40 research outputs found
Mechanistic investigation of human cytochromes P450 involved in hormone biosynthesis
Get PDFCYP17A1 and CYP19A1 are the key cytochromes P450 involved in steroidogenesis. Mutations causing hypo- or hyper-activation of both these enzymes results in diseased states, including cancers. In addition to carrying out mono-oxygenation reactions, both CYP17A1 and CYP19A1 also catalyze carbon-carbon lyase reactions. There is significant interest in developing therapeutics capable of specifically targeting the CYP17A1 lyase reaction to target androgen synthesis without impacting glucocorticoid production, and understanding of CYP19A1 reaction mechanism could similarly help guide design of mechanism based inhibitors for certain breast cancers. To interrogate mechanistic aspects of both these enzymes, I used Nanodisc technology for reconstituting active forms of these proteins in functional complexes with their effector proteins in a native-like membrane environment.
Cytochrome P450 17A1 (CYP17A1) is a multi-functional enzyme, catalyzing synthesis of glucocorticoid precursors by hydroxylation of pregnene nucleus, and androgen biosynthesis by a second C-C lyase step, at the expense of glucocorticoid production. It has been recently confirmed in our lab that when 17-hydroxy-pregnenolone is a substrate, CYP17A1 lyase reaction proceeds through a non-conventional hemiketal intermediate. However, it remains unknown if the alternate substrate, 17-hydroxy-progesterone (OH-PROG) is transformed to androstenedione via this novel mechanism, by the traditional compound I (Cpd I) mechanism, or by a combination of the two. To this end, I investigated kinetic solvent isotope effects on steady state turnover for lyase reaction on OH-PROG. Further, cryo-trapped peroxo-anion intermediate was spectroscopically followed as the samples were thermally annealed. Resonance Raman spectroscopy (rR) on the trapped intermediate was also performed. Our results strongly indicate that the CYP17A1 mediated OH-PROG lyase proceeds through a hemiketal intermediate.
Cytochrome b5 (cyt b5) is a small heme-protein, known to enhance the rate of the CYP17A1 lyase reaction by ~5 fold, while having little effect on hydroxylation. Investigation with a redox inactive form of cyt b5 indicated that for lyase enhancement, cyt b5 critically requires a redox effector role. In order to investigate the electron transfer by cyt b5 to CYP17A1, I employed stopped flow spectroscopy and compared the CYP17A1 reduction rates by cyt b5 that by cytochrome P450 reductase (CPR). RR was also performed on CYP17A1—cyt b5 complex investigate any conformational perturbations. Taken together, our results suggest that cyt b5 critically requires a redox transfer role is essential to enhance lyase reaction, and it reduces CYP17A1 oxy-complex ~10fold faster than CPR. RR on oxyferrous CYP17A1—cyt b5 complex indicated subtle conformational changes in the active site, which were heavily dependent on the identity of the substrate.
CYP19A1 produces estrogens in a three-step reaction, the first two being hydroxylation steps, while the third step comprises a lyase step. The hydroxylation reactions are expected to proceed through the conventional Cpd I intermediate, but the active intermediate for the lyase step is not known. We investigated kinetic solvent isotope effects on the steady state turnover for the three-step reaction, and our results suggest that CYP19A1 lyase chemistry, unlike CYP17A1, proceeds through the Cpd I intermediate.
