor-made supports to immobilize two different enzymes, lipase and laccase. These materials provide an environment where abundant organic groups in close vicinity of the enzyme surface generates a high chemical affinity, which results in high values of enzyme loading, catalytic activity and stabilization. A hydrophobic periodic mesoporous organosilica support (PMO) with a highly ordered hexagonal arrangement of parallel pore channels with diameter around 7 nm, containing framework hydrocarbon groups (ethylene), was used for lipase immobilization. A novel periodic mesoporous aminosilica (PMA), containing secondary amine groups in its framework and having expanded pores was synthesized and studied for immobilization of a larger enzyme, namely laccase. The synthesis conditions were adjusted, using bis[(3- trimethoxysilyl)propyl]amine and 1,2-bis(trimethoxysilyl)ethane as framework co-precursors and 1,3,5- triisopropylbenzene as micelle expander for producing large pores (> 10 nm). The properties of this multifunctional PMA having hydrophilic amino groups and hydrophobic ethylene/propylene groups within the framework were studied. This work compares the confinement of lipase and laccase enzymes in the pores of these hybrid organosilica materials and its effect on immobilization and stabilization parameters. Laccase immobilized on PMA and lipase immobilized on PMO exhibited higher stability in solvents (ethanol and methanol, respectively) compared to enzymes supported on functionalized silica materials with pending organic groups on the surface. High retention of enzymes inside the pores of these materials has been achieved and leaching has been fully prevented. These results can be attributed to the different interactions (hydrophobic, electrostatic and hydrogen bonding) established between the surfaces of enzyme and the PMO/PMA support, which are enhanced by an optimum pore size adjusted to the enzyme dimensions., Authors thank the Spanish MINECO for the nancial support through the project MAT 2012-31127. We thank D. Ramiro Martinez (Novozymes, Spain) for his kind help with the supply of enzymes. V. G. acknowledges Ministerio de Educación, Cultura y Deporte for a FPU PhD fellowship (AP2010-2145).

The functionalization with amine groups was developed on the SBA-15, and its effect in the laccase immobilization was compared with that of a Periodic Mesoporous Aminosilica. A method to encapsulate the laccase in situ has now been developed. In this work, spherical aberration (Cs) corrected scanning transmission electron microscopy combined with high angle annular dark field detector and electron energy loss spectroscopy were applied to identify the exact location of the enzyme in the matrix formed by the ordered mesoporous solids., The authors would like to acknowledge the Spanish Government (MAT 2012-31127) for funding and V.G. for a FPU Ph.D. fellowship (AP2010-2145). A.M. acknowledges the European Union Seventh Frame-work Programme under Grant Agreement No. 312483—ESTEEM2 (Integrated Infrastructure Initiative—I3).

Immobilized enzyme derivatives, in organic media at neutral pH and moderate temperatures, should be mainly and perhaps uniquely inactivated by promotion of conformational changes on their 3D structure. Subsequent irreversible inactivation mechanisms (intermolecular aggregations, chemical modifications, thiol–disulfide exchanges) are thus impossible. However, simple reincubation in aqueous medium of enzymes previously inactivated by solvents usually yields significant but slow and incomplete reactivations. Disruption of incorrect protein structures by denaturing agents (urea, guanidine) is proposed as a new strategy to get rapid, complete and technologically feasible reactivations. By using multipoint immobilized chymotrypsin derivatives, we have evaluated the possibility of unfolding and further refolding of native (non-inactivated) derivatives by different denaturing conditions. After unfolding in 8 M guanidine, derivatives were quickly and completely refolded up to 100% of catalytic activity in 10 minutes. Besides, successive cycles of unfolding and refolding could be exactly reproduced. Finally we checked the possibility to reactivate chymotrypsin derivatives inactivated by dioxane. Simple reincubations in aqueous media yielded a poor reactivation even after 24 hours. However, unfolding in 8 M guanidine enabled complete reactivation in less than 2 hours. From this point of view, by working under `chemically inert conditions' (moderate pH and temperatures), fully dispersed covalently immobilized enzyme derivatives seem to behave as almost everlasting catalysts despite the very deleterious effect of organic media.

In the current manuscript we report a detailed characterization based on spherical aberration (Cs) corrected scanning transmission electron microscopy (STEM) of enzyme (lipase) loaded ordered mesoporous silica (SBA-12) at an accelerating voltage of 80 kV. The extremely high resolution images combined with electron energy loss spectroscopy (EELS) analysis have allowed a complete and unambiguous determination of the presence of the enzyme inside the pores., Spanish Government (MAT2009-13569 project number) is acknowledged for funding.

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., The authors acknowledge the Spanish Government (MAT2009‐13569 project) for funding.

