1,720,973 research outputs found
Potent inhibitory activity of taniborbactam towards NDM-1 and NDM-1Q119X mutants, and in vitro activity of cefepime/taniborbactam against MBLs producing Enterobacterales
No full textObjective: This study aimed to investigate the in vitro activity of taniborbactam (VNRX-5133), a novel broad-spectrum bicyclic boronate, against NDM-1 and Q119E, Q119K, Q119C, Q119F, Q119V, and Q119Y NDM-1 variants, which showed an increased activity towards some β-lactams, including cefepime. Methods: Inhibition kinetic assays were spectrophotometrically performed using cefepime (50 μM) as the reporter substrate and 80 nM of each enzyme. Taniborbactam behaves as a competitive inhibitor towards NDM-1 and NDM-1 Q119 variants with lower Ki values (range 3–16 nM). The phenotypic profile was assessed in both Enterobacterales clinical isolates and engineered Escherichia coli BL21(DE3) strains by conventional broth microdilution procedures according to the Clinical and Laboratory Standards Institute (CLSI). Results: Taniborbactam at a fixed concentration of 4 mg/L was able to restore activity of cefepime in 24 of 26 Enterobacterales clinical isolates harbouring metallo-β-lactamases with MIC50/MIC90 values of 14 mg/L. Cefepime MICs were drastically reduced in all clinical isolates and in NDM-1 and Q119X producing Escherichia coli BL21(DE3). Taniborbactam was unable to restore susceptibility to cefepime in two IMP variants producing clinical isolates. Conclusion: The inhibition level of NDM enzymes provided by taniborbactam protects the antibacterial activity of cefepime from this important metallo-β-lactamase
Kinetic properties of native and mutagenized isoforms of mitochondrial alcohol dehydrogenase III purified from Kluyveromices lactis
No full textBy computer modelling and protein engineering we have investigated changes in two amino acid residues located in the coenzyme pocket
of the yeast Kluyveromyces lactis mitochondrial alcohol dehydrogenase III. These two residues, Gly 225 and Ala 274, were hypothesized to
be involved in the enzyme discrimination between NAD(H) and NADP(H). Upon changing Gly 225 to Ala we produced an enzyme (mutant
G225A) showing very little difference from the wild-type. On the contrary, change at position 274 of Phe instead ofAla (mutantA274F) caused
a significant increase of Km values for NAD(P) and for NADPH and even a more marked decrease in catalytic activity. The kcat/Km rates for
NADP(H) were also decreased in this mutant. Enzymes with the double changes at 225 and 274 (mutant G225A-A274F) showed, apart the
substantial low Km value for NADPH and its high catalytic efficiency, kinetic parameters relative to coenzymes which were not additive over
the single substitutions. Surprisingly, enzymes with changes at the two positions reduced efficiently acetaldehyde, displaying a Km value
10-fold lower and a catalytic efficiency sevenfold higher with respect to parent or singularly mutated enzymes. None of the engineered
enzymes would convert formaldehyde, glutaraldehyde or aromatic aldehydes but all enzymes reduced propionaldehyde and butyraldehyde at
relative reaction rates approximately half of that exhibited by acetaldehyde. Interestingly only mutant A274F was able to oxidize methanol
almost as well as ethanol. In addition, this mutant was capable to convert secondary and cyclic alcohols, at a rate not detected in the other
isoforms. These results are in general agreement with the prediction that increasing the size of amino acids in the proximity of the coenzyme
pocket would hamper the accommodation of NADP but discord the increased affinity for NADPH as well as for alcoholic or aldehydic
substrates with high steric hindrance
Protein glycans alteration and a different distribution of some enzymatic activities involved in the glycan processing are found in AZT-treated K562 cells
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Kinetic profile and molecular dynamic studies show that Y229W substitution in an NDM-1/L209F variant restores the hydrolytic activity of the enzyme toward penicillins, cephalosporins, and carbapenems
