Copper, Zinc-Superoxide Dismutase from Clinically Isolated Escherichia coli: Cloning, Analysis of sodC and Its Possible Role in Pathogenicity

Superoxide dismutase has been discovered within the periplasm of several Gram-negative pathogens. We studied the Cu,Zn-SOD enzyme in Escherichia coli isolated from clinical samples (stool samples) collected from patients suffering from diarrhea. Antibiogram studies of the isolates were carried out to determine the sensitive and resistant strains. The metal co-factor present in the enzyme was confirmed by running samples in native gels and inhibiting with 2 mM potassium cyanide. A 519 bp sodC gene was amplified from resistant and sensitive strains of Escherichia coli. Cloning and sequencing of the sodC gene indicated variation in the protein and amino acid sequences of sensitive and resistant isolates. The presence of sodC in highly resistant Escherichia coli isolates from diarrheal patients indicates that sodC may play role in enhancing the pathogenicity by protecting cells from exogenous sources of superoxide, such as the oxidative burst of phagocytes. The presence of SodC could be one of the factors for bacterial virulence.

Cloning, expression, purification and preliminary X-ray diffraction studies of a putative Mycobacterium smegmatis thiolase

Thiolases are important in fatty-acid degradation and biosynthetic pathways. Analysis of the genomic sequence of Mycobacterium smegmatis suggests the presence of several putative thiolase genes. One of these genes appears to code for an SCP-x protein. Human SCP-x consists of an N-terminal domain (referred to as SCP2 thiolase) and a C-terminal domain (referred as sterol carrier protein 2). Here, the cloning, expression, purification and crystallization of this putative SCP-x protein from M. smegmatis are reported. The crystals diffracted X-rays to 2.5 angstrom resolution and belonged to the triclinic space group P1. Calculation of rotation functions using X-ray diffraction data suggests that the protein is likely to possess a hexameric oligomerization with 32 symmetry which has not been observed in the other six known classes of this enzyme.

Generation of Minimum Leakage Input Vectors with Constrained NBTI Degradation

Technology scaling has caused Negative Bias Temperature Instability (NBTI) to emerge as a major circuit reliability concern. Simultaneously leakage power is becoming a greater fraction of the total power dissipated by logic circuits. As both NBTI and leakage power are highly dependent on vectors applied at the circuit’s inputs, they can be minimized by applying carefully chosen input vectors during periods when the circuit is in standby or idle mode. Unfortunately input vectors that minimize leakage power are not the ones that minimize NBTI degradation, so there is a need for a methodology to generate input vectors that minimize both of these variables.This paper proposes such a systematic methodology for the generation of input vectors which minimize leakage power under the constraint that NBTI degradation does not exceed a specified limit. These input vectors can be applied at the primary inputs of a circuit when it is in standby/idle mode and are such that the gates dissipate only a small amount of leakage power and also allow a large majority of the transistors on critical paths to be in the “recovery” phase of NBTI degradation. The advantage of this methodology is that allowing circuit designers to constrain NBTI degradation to below a specified limit enables tighter guardbanding, increasing performance. Our methodology guarantees that the generated input vector dissipates the least leakage power among all the input vectors that satisfy the degradation constraint. We formulate the problem as a zero-one integer linear program and show that this formulation produces input vectors whose leakage power is within 1% of a minimum leakage vector selected by a search algorithm and simultaneously reduces NBTI by about 5.75% of maximum circuit delay as compared to the worst case NBTI degradation. Our paper also proposes two new algorithms for the identification of circuit paths that are affected the most by NBTI degradation. The number of such paths identified by our algorithms are an order of magnitude fewer than previously proposed heuristics.

Fast Direct Torque Control of an Open-End Induction Motor Drive Using 12-Sided Polygonal Voltage Space Vectors

A torque control scheme, based on a direct torque control (DTC) algorithm using a 12-sided polygonal voltage space vector, is proposed for a variable speed control of an open-end induction motor drive. The conventional DTC scheme uses a stator flux vector for the sector identification and then the switching vector to control stator flux and torque. However, the proposed DTC scheme selects switching vectors based on the sector information of the estimated fundamental stator voltage vector and its relative position with respect to the stator flux vector. The fundamental stator voltage estimation is based on the steady-state model of IM and the synchronous frequency of operation is derived from the computed stator flux using a low-pass filter technique. The proposed DTC scheme utilizes the exact positions of the fundamental stator voltage vector and stator flux vector to select the optimal switching vector for fast control of torque with small variation of stator flux within the hysteresis band. The present DTC scheme allows full load torque control with fast transient response to very low speeds of operation, with reduced switching frequency variation. Extensive experimental results are presented to show the fast torque control for speed of operation from zero to rated.

On the structure of analytic vectors for the Schrodinger representation

This article deals with the structure of analytic and entire vectors for the Schrodinger representations of the Heisenberg group. Using refined versions of Hardy's theorem and their connection with Hermite expansions we obtain very precise representation theorems for analytic and entire vectors.

