Analysis of the impact of a uracil DNA glycosylase attenuated in AP-DNA binding in maintenance of the genomic integrity in Escherichia coli

Uracil DNA glycosylase (Ung)initiates the uracil excision repair pathway. We have earlier characterized the Y66W and Y66H mutants of Ung and shown that they are compromised by similar to 7- and similar to 170-fold, respectively in their uracil excision activities. In this study, fluorescence anisotropy measurements show that compared with the wild-type, the Y66W protein is moderately compromised and attenuated in binding to AP-DNA. Allelic exchange of ung in Escherichia coli with ung::kan, ungY66H:amp or ungY66W:amp alleles showed similar to 5-, similar to 3.0- and similar to 2.0-fold, respectively increase in mutation frequencies. Analysis of mutations in the rifampicin resistance determining region of rpoB revealed that the Y66W allele resulted in an increase in A to G (or T to C) mutations. However, the increase in A to G mutations was mitigated upon expression of wild-type Ung from a plasmid borne gene. Biochemical and computational analyses showed that the Y66W mutant maintains strict specificity for uracil excision from DNA. Interestingly, a strain deficient in AP-endonucleases also showed an increase in A to G mutations. We discuss these findings in the context of a proposal that the residency of DNA glycosylase(s) onto the AP-sites they generate shields them until recruitment of AP-endonucleases for further repair.

Mass Spectrometry-Based Systems Approach for Identification of Inhibitors of Plasmodium falciparum Fatty Acid Synthase{triangledown}

The emergence of strains of Plasmodium falciparum resistant to the commonly used antimalarials warrants the development of new antimalarial agents. The discovery of type II fatty acid synthase (FAS) in Plasmodium distinct from the FAS in its human host (type I FAS) opened up new avenues for the development of novel antimalarials. The process of fatty acid synthesis takes place by iterative elongation of butyryl-acyl carrier protein (butyryl-ACP) by two carbon units, with the successive action of four enzymes constituting the elongation module of FAS until the desired acyl length is obtained. The study of the fatty acid synthesis machinery of the parasite inside the red blood cell culture has always been a challenging task. Here, we report the in vitro reconstitution of the elongation module of the FAS of malaria parasite involving all four enzymes, FabB/F (β-ketoacyl-ACP synthase), FabG (β-ketoacyl-ACP reductase), FabZ (β-ketoacyl-ACP dehydratase), and FabI (enoyl-ACP reductase), and its analysis by matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS). That this in vitro systems approach completely mimics the in vivo machinery is confirmed by the distribution of acyl products. Using known inhibitors of the enzymes of the elongation module, cerulenin, triclosan, NAS-21/91, and (–)-catechin gallate, we demonstrate that accumulation of intermediates resulting from the inhibition of any of the enzymes can be unambiguously followed by MALDI-TOF MS. Thus, this work not only offers a powerful tool for easier and faster throughput screening of inhibitors but also allows for the study of the biochemical properties of the FAS pathway of the malaria parasite.

Mass Spectrometry-Based Systems Approach for Identification of Inhibitors of Plasmodium falciparum Fatty Acid Synthase{triangledown}

The emergence of strains of Plasmodium falciparum resistant to the commonly used antimalarials warrants the development of new antimalarial agents. The discovery of type II fatty acid synthase (FAS) in Plasmodium distinct from the FAS in its human host (type I FAS) opened up new avenues for the development of novel antimalarials. The process of fatty acid synthesis takes place by iterative elongation of butyryl-acyl carrier protein (butyryl-ACP) by two carbon units, with the successive action of four enzymes constituting the elongation module of FAS until the desired acyl length is obtained. The study of the fatty acid synthesis machinery of the parasite inside the red blood cell culture has always been a challenging task. Here, we report the in vitro reconstitution of the elongation module of the FAS of malaria parasite involving all four enzymes, FabB/F (β-ketoacyl-ACP synthase), FabG (β-ketoacyl-ACP reductase), FabZ (β-ketoacyl-ACP dehydratase), and FabI (enoyl-ACP reductase), and its analysis by matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS). That this in vitro systems approach completely mimics the in vivo machinery is confirmed by the distribution of acyl products. Using known inhibitors of the enzymes of the elongation module, cerulenin, triclosan, NAS-21/91, and (–)-catechin gallate, we demonstrate that accumulation of intermediates resulting from the inhibition of any of the enzymes can be unambiguously followed by MALDI-TOF MS. Thus, this work not only offers a powerful tool for easier and faster throughput screening of inhibitors but also allows for the study of the biochemical properties of the FAS pathway of the malaria parasite.

