Elena M. Sánchez-Fernández

Investigations on the Oxygen Dependence of a 2-Oxoglutarate Histone Demethylase

Histone N(ϵ)-methyl lysine demethylases are important in epigenetic regulation. KDM4E (histone lysine demethylase 4E) is a representative member of the large Fe(II)/2-oxoglutarate- dependent family of human histone demethylases. In the present study we report kinetic studies on the reaction of KDM4E with O2. Steady-state assays showed that KDM4E has a graded response to O2 over a physiologically relevant range of O2 concentrations. Pre-steady state assays implied that KDM4E reacts slowly with O2 and that there are variations in the reaction kinetics which are dependent on the methylation status of the substrate. The results demonstrate the potential for histone demethylase activity to be regulated by oxygen availability.

sp2-Iminosugar O-, S-, and N-glycosides as conformational mimics of α-linked disaccharides; implications for glycosidase inhibition

The synthesis of mimics of the α(1→6)- and α(1→4)-linked disaccharides isomaltose and maltose featuring a bicyclic sp(2)-iminosugar nonreducing moiety O-, S-, or N-linked to a glucopyranoside residue is reported. The strong generalized anomeric effect operating in sp(2)-iminosugars determines the α-stereochemical outcome of the glycosylation reactions, independent of the presence or not of participating protecting groups and of the nature of the heteroatom. It also imparts chemical stability to the resulting aminoacetal, aminothioacetal, or gem-diamine functionalities. All the three isomaltose mimics behave as potent and very selective inhibitors of isomaltase and maltase, two α-glucosidases that bind the parent disaccharides either as substrate or inhibitor. In contrast, large differences in the inhibitory properties were observed among the maltose mimics, with the O-linked derivative being a more potent inhibitor than the N-linked analogue; the S-linked pseudodisaccharide did not inhibit either of the two target enzymes. A comparative conformational analysis based on NMR and molecular modelling revealed remarkable differences in the flexibility about the glycosidic linkage as a function of the nature of the linking atom in this series. Thus, the N-pseudodisaccharide is more rigid than the O-linked derivative, which exhibits conformational properties very similar to those of the natural maltose. The analogous pseudothiomaltoside is much more flexible than the N- or O-linked derivatives, and can access a broader area of the conformational space, which probably implies a strong entropic penalty upon binding to the enzymes. Together, the present results illustrate the importance of taking conformational aspects into consideration in the design of functional oligosaccharide mimetics.

Efficient Transfection of Hepatocytes Mediated by mRNA Complexed to Galactosylated Cyclodextrins

In this study, we aimed at specific targeting of polycationic amphiphilic cyclodextrins (paCDs) to HepG2 cells via the asialoglycoprotein receptor (ASGPr). The transfection efficiencies of paCDs modified with galactose moieties were evaluated. In preliminary experiments, attempts to transfect HepG2 cells with pDNA complexed with different modified paCDs resulted in very low transfection levels. In additional series of experiments, we found out that nucleic acid/cyclodextrin complexes (CDplexes) were efficiently taken up by the cells and that photochemical internalization, which facilitates release from endosomes, did not improve transfection. Further experiments showed that pDNA can be readily released from the CDplexes when exposed to negatively charged vesicles. These observations imply that the lack of transfection cannot be attributed to a lack of internalization, release of CDplexes from the endosomal compartment, or release of free pDNA from the CDplexes. This in turn suggests that the nuclear entry of the pDNA represents the main limiting factor in the transfection process. To verify that HepG2 cells were transfected with targeted CDplexes containing mRNA, which does not require entry into the nucleus for being translated. With mRNA encoding the green fluorescent protein, fractions of GFP-positive cells of up to 31% were obtained. The results confirmed that the galactosylated complexes are specifically internalized via the ASGPr.

Generalized anomeric effect in gem-diamines: stereoselective synthesis of alpha-N-linked disaccharide mimics.

The orbital (negative hyperconjugation) contribution to the generalized anomeric effect is highly increased in bicyclic gem-diamines with a pseudoamide-type endocyclic nitrogen atom, which has been exploited for the stereoselective synthesis of configurationally stable alpha-N-linked azadisaccharide heteroanalogues of the natural disaccharides maltose and isomaltose as aglycon-sensitive inhibitors of isomaltase.

Generalized Anomeric Effect in gem-Diamines: Stereoselective Synthesis of α-N-Linked Disaccharide Mimics

The orbital (negative hyperconjugation) contribution to the generalized anomeric effect is highly increased in bicyclic gem-diamines with a pseudoamide-type endocyclic nitrogen atom, which has been exploited for the stereoselective synthesis of configurationally stable alpha-N-linked azadisaccharide heteroanalogues of the natural disaccharides maltose and isomaltose as aglycon-sensitive inhibitors of isomaltase.

Chemical Composition of the Essential Oil from the Leaves of Juniperus phoenicea L. from North Africa

Abstract The chemical study of the essential oil extracted from leaves of Juniperus phoenicea growing in Morocco has been carried out. The main components of J. phoenicea oil were the monoterpene hydrocarbons α-pinene (45.5%) and ω-3-carene (13.0%). Our results show a number of differences in composition with respect to a previous study of the leaf oils of J. phoenicea (Greece and Spain).