No full textThe New Delhi metallo-β-lactamase-1 (NDM-1) enzyme is the most common metallo-β-lactamase identified in many Gram-negative bacteria causing severe nosocomial infections. The aim of this study was to focus the attention on non-active-site residues L209 and Y229 of NDM-1 and to investigate their role in the catalytic mechanism. Specifically, the effect of the Y229W substitution in the L209F variant was evaluated by antimicrobial susceptibility testing, kinetic, and molecular dynamic (MD) studies. The Y229W single mutant and L209F-Y229W double mutant were generated by site-directed mutagenesis. The Km , k cat, and k cat/Km kinetic constants, calculated for the two mutants, were compared with those of (wild-type) NDM-1 and the L209F variant. Compared to the L209F single mutant, the L209F-Y229W double mutant showed a remarkable increase in k cat values of 100-, 240-, 250-, and 420-fold for imipenem, meropenem, benzylpenicillin, and cefepime, respectively. In the L209F-Y229W enzyme, we observed a remarkable increase in k cat/Km of 370-, 140-, and 80-fold for cefepime, meropenem, and cefazolin, respectively. The same behavior was noted using the antimicrobial susceptibility test. MD simulations were carried out on both L209F and L209F-Y229W enzymes complexed with benzylpenicillin, focusing attention on the overall mechanical features and on the differences between the two systems. With respect to the L209F variant, the L209F-Y229W double mutant showed mechanical stabilization of loop 10 and the N-terminal region. In addition, Y229W substitution destabilized both the C-terminal region and the region from residues 149 to 154. The epistatic effect of the Y229W mutation jointly with the stabilization of loop 10 led to a better catalytic efficiency of β-lactams. NDM numbering is used in order to facilitate the comparison with other NDM-1 studies
Effects of AZT on cellular iron homeostasis
No full text3'-azido-3'-deoxythymidine (AZT), the first chemotherapeutic drug approved by FDA for treatment of HIV-infected patients and still used in combination therapy, has been shown to induce, upon prolonged exposure, severe bone marrow toxicity manifested as anemia, neutropenia and siderosis. These toxic effects are caused by inhibition of heme synthesis and, as a consequence, transferrin receptor (TfR) number appears increased and so iron taken up by cells. Since iron overload can promote the frequency and severity of many infections, siderosis is viewed as a further burden for AIDS patients. We have previously demonstrated that AZT-treated K562 cells showed an increase of the number of TfRs located on the surface of the plasma membrane without affecting their biosynthesis, but slowing down their endocytotic pathway. In spite of the higher number of receptors on the plasma-membrane of AZT-treated cells, intracellular accumulation of iron showed a similar level in control and in drug-exposed cells. The chelating ability of AZT and of its phosphorylated derivatives, both in an acellular system and in K562 cells, was also checked. The results demonstrated that AZT and AZTMP were uneffective as iron chelators, while AZTTP displayed a significant capacity to remove iron from transferrin (Tf). Our results suggest that AZT may be not directly involved in the iron overloading observed upon its prolonged use in AIDS therapy. The iron accumulation found in these patients is instead caused by other unknown mechanisms that need further studies to be clarified
3 '-Azido-3 '-deoxythymidine reduces the rate of transferrin receptor endocytosis in K562 cells
No full textK562 cells, exposed for at least 24 h to 5 mu M 3'-azido-3'-deoxythymidine (AZT), gave rise to an overall increase in the number of cell surface transferrin binding receptors (18-20%). This effect was ascertained either with binding experiments by using I-125-transferrin and with immunoprecipitation by using a specific monoclonal antibody against the transferrin receptor. At higher AZT concentrations (20 and 40 mu M), a further increase was found, that is, up to 23% by binding experiments and up to 110% by immunoprecipitation. However, Scatchard analysis of the binding data indicated that although the number of cell surface transferrin receptors increased, the affinity of transferrin for its receptor did not change (K-a = 4.0 x 10(8) M). Surprisingly, immunoprecipitation of total receptor molecules showed that the synthesis of receptor was not enhanced by the drug treatment. The effect of AZT on transferrin internalization and receptor recycling was also investigated. In this case, data indicated that the increase in the number of receptors at the cell surface was probably due to a slowing down of endocytosis rate rather than to an increased recycling rate of the receptor to cell surface. In fact, the time during which half the saturated amount of transferrin had been endocytosed (t(1/2)) was 2.15 min for control cells and 3.41, 3.04, and 3.74 min for 5, 20, and 40 mu M AZT-treated cells, respectively. Conversely, recycling experiments did not show any significant differences between control and treated cells. A Likely mechanism through which AZT could interfere with the transferrin receptor trafficking, together with the relevance of our findings, is extensively discussed
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Evaluation of cefiderocol stability against clinically relevant serine- and metallo-β-lactamases
No full textVariations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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