My thesis is a mechanistic study of the androgenic CYP17A1 and the estrogenic CYP19A1. I investigated the active intermediates in the final lyase step by employing various biochemical and biophysical techniques to Nanodisc reconstituted enzymes. While CYP19A1 lyase step is suggested to proceed through the Cpd I intermediate, in the case of CYP17A1 lyase reaction, a hemiketal intermediate is implicated. Furthermore, the biological regulator of androgen synthesis, cyt b5, acts as a faster redox donor than CPR in the lyase reaction cycle, and also exerts subtle conformational changes dependent on the identity of the substrate in the active site.Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2019-12-01The student, Ruchia Duggal, accepted the attached license on 2017-10-16 at 14:29.The student, Ruchia Duggal, submitted this Dissertation for approval on 2017-10-16 at 14:46.This Dissertation was approved for publication on 2017-10-20 at 11:09.DSpace SAF Submission Ingestion Package generated from Vireo submission #11688 on 2018-03-13 at 10:33:02Made available in DSpace on 2018-03-13T17:29:09Z (GMT). No. of bitstreams: 4
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Previous issue date: 2017-10-20Embargo set by: Seth Robbins for item 105441
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Ethanol administration results in a prolonged decrease in blood ionized calcium levels in the rat
No full textPrevious studies have shown that ethanol decreases the level of ionized calcium (iCa) in the blood, and appears to prevent a compensatory increase in parathyroid hormone level. We have shown, however, that the presence of ethanol interferes with the measurement of blood iCa by the most commonly used iCa analyzer. It is impossible to interpret ethanol-induced alterations in Ca-regulating hormone levels without accurate measurement of blood iCa, thus the purpose of this study was to determine if ethanol decreases blood iCa levels independent of methodological artifacts. The time course of ethanol's effect and the relationship between iCa and blood ethanol concentration (BEC) were also examined. Rats (n = 22) received ethanol (1.5 or 3 g/kg body weight) or saline by intraperitoneal injection. Blood samples were obtained by tail nick at 0, 2, 6, 24, 48 h and 8 days postinjection, and analyzed for iCa, pH, and BEC. Blood iCa and pH were measured using the I-Stat Clinical Analyzer, whose performance is not affected by the presence of ethanol. Ethanol administration resulted in a decrease in blood iCa levels. The magnitude and time course of the decrease varied with dose of ethanol, being greater and more prolonged with the higher dose, and blood iCa levels were not fully recovered at 48 h postinjection. No significant relationship was found between individual iCa and BEC values. This study confirms that ethanol decreases blood iCa levels, independent of methodological artifacts. Prolonged disruptions in Ca homeostasis resulting from ethanol consumption could have implications for long-term bone health.Peer reviewedAlcohol; Ethanol; Calcium; Ionized calcium; Ra
Linearization of Quadratic Drag to Estimate CALM Buoy Pitch Motion in Frequency-Domain and Experimental Validation
No full textLinearization of Quadratic Drag to Estimate CALM Buoy Pitch Motion in Frequency-Domain and Experimental Validation
No full textThe dynamics of an oil offloading catenary anchor leg mooring (CALM) buoy coupled with mooring and flow lines are directly related to the fatigue life of a mooring system, necessitating an accurate estimate of the buoy hydrodynamic response. Linear wave theory is used for modeling the surface boundary value problem, and the boundary element method is used to solve the fluid-structure interaction between the buoy hull and the incident waves in the frequency-domain. The radiation problem is solved to estimate the added mass and radiation damping coefficients, and the diffraction problem is solved to determine the linear wave exciting loading. The buoy pitch motion is investigated, and linearizations of the quadratic drag/damping term are performed in the frequency-domain. The pitch motion response is calculated by considering an equivalent linearized drag/damping. Quadratic, cubic, and stochastic linearizations of the nonlinear drag term are employed to derive the equivalent drag/damping. Comparisons between the linear and nonlinear damping effects are presented. Time-domain simulations of the buoy motions are performed in conjunction with Morison’s equation to validate the floating buoy response. The time- and frequency-domain results are finally compared with the experimental model test results for validations. The linearization methods applied result in good estimates for the peak pitch response. However, only the stochastic linearization method shows a good agreement for the period range of the incident wave where typical pitch response estimate has not been correctly estimated.</jats:p
Functions, structure, and read-through alternative splicing of feline APOBEC3 genes