Studies on a chitooligosaccharide-specific lectin from Coccinia indica. Thermodynamics and kinetics of umbelliferyl glycoside binding.

Coccinia indica agglutinin (CIA) is a chitooligosaccharide-specific lectin with two binding sites/homodimer of M(r) 32,000. Quenching studies implied tryptophan involvement in binding activity, which was confirmed by chemical modification experiments (A. R. Sanadi and A. Surolia, submitted for publication). Binding of 4-methylumbelliferyl chitooligosaccharides has been carried out to study their binding by CIA. Reversal experiments confirm the validity of the data previously obtained (A. R. Sanadi and A. Surolia, submitted for publication) from intrinsic fluorescence studies. Surprisingly, unlike wheat germ agglutinin, there is no consistent thermodynamic effect of the chromophoric label on binding activities as compared with the native sugars. From the changes in the optical properties of the chromophoric group upon binding to CIA, it has been possible to confirm that the tryptophan located in the binding site is closest to the fourth subsite. Thermodynamic analysis shows that the binding of the labeled tetrasaccharide is very strongly entropically driven, with the terminal, nonreducing sugar residue protruding from the binding pocket. The results of stopped-flow kinetic studies on the binding of the chromophoric trisaccharide by CIA show that the mechanism of binding is a one-step process.

N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yI)colcemid, a probe for different classes of colchincine-binding site on tubulin

The nature of binding of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-colcemid (NBD-colcemid), an environment-sensitive fluorescent analogue of colchicine, to tubulin was tested. This article reports the first fluorometric study where two types of binding site of colchincine analogue on tubulin were detected. Binding of NBD-colcemid to one of these sites equilibrates slsowly. NBD-colcemid competes with colchicine for this site. Binding of NBD-colcemid to this site also causes inhibition of tubulin self-assembly. In contrast, NBD-colcemid binding to the other site is characterised by rapid equilibration and lack of competition with colchicine. Nevertheless, binding to this site is highly specific for the cholchicine nucleus, as alkyl-NBD analogues have no significant binding activity. Fast-reaction-kinetic studies gave 1.76 × 105 M–1 s–1 for the association and 0.79 s–1 for the dissociation rate constants for the binding of NBD-colcemid to the fast site of tubulin. The association rate constants for the two phases of the slow site are 0.016 × 10–4 M–1 s–1 and 3.5 × 10–4 M–1 respectively. These two sites may be related to the two sites of colchicine reported earlier, with binding characteristics altered by the increased hydrophobic nature of NBD-colcemid.

Approximate methods for step function response of undamped non-linear spring mass systems with arbitrary hardening type spring characteristic

The possibility of applying two approximate methods for determining the salient features of response of undamped non-linear spring mass systems subjected to a step input, is examined. The results obtained on the basis of these approximate methods are compared with the exact results that are available for some particular types of spring characteristics. The extension of the approximate methods for non-linear systems with general polynomial restoring force characteristics is indicated.

Studies of the polarographic and coulometric behaviour of aromatic nitro-compounds: I. Nitrobenzene in ethanol

A detailed polarographic (a.c. and d.c.) and coulometric investigation of nitrobenzene has been made at various pH values in the presence of different concentrations of ethanol. Below pH 4.7, two waves are apparent but above this pH, the second wave does not appear. Coulometric evidence indicates that the first and second waves correspond to the four-and two-electron processes, respectively. The coulometric method was not applicable in sodium hydroxide and sodium acetate solutions. When the diffusion coefficients (from the diaphragm cell) are used in the Ilkovic equation, no reliable conclusions can be reached for the number of electrons involved in the reduction process in alkaline solutions. The a.c. polarographic method gives evidence for the formation of species such as: C6H5NO2H22+, C6H5NO2− and C6H5NO22−. Analysis of d.c. polarographic data by Delahay's treatment of irreversible waves, indicates that the number of electrons involved in the rate-determining step is 2. In sodium hydroxide solutions, however, the first main wave is split indicating more than one rate-determining step. The results presented in this paper indicate that the first wave in the reduction of nitrobenzene is a four-electron process at all pH values. The second wave, which appears below pH 4.7, corresponds to a two-electron process irrespective of wave heights. The difference in the a.c. polarographic behaviour in acid and alkaline solutions has given evidence for the formation of species like C6H5NO2H2, C6H5NO2−, and C6H5NO22.