Badly approximable numbers and vectors in Cantor-like sets

We show that a large class of Cantor-like sets of R-d, d >= 1, contains uncountably many badly approximable numbers, respectively badly approximable vectors, when d >= 2. An analogous result is also proved for subsets of R-d arising in the study of geodesic flows corresponding to (d+1)-dimensional manifolds of constant negative curvature and finite volume, generalizing the set of badly approximable numbers in R. Furthermore, we describe a condition on sets, which is fulfilled by a large class, ensuring a large intersection with these Cantor-like sets.

Cloning, expression, purification, and biochemical characterisation of the FIC motif containing protein of Mycobacterium tuberculosis

The role of FIC (Filamentation induced by cAMP)(2) domain containing proteins in the regulation of many vital pathways, mostly through the transfer of NMPs from NTPs to specific target proteins (NMPylation), in microorganisms, higher eukaryotes, and plants is emerging. The identity and function of FIC domain containing protein of the human pathogen, Mycobacterium tuberculosis, remains unknown. In this regard, M. tuberculosis fic gene (Mtfic) was cloned, overexpressed, and purified to homogeneity for its biochemical characterisation. It has the characteristic FIC motif, HPFREGNGRSTR (HPFxxGNGRxxR), spanning 144th to 155th residue. Neither the His-tagged nor the GST-tagged MtFic protein, overexpressed in Escherichia coil, nor expression of Mtfic in Mycobacterium smegmatis, yielded the protein in the soluble fraction. However, the maltose binding protein (MBP) tagged MtFic (MBP-MtFic) could be obtained partly in the soluble fraction. The cloned, overexpressed, and purified recombinant MBP-MtFic showed conversion of ATP, GTP, CTP, and UTP into AMP. GMP, CMP, and UMP, respectively. Sequence alignment with several FIC motif containing proteins, complemented with homology modeling on the FIC motif containing protein, VbhT of Bartonella schoenbuchensis as the template, showed conservation and interaction of residues constituting the FIC domain. Site-specific mutagenesis of the His144, or Glu148, or Asn150 of the FIC motif, or of Arg87 residue that constitutes the FIC domain, or complete deletion of the FIC motif, abolished the NTP to NMP conversion activity. The design of NMP formation assay using the recombinant, soluble MtFic would enable identification of its target substrate for NMPylation. (C) 2012 Elsevier Inc. All rights reserved.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of thymidylate kinase (TTHA1607) from Thermus thermophilus HB8

Nucleotide biosynthesis plays a key role in cell survival and cell proliferation. Thymidylate kinase is an enzyme that catalyses the conversion of dTMP to dTDP using ATP-Mg2+ as a phosphoryl-donor group. This enzyme is present at the junction of the de novo and salvage pathways; thus, any inhibitor designed against it will result in cell death. This highlights the importance of this enzyme as a drug target. Thymidylate kinase from the extremely thermophilic organism Thermus thermophilus HB8 has been expressed, purified and crystallized using the microbatch method. The crystals diffracted to a resolution of 1.83 angstrom and belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 39.50, b = 80.29, c = 122.55 angstrom. Preliminary studies revealed the presence of a dimer in the asymmetric unit with a Matthews coefficient (V-M) of 2.18 angstrom(3) Da(-1).

A Hybrid Multilevel Inverter System Based on Dodecagonal Space Vectors for Medium Voltage IM Drives

Dodecagonal (12-sided) space vector pulsewidth modulation (PWM) schemes are characterized by the complete absence of (6n +/- 1)th-order harmonics (for odd n) in the phase voltages, within the linear modulation range and beyond, including over-modulation. This paper presents a new topology suitable for the realization of such multilevel inverter schemes for induction motor (IM) drives, by cascading two-level inverters with flying-capacitor-inverter fed floating H-bridge cells. Now, any standard IM may be used to get the dodecagonal operation which hitherto was possible only with open-end winding IM. To minimize the current total harmonic distortion (THD), a strategy for synchronous PWM is also proposed. It is shown that the proposed method is capable of obtaining better THD figures, compared to conventional dodecagonal schemes. The topology and the PWM strategy are validated through analysis and subsequently verified experimentally.

Medium Voltage Drive for Induction Motors Using Multilevel Octadecagonal Voltage Space Vectors

Multilevel inverters with hexagonal and dodecagonal voltage space vector structures have improved harmonic profile compared to two-level inverters. Further improvement in the quality of the waveform is possible using multilevel octadecagonal (18-sided polygon) voltage space vectors. This paper proposes an inverter circuit topology capable of generating multilevel octadecagonal voltage space vectors, by cascading two asymmetric three-level inverters. By the proper selection of dc-link voltages and the resultant switching states for the inverters, voltage space vectors, whose tips lie on three concentric octadecagons, are obtained. The advantages of octadecagonal voltage space vector-based pulsewidth modulation (PWM) techniques are the complete elimination of fifth, seventh, eleventh, and thirteenth harmonics in phase voltages and the extension of linear modulation range. In this paper, a simple PWM timing calculation method is also proposed. Experimental results have been presented in this paper to validate the proposed concept.

