Analogs of farnesylcysteine induce apoptosis in HL-60 cells

AbstractS-Farnesyl-thioacetic acid (FTA), a competitive inhibitor of isoprenylated protein methyltransferase, potently suppressed the growth of HL-60 cells and induced apoptosis, as evidenced by the development of increased annexin-V binding, decreased binding of DNA dyes and internucleosomal DNA degradation. FTA did not impair the membrane association of ras proteins, conversely, it brought about a decrease in the proportion of ras present in the cytosolic fraction. Farnesylated molecules which are weak inhibitors of the methyltransferase also induced DNA laddering and reduced the proportion of cytosolic ras. These findings suggest that neither inhibition of isoprenylated protein methylation nor impairment of ras membrane association are essential for apoptosis induced by farnesylcysteine analogs

The C-terminal peptide of H-Ras as a target for the covalent binding of drugs modulating ras-dependent pathways

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CARB-ES-19 Multicenter Study of Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli From All Spanish Provinces Reveals Interregional Spread of High-Risk Clones Such as ST307/OXA-48 and ST512/KPC-3

ObjectivesCARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain.MethodsIn total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis.ResultsIn total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were blaOXA–48 (263/377), blaKPC–3 (62/377), blaVIM–1 (28/377), and blaNDM–1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5).ConclusionThis study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3

Effect of irrigation water quality on soil properties and infrared spectroscopic signatures

Aim of study: To study the effect of irrigation with medium-to-low-quality water on an olive farm that seems to be causing salinity and/or sodicity problems in soils, and the ability of infrared spectroscopy to detect this problem.Area of study: The study was conducted in an olive (Olea europaea L.) grove located in Guarromán (Jaen, Spain), on the boundary of the Sierra Morena Mountains and the Guadalquivir Depression.Material and methods: The olive farm is cultivated over two soil typologies, a calcareous area (carbonated) dominated by Regosols and a siliceous area with Leptosols. Typical soil physical and chemical parameters were determined, as well as near and mid infrared spectra were collected for analysis.Main results: Soil physical properties were affected by irrigation, with low infiltration rates and symptoms of structural degradation. Chemical properties were also altered, showing high pH, low amounts of organic carbon and N, and high sodium concentrations. These effects were stronger in the samples directly affected by the irrigation bulb, with the siliceous soils more affected than carbonated, probably due to the positive effect of the higher amounts of calcium in the latter. Using infrared spectroscopy, it was possible to discriminate the samples of this farm affected by sodicity from similar soil samples in Jaen province not affected.Research highlights: the use of medium-to-low quality irrigation water affected soil physical and chemical properties. Infrared spectroscopy could be useful for quick assessment of soil quality and soil degradation from salinity and sodicity

DLG differentially localizes Shaker K+-channels in the central nervous system and retina of Drosophila

Subcellular localization of ion channels is crucial for the transmission of electrical signals in the nervous system. Here we show that Discs-Large (DLG), a member of the MAGUK (membrane-associated guanylate kinases) family in Drosophila, co-localizes with Shaker potassium channels (Sh Kch) in most synaptic areas of the adult brain and in the outer membrane of photoreceptors. However, DLG is absent from axonal tracts in which Sh channels are concentrated. Truncation of the C-terminal of Sh (including the PDZ binding site) disturbs its pattern of distribution in both CNS and retina, while truncation of the guanylate kinase/C-terminal domain of DLG induces ectopic localization of these channels to neuronal somata in the CNS, but does not alter the distribution of channels in photoreceptors. Immunocytochemical, membrane fractionation and detergent solubilization analysis indicate that the C-terminal of Sh Kch is required for proper trafficking to its final destination. Thus, several major conclusions emerge from this study. First, DLG plays a major role in the localization of Shchannels in the CNS and retina. Second, localization of DLG in photoreceptors but not in the CNS seems to depend on its interaction with Sh. Third, the guanylate kinase/C-terminal domain of DLG is involved in the trafficking of Shaker channels but not of DLG in the CNS. Fourth, different mechanisms for the localization of Sh Kch operate in different cell types.This work was supported by RO1 grants by the National Institutes of Health and by a Research Career Development Award to VB, by a Generalitat Valenciana grant and by an European Union QLRT grant to FJT, and a pre-doctoral fellowship of Generalitat Valenciana to CR.Peer reviewe

Effect of soil type and management (organic vs. conventional) on soil organic matter quality in olive groves in a semi-arid environment in Sierra Mágina Natural Park (S Spain)

Organic matter from surface horizons of olive grove soils under organic and conventional management, compared to natural vegetation (undisturbed), on two contrasting geological substrates (colluvial limestones and marls) in a semi-arid Mediterranean area were characterized to evaluate the influence of management and soil type. Higher percentages of total carbon, total humic extract and humin fractions were observed in the organic management and undisturbed soil samples. The main qualitative functional and compositional changes induced by management were related to the aromaticity of humic acids (HA), which were lower in organically managed samples than conventional management. This may be due to continuous effective incorporation of alkyl compounds from spontaneous vegetation, which would explain its typical lignin patterns and higher E4/E6 ratios (visible spectra absorbance to 465 and 665nm). The main qualitative changes affecting organic matter were induced by the geological substrate (soil type). Soil samples from marls had a lower humic to fulvic acids ratio, and their HA were more aliphatic (with respect to the 2920cm-1 infrared band) and more intense with organic management. These samples also had well-defined amide and carbohydrate infrared bands (labile compounds) and lower E4 optical density. These results showed less efficient biodegradation of aliphatic components. On the contrary, greater decomposition of organic matter was detected in the HA fraction from colluvial limestones, which was transformed into more stable and evolved forms of humus. This may be due to overlapping of current soil management and its biogenic background, representative of the original soil in this area, where pedogenic development is more intense. Characteristics of soil organic matter in the undisturbed soil were between conventional and organic management, which shows how difficult it is to find natural, well-conserved soil to serve as a reference in the agricultural context studied. Our findings reveal that more sustainable soil management and development of management practices more in harmony with the original evolution of the soil and its relationship to the parent material are especially important in this area. © 2011 Elsevier B.V.Peer Reviewe