The interest of epoxidation of fatty esters lies in the production of renewable and biodegradable intermediates. A plethora of epoxidizing conditions have been reported in the literature, but it remains difficult to evaluate their relative efficiency and sustainability. To this aim, we present here an evaluation of several catalytic systems for the epoxidation of methyl oleate, together with a green metrics study using atom economy (AE), reaction mass efficiency (RME), material recovery parameter (MRP), yield and reaction time as main parameters. This study reveals the distinct efficiency of the fatty ester epoxidation systems presented in this work. The use of hydrogen peroxide (H2O2) together with fluorinated solvents and aryldiselenide compounds as catalysts was found to be the most efficient methodology. Enzymatic epoxidation also arises as an efficient alternate way to obtain methyl 9,10-epoxystearate. CALB immobilized onto silica has shown a good activity and improved recyclability with respect to Novozym 435., The authors acknowledge E. L.A. (Energías Limpias Alternativas S. L.) (Project OTRI 2010/0529), CICYT (Project CTQ 2011-28124-C02-01), Diputación General de Aragón.

Two immobilized lipases from Candida antarctica have been compared for the direct esterification of tyrosol with oleic acid in equimolar conditions and in the absence of organic solvent. Candida antarctica lipase B was immobilized on octyl-silica agglomerates and compared with commercial Novozym 435. Reduction of tyrosol particle size to 0.1 mm significantly increased the reaction rate with both immobilized lipases, and reduced pressure improved the final tyrosyl oleate yield up to 95% (w/w) in both cases. Immobilized lipases were recovered and reutilized in three consecutive trials with negligible inactivation. Under optimum conditions, a product mixture comprising more than 95% of tyrosyl oleate (w/w) was attained in less than 2 hours. Finally, the index of antioxidant activity obtained, according to the Rancimat method, indicated that tyrosyl oleate was slightly more effective than tyrosol as an antioxidant in a low polar matrix. © 2012 Informa UK, Ltd.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., 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., This study was supported by grants SAF08-01581 and ISCIII-RETIC RD06/0020/0017 from the Spanish Ministerio de Ciencia e Innovacion (MICINN) to JL; FIS 08/0829 to MDD; BFU2008-00238 and CSD2006-00049 from MICINN, and P06-CVI-4844 from Junta de Andalucía to JCR. NF was the recipient of a postdoctoral fellowship of the University of Cantabria.

El pdf del artículo es la versión pre-print.-- et al., Imatinib is a Bcr-Abl inhibitor used as first-line therapy of chronic myeloid leukemia (CML). p21Cip1, initially described as a cell cycle inhibitor, also protects from apoptosis in some models. We describe that imatinib down-regulates p21Cip1 expression in CML cells. Using K562 cells with inducible p21 expression and transient transfections we found that p21 confers partial resistance to imatinib-induced apoptosis. This protection is not related to the G2-arrest provoked by p21, a decrease in the imatinib activity against Bcr-Abl or a cytoplasmic localization of p21. The results suggest an involvement of p21Cip1 in the response to imatinib in CML., This work was supported by Grants SAF08-01581 and ISCIII-RETIC-RD06/0020/0017 to J.L. and Grant FIS08/0829 to M.D.D. NF and JMC are supported by fellowships from University of Cantabria.

6 páginas, 4 figuras, 2 tablas., Lipase-catalyzed ethanolysis of two short-chain triradylglycerols, namely tributyrin and 2,3-dibutyroil-1-O-alkylglycerols, have been studied. Much faster rate of reaction for the ethanolysis of tributyrin than that of 2,3-dibutyroil-1-O-alkylglycerols was attained. A kinetic model for the rate of release of ethyl butyrate and for the inactivation of the lipase has been also studied. The parameter corresponding to the release of ethyl butyrate was one order of magnitude higher for ethanolysis of tributyrin than the corresponding of 2,3-dibutyroil-1-O-alkylglycerols. On the contrary, the stability of Novozym 435 during ethanolysis of 2,3-dibutyroil-1-O-alkylglycerols was higher than the corresponding of tributyrin. At the reaction conditions under study, both ethanolysis reactions take place with high selectivity and yield monoesterified alkylglycerols and sn-2 monobutyrin as the main acylglycerols in the reaction mixtures., This work was supported by the projects AGL2006-02031/ALI and AGL2008-05655 by Ministerio de Ciencia (Spain) and also by Comunidad Autonoma de Madrid (ALIBIRD, project number S-505/AGR-0153) and Consolider-Ingenio FUN-C-FOOD (CSD2007- 00063). A predoctoral fellowship for Luis Vázquez, provided by Comunidad Autonoma de Madrid, is also acknowledged.

Chymotrypsin linked to agarose beads by multi-point covalent attachment catalyzes synthesis of Ac-Trp-OEt in 3-pentanone even when the thermodynamic water activity (aw) of the system is reduced to as low as 0.4. If fully hydrated catalyst is added to the reaction mixture before removal of water, product is formed linearly once aw has stabilized. The initial rate is reduced from that if aw is kept close to 1 (0.47 mmol s-1 (kg enzyme)-1), to 50% (aw 0.9), 25% (aw 0.4) and < 1% (aw 0.25). The large drop between aw of 1 and 0.9 probably reflects the effects of water removal on the agarose gel structure. Catalyst partly dried (even only to aw 0.86) before adding to the organic phase is inactive. At reduced aw, the equilibrium (when reached) is shifted in favor of the ester, as expected.