Influence of the configurational pattern of sp2-iminosugar pseudo N-, S-, O- and C-glycosides on their glycoside inhibitory and antitumor properties

The synthesis of a complete series of cyclic carbamate-type sp(2)-iminosugar N-, S-, O- and C-octyl pseudoglycosides related to nojirimycin, mannojirimycin and galactonojirimycin, all having the α-pseudoanomeric configuration, is reported. The gem-diamine-type N-pseudoglycosides can be accessed directly from the corresponding reducing sp(2)-imisosugar precursors by reaction with octylamine in methanol, whereas per-O-acetyl or 1-fluoro derivatives were used as pseudoglycosyl donors for the preparation of S-pseudoglycosides or O- and C-pseudoglycosides, respectively. Evaluation of their inhibitory properties against a panel of glycosidases evidenced selectivity profiles that strongly depend on the configurational pattern and the nature of the glycosidic linkage. On the contrary, the antiproliferative activity determined against a panel of tumor cell lines was largely independent of the relative orientation of the hydroxyl groups in the sp(2)-iminosugar moiety. Indeed, sp(2)-iminosugar representatives exhibiting significant growth inhibition potencies were identified in all three configurationally different types of compounds studied, namely α-d-gluco, α-d-manno and α-d-galacto glycoside analogs. Interestingly, none of the compounds affected viability and mortality of normal cells at the used concentrations. Altogether, the results strongly suggest that the anticancer activity of amphiphilic sp(2)-iminosugar glycosides might be unrelated, or not solely related, to their glycosidase inhibitory activity.

Antileishmanial activity of sp2-iminosugar derivatives

A series of sp2-iminosugar-type glycomimetics bearing S-linked pseudoglycoside substituents (sulfide, sulfoxide and sulfone derivatives) has been synthesized and evaluated as new potential drugs against the protozoan parasite Leishmania, responsible of leishmaniasis, the second most relevant parasitic disease after malaria. All the prepared compounds share a bicyclic 5N,6O-oxomethylidenenojirimycin glycone-like moiety bearing a substitution pattern of configurational complementarity with the natural α-glucosides and incorporate either an n-octyl or n-dodecyl aglycone-like substituent. Not surprisingly, they behaved as potent to moderate competitive inhibitors of α-glucosidase (inhibition constants, Ki, in the range 1.3 to 447 μM). Evaluation of the antileishmanial activity indicated that the dodecyl pseudoglycosides present a significant antiparasitic activity in intracellular amastigotes of Leishmania donovani, the clinically relevant form of the parasite. The antileishmanial effect seems to be associated with the anticancer and proapoptotic activity of the glycomimetics, but not with the α-glucosidase inhibitory efficiency. The (SS)-configured dodecylsulfoxide derivative 4, exhibiting the most favourable activity/toxicity profile, was further assayed in combination treatment with miltefosine, the first oral antileishmanial drug, using the fixed ratio isobologram method. The interaction between derivative 4 and 0.1, 0.2 and 0.3 μM miltefosine was classified as synergistic, showing combination indices of 0.78, 0.76 and 0.80, respectively. Additionally, a miltefosine resistant Leishmania line and the wild-type strain showed similar susceptibility to derivative 4. The results illustrate the potential of sp2-iminosugar pseudoglycosides as promising prototypes for the development of new therapeutic strategies for leishmaniasis.

Synthesis of Multibranched Australine Derivatives from Reducing Castanospermine Analogues through the Amadori Rearrangement of gem-Diamine Intermediates: Selective Inhibitors of β-Glucosidase

A practical one-pot synthesis of bi- and triantennated australine analogues from a pivotal sp(2)-iminosugar-type reducing castanospermine precursor is reported. The transformation involves a gem-diamine intermediate that undergoes the indolizidine → pyrrolizidine Amadori-type rearrangement and proceeds under strict control of the generalized anomeric effect to afford a single diastereomer. The final compounds behave as selective competitive inhibitors of β-glucosidase and are promising candidates as pharmacological chaperones for Gaucher disease.

Marginal bone loss in implants placed in grafted maxillary sinus.

PURPOSE The purpose of this study is to evaluate the vertical and horizontal graft bone resorption (GR) in grafted maxillary sinuses and the marginal bone loss (MBL) around implants placed in the sinuses with different prosthetic connections and to determine the effect of other clinical factors on these tissue responses at 6 and 18 months postloading. MATERIAL AND METHOD A total of 254 implants were placed in 150 grafted maxillary sinuses of 101 patients (51.5% female) with mean age of 52.2 years (range, 32-82 years). GR and MBL measurements were made in implants placed with two different prosthetic connections (internal and external) at 6 and 18 months postloading. The complex samples general linear model was used to analyze the influence of patient age, gender, smoking habit, history of periodontal disease, implantation timing (simultaneous vs deferred), and prosthetic abutment length on radiographic GR and MBL values. RESULTS At 18 months postloading, the MBL ranged from 0 mm to 5.89 mm; less than 1 mm was lost around 49.0% (mesial) and 44.3% (distal) of the implants, while no bone was lost around 32.9% (mesial) and 26.7% (distal). The GR was significantly affected by smoking, remnant alveolar bone height, graft length, graft height, gender, and age, and it significantly decreased over time. The MBL was influenced by the type of connection, implantation timing, and prosthetic abutment length. The MBL was greater with longer postloading interval and higher patient age and in smokers. CONCLUSION Resorption of grafts that combine autogenous cortical bone with anorganic bovine bone is dependent on the anatomic features of the sinus and is not affected by the time elapsed after the first 6 months. The MBL in implants placed in these grafted areas is time dependent and mainly related to potentially modifiable clinical decisions and patient habits.