A Multilevel Inverter Scheme With Dodecagonal Voltage Space Vectors Based on Flying Capacitor Topology for Induction Motor Drives

This paper presents a multilevel inverter topology suitable for the generation of dodecagonal space vectors instead of hexagonal space vectors as in the case of conventional schemes. This feature eliminates all the 6n +/- 1 (n = odd) harmonics from the phase voltages and currents in the entire modulation range with an increase in the linear modulation range. The topology is realized by flying capacitor-based three-level inverters feeding from two ends of an open-end winding induction motor with asymmetric dc links. The flying capacitor voltages are tightly controlled throughout the modulation range using redundant switching states for any load power factor. A simple and fast carrier-based space-vector pulsewidth modulation (PWM) scheme is also proposed for the topology which utilizes only the sampled amplitudes of the reference wave for the PWM timing computation.

Molecular cloning, overexpression, and characterization of autophosphorylation in calcium-dependent protein kinase 1 (CDPK1) from Cicer arietinum

In plants, calcium-dependent protein kinases (CDPKs) are key intermediates in calcium-mediated signaling that couple changes in Ca2+ levels to a specific response. In the present study, we report the high-level soluble expression of calcium-dependent protein kinase1 from Cicer arietinum (CaCDPK1) in Escherichia coli. The expression of soluble CaCDPK1 was temperature dependent with a yield of 3-4 mg/l of bacterial culture. CaCDPK1 expressed as histidine-tag fusion protein was purified using Ni-NTA affinity chromatography till homogeneity. The recombinant CaCDPK1 protein exhibited both calcium-dependent autophosphorylation and substrate phosphorylation activities with a V (max) and K (m) value of 13.2 nmol/min/mg and 34.3 mu M, respectively, for histone III-S as substrate. Maximum autophosphorylation was seen only in the presence of calcium. Optimum temperature for autophosphorylation was found to be 37 A degrees C. The recombinant protein showed optimum pH range of 6-9. The role of autophosphorylation in substrate phosphorylation was investigated using histone III-S as exogenous substrate. Our results show that autophosphorylation happens before substrate phosphorylation and it happens via intra-molecular mechanism as the activity linearly depends on enzyme concentrations. Autophosphorylation enhances the kinase activity and reduces the lag phase of activation, and CaCDPK1 can utilize both ATP and GTP as phosphodonor but ATP is preferred than GTP.

A nearly constant switching frequency current hysteresis controller with generalized parabolic boundaries using 12-sided polygonal voltage space vectors for IM drives

In this paper, a current hysteresis controller with parabolic boundaries for a 12-sided polygonal voltage space vector inverter fed induction motor (IM) drive is proposed. Parabolic boundaries with generalized vector selection logic, valid for all sectors and rotational direction, is used in the proposed controller. The current error space phasor boundary is obtained by first studying the drive scheme with space vector based PWM (SVPWM) controller. Four parabolas are used to approximate this current error space phasor boundary. The system is then run with space phasor based hysteresis PWM controller by limiting the current error space vector (CESV) within the parabolic boundary. The proposed controller has simple controller implementation, nearly constant switching frequency, extended modulation range and fast dynamic response with smooth transition to the over modulation region.

A Medium-Voltage Inverter-Fed IM Drive Using Multilevel 12-Sided Polygonal Vectors, With Nearly Constant Switching Frequency Current Hysteresis Controller

In this paper, a current error space vector (CESV)-based hysteresis current controller for a multilevel 12-sided voltage space vector-based inverter-fed induction motor (IM) drive is proposed. The proposed controller gives a nearly constant switching frequency operation throughout different speeds in the linear modulation region. It achieves the elimination of 6n +/- 1, n = odd harmonics from the phase voltages and currents in the entire modulation range, with an increase in the linear modulation range. It also exhibits fast dynamic behavior under different transient conditions and has a simple controller implementation. Nearly constant switching frequency is obtained by matching the steady-state CESV boundaries of the proposed controller with that of a constant switching frequency SVPWM-based drive. In the proposed controller, the CESV reference boundaries are computed online, using the switching dwell time and voltage error vector of each applied vector. These quantities are calculated from estimated sampled reference phase voltages. Vector change is decided by projecting the actual current error along the computed hysteresis space vector boundary of the presently applied vector. The estimated reference phase voltages are found from the stator current error ripple and the parameters of the IM.

Cloning, expression, purification, crystallization and preliminary X-ray studies of argininosuccinate lyase (Rv1659) from Mycobacterium tuberculosis

The last enzyme in the arginine-biosynthesis pathway, argininosuccinate lyase, from Mycobacterium tuberculosis has been cloned, expressed, purified and crystallized, and preliminary X-ray studies have been carried out on the crystals. The His-tagged tetrameric enzyme with a subunit molecular weight of 50.9 kDa crystallized with two tetramers in the asymmetric unit of the orthorhombic unit cell, space group P2(1)2(1)2(1). Molecular-replacement calculations and self-rotation calculations confirmed the space group and the tetrameric nature of the molecule.