Get PDFBackground Over the past years a variety of host restriction genes have been identified in human and mammals that modulate retrovirus infectivity, replication, assembly, and/or cross-species transmission. Among these host-encoded restriction factors, the APOBEC3 (A3; apolipoprotein B mRNA-editing catalytic polypeptide 3) proteins are potent inhibitors of retroviruses and retrotransposons. While primates encode seven of these genes (A3A to A3H), rodents carry only a single A3 gene. Results Here we identified and characterized several A3 genes in the genome of domestic cat (Felis catus) by analyzing the genomic A3 locus. The cat genome presents one A3H gene and three very similar A3C genes (a-c), probably generated after two consecutive gene duplications. In addition to these four one-domain A3 proteins, a fifth A3, designated A3CH, is expressed by read-through alternative splicing. Specific feline A3 proteins selectively inactivated only defined genera of feline retroviruses: Bet-deficient feline foamy virus was mainly inactivated by feA3Ca, feA3Cb, and feA3Cc, while feA3H and feA3CH were only weakly active. The infectivity of Vif-deficient feline immunodeficiency virus and feline leukemia virus was reduced only by feA3H and feA3CH, but not by any of the feA3Cs. Within Felidae, A3C sequences show significant adaptive selection, but unexpectedly, the A3H sequences present more sites that are under purifying selection. Conclusion Our data support a complex evolutionary history of expansion, divergence, selection and individual extinction of antiviral A3 genes that parallels the early evolution of Placentalia, becoming more intricate in taxa in which the arms race between host and retroviruses is harsher
Correction: Epidemiology and outcomes of early-onset AKI in COVID-19-related ARDS in comparison with non-COVID-19-related ARDS: insights from two prospective global cohort studies (Critical Care, (2023), 27, 1, (3), 10.1186/s13054-022-04294-5)
No full textFollowing publication of the original article [1], the authors identified that the collaborating authors part of the collaborating author group CCCC Consortium was missing. The collaborating author group is available and included as Additional file 1 in this article
Exploration of jet substructure using iterative declustering in pp and Pb-Pb collisions at LHC energies
No full textThe ALICE collaboration at the CERN LHC reports novel measurements of jet substructure in pp collisions at root s = 7 TeV and central Pb-Pb collisions at root s(NN) = 2.76 TeV. Jet substructure of track-based jets is explored via iterative declustering and grooming techniques. We present the measurement of the momentum sharing of two-prong substructure exposed via grooming, the z(g), and its dependence on the opening angle, in both pp and Pb-Pb collisions. We also present the measurement of the distribution of the number of branches obtained in the iterative declustering of the jet, which is interpreted as the number of its hard splittings. In Pb-Pb collisions, we observe a suppression of symmetric splittings at large opening angles and an enhancement of splittings at small opening angles relative to pp collisions, with no significant modification of the number of splittings. The results are compared to predictions from various Monte Carlo event generators to test the role of important concepts in the evolution of the jet in the medium such as colour coherence. (C) 2020 The Author(s). Published by Elsevier B.V
International Journal of Mathematical Combinatorics, Vol.4
Get PDFThe International J.Mathematical Combinatorics (ISSN 1937-1055) is a fully refereed international journal, sponsored by the MADIS of Chinese Academy of Sciences and published in USA quarterly comprising 460 pages approx. per volume, which publishes original research papers and survey articles in all aspects of Smarandache multi-spaces, Smarandache geometries, mathematical combinatorics, non-euclidean geometry and topology and their applications to other sciences
Measuring (KSK +/-)-K-0 interactions using Pb-Pb collisions at root S-NN=2.76 TeV
No full textWe present the first ever measurements of femtoscopic correlations between the K-S(0) and K-+/- particles. The analysis was performed on the data from Pb-Pb collisions at root S-NN = 2.76 TeV measured by the ALICE experiment. The observed femtoscopic correlations are consistent with final-state interactions proceeding via the a(0)(980) resonance. The extracted kaon source radius and correlation strength parameters for (KSK-)-K-0 are found to be equal within the experimental uncertainties to those for (KSK+)-K-0. Comparing the results of the present study with those from published identical-kaon femtoscopic studies by ALICE, mass and coupling parameters for the a(0) resonance are tested. Our results are also compatible with the interpretation of the a(0) having a tetraquark structure instead of that of a diquark. (c) 2017 The Author. Published by Elsevier B.V
Measurement of the production of high-p(T) electrons from heavy-flavour hadron decays in Pb-Pb collisions at root s(NN)=2.76 TeV
No full textElectrons from heavy-flavour hadron decays (charm and beauty) were measured with the ALICE detector in Pb-Pb collisions at a centre-of-mass of energy root s(NN) = 2.76 TeV. The transverse momentum (pT) differential production yields at mid-rapidity were used to calculate the nuclear modification factor R-AA in the interval 3 < p(T) < 18 GeV/c. The R-AA shows a strong suppression compared to binary scaling of pp collisions at the same energy (up to a factor of 4) in the 10% most central Pb-Pb collisions. There is a centrality trend of suppression, and a weaker suppression (down to a factor of 2) in semi-peripheral (50-80%) collisions is observed. The suppression of electrons in this broad p(T) interval indicates that both charm and beauty quarks lose energy when they traverse the hot medium formed in Pb-Pb collisions at LHC. (C) 2017 The Author. Published by Elsevier B.V