Effect of dietary cholesterol and ubiquinone on isoprene synthesis in rat liver

The effect of dietary cholesterol and ubiquinone on the synthesis of isoprene compounds in the liver, as tested by the incorporation of acetate-1-14C and mevalonate-2-14C, was studied in rats. In cholesterol feeding, there appears to be a second site of inhibition after squalene in addition to the previously known primary site of inhibition at the β-hydroxy-β-methyl glutaryl-CoA reductase. Feeding ubiquinone inhibited at some common step between acetate and mevalonate in the synthesis of both cholesterol and ubiquinone, without affecting the acetate activation or fatty acid synthesis, and also at a step in the synthesis of ubiquinone not common with the synthesis of cholesterol. These results are suggestive of a role for ubiquinone in the regulation of isoprene synthesis.

Studies in oxasteroids—I : Synthesis of 3-cyano-3-methyl-7-methoxychroman-4-one and the structure of an “abnormal” product

A synthesis of 3-cyano-3-methyl-7-methoxychroman-4-one is reported. The structure of an “abnormal” product obtained during isomerization (III) with potassium t-butoxide in t-butanol, followed by alkylation with methyl iodide has been proved to be 3-t-butoxy-2-cyano- 2-mehthyl-2′,4′-dimethoxypropiophenone (IVa).

Self-regularized pseudo time-marching schemes for structural system identification with static measurements

We explore the application of pseudo time marching schemes, involving either deterministic integration or stochastic filtering, to solve the inverse problem of parameter identification of large dimensional structural systems from partial and noisy measurements of strictly static response. Solutions of such non-linear inverse problems could provide useful local stiffness variations and do not have to confront modeling uncertainties in damping, an important, yet inadequately understood, aspect in dynamic system identification problems. The usual method of least-square solution is through a regularized Gauss-Newton method (GNM) whose results are known to be sensitively dependent on the regularization parameter and data noise intensity. Finite time,recursive integration of the pseudo-dynamical GNM (PD-GNM) update equation addresses the major numerical difficulty associated with the near-zero singular values of the linearized operator and gives results that are not sensitive to the time step of integration. Therefore, we also propose a pseudo-dynamic stochastic filtering approach for the same problem using a parsimonious representation of states and specifically solve the linearized filtering equations through a pseudo-dynamic ensemble Kalman filter (PD-EnKF). For multiple sets of measurements involving various load cases, we expedite the speed of thePD-EnKF by proposing an inner iteration within every time step. Results using the pseudo-dynamic strategy obtained through PD-EnKF and recursive integration are compared with those from the conventional GNM, which prove that the PD-EnKF is the best performer showing little sensitivity to process noise covariance and yielding reconstructions with less artifacts even when the ensemble size is small.

Self-regularized pseudo time-marching schemes for structural system identification with static measurements

We explore the application of pseudo time marching schemes, involving either deterministic integration or stochastic filtering, to solve the inverse problem of parameter identification of large dimensional structural systems from partial and noisy measurements of strictly static response. Solutions of such non-linear inverse problems could provide useful local stiffness variations and do not have to confront modeling uncertainties in damping, an important, yet inadequately understood, aspect in dynamic system identification problems. The usual method of least-square solution is through a regularized Gauss-Newton method (GNM) whose results are known to be sensitively dependent on the regularization parameter and data noise intensity. Finite time, recursive integration of the pseudo-dynamical GNM (PD-GNM) update equation addresses the major numerical difficulty associated with the near-zero singular values of the linearized operator and gives results that are not sensitive to the time step of integration. Therefore, we also propose a pseudo-dynamic stochastic filtering approach for the same problem using a parsimonious representation of states and specifically solve the linearized filtering equations through apseudo-dynamic ensemble Kalman filter (PD-EnKF). For multiple sets ofmeasurements involving various load cases, we expedite the speed of the PD-EnKF by proposing an inner iteration within every time step. Results using the pseudo-dynamic strategy obtained through PD-EnKF and recursive integration are compared with those from the conventional GNM, which prove that the PD-EnKF is the best performer showing little sensitivity to process noise covariance and yielding reconstructions with less artifacts even when the ensemble size is small. Copyright (C) 2009 John Wiley & Sons, Ltd.