Amoxicillin inactivation by thiol-catalyzed cyclization reduces protein haptenation and antibacterial potency

16 p.-7 fig.-2 tab.-1 graph. abst.Serum and cellular proteins are targets for the formation of adducts with the β-lactam antibiotic amoxicillin. This process could be important for the development of adverse, and in particular, allergic reactions to this antibiotic. In studies exploring protein haptenation by amoxicillin, we observed that reducing agents influenced the extent of amoxicillin-protein adducts formation. Consequently, we show that several thiol-containing compounds, including dithiothreitol, N-acetyl-L-cysteine, and glutathione, perform a nucleophilic attack on the amoxicillin molecule that is followed by an internal rearrangement leading to amoxicillin diketopiperazine, a known amoxicillin metabolite with residual activity. Increased diketopiperazine conversion is also observed with human serum albumin but not with L-cysteine, which mainly forms the amoxicilloyl amide. The effect of thiols is catalytic and can render complete amoxicillin conversion. Interestingly, this process is dependent on the presence of an amino group in the antibiotic lateral chain, as in amoxicillin and ampicillin. Furthermore, it does not occur for other β-lactam antibiotics, including cefaclor or benzylpenicillin. Biological consequences of thiol-mediated amoxicillin transformation are exemplified by a reduced bacteriostatic action and a lower capacity of thiol-treated amoxicillin to form protein adducts. Finally, modulation of the intracellular redox status through inhibition of glutathione synthesis influenced the extent of amoxicillin adduct formation with cellular proteins. These results open novel perspectives for the understanding of amoxicillin metabolism and actions, including the formation of adducts involved in allergic reactions.This work was supported by grant SAF2015-68590-R from MINECO/FEDER, RTI2018-097624-B-I00 and RETIC Aradyal from ISCIII/FEDER RD16/0006/0021 to DP-S; RD16/0006/0001 to MT, RD16/0006/0024 to MB; grants CP15/00103 and PI17/ 01237 from ISCIII/ERDF and PI-0179-2014 from Andalusian Regional Ministry Health to MM. AA holds a “Sara Borrell” research contract (CD17/0146) supported by ISCIII from MINECO [confounded by the European Social Fund (ESF)]. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)Peer reviewe

Enzymatic fine-tuning for 2-(6-hydroxynaphthyl) β-d-xylopyranoside synthesis catalyzed by the recombinant β-xylosidase BxTW1 from Talaromyces amestolkiae

Abstract Background Glycosides are compounds displaying crucial biological roles and plenty of applications. Traditionally, these molecules have been chemically obtained, but its efficient production is limited by the lack of regio- and stereo-selectivity of the chemical synthesis. As an interesting alternative, glycosidases are able to catalyze the formation of glycosides in a process considered green and highly selective. In this study, we report the expression and characterization of a fungal β-xylosidase in Pichia pastoris. The transglycosylation potential of the enzyme was evaluated and its applicability in the synthesis of a selective anti-proliferative compound demonstrated. Results The β-xylosidase BxTW1 from the ascomycete fungus Talaromyces amestolkiae was cloned and expressed in Pichia pastoris GS115. The yeast secreted 8 U/mL of β-xylosidase that was purified by a single step of cation-exchange chromatography. rBxTW1 in its active form is an N-glycosylated dimer of about 200 kDa. The enzyme was biochemically characterized displaying a K m and k cat against p-nitrophenyl-β-d-xylopyranoside of 0.20 mM and 69.3 s−1 respectively, and its maximal activity was achieved at pH 3 and 60 °C. The glycan component of rBxTW1 was also analyzed in order to interpret the observed loss of stability and maximum velocity when compared with the native enzyme. A rapid screening of aglycone specificity was performed, revealing a remarkable high number of potential transxylosylation acceptors for rBxTW1. Based on this analysis, the enzyme was successfully tested in the synthesis of 2-(6-hydroxynaphthyl) β-d-xylopyranoside, a well-known selective anti-proliferative compound, enzymatically obtained for the first time. The application of response surface methodology, following a Box-Behnken design, enhanced this production by eightfold, fitting the reaction conditions into a multiparametric model. The naphthyl derivative was purified and its identity confirmed by NMR. Conclusions A β-xylosidase from T. amestolkiae was produced in P. pastoris and purified. The final yields were much higher than those attained for the native protein, although some loss of stability and maximum velocity was observed. rBxTW1 displayed remarkable acceptor versatility in transxylosylation, catalyzing the synthesis of a selective antiproliferative compound, 2-(6-hydroxynaphthyl) β-d-xylopyranoside. These results evidence the interest of rBxTW1 for transxylosylation of relevant products with biotechnological interest.Science, Faculty ofNon UBCChemistry, Department ofReviewedFacult