Studies on Aspergillus niger glutamine synthetase: Regulation of enzyme levels by nitrogen sources and identification of active site residues

The specific activity of glutamine synthetase (L-glutamate: ammonia ligase, EC 6.3.1.2) in surface grown Aspergillus niger was increased 3-5 fold when grown on L-glutamate or potassium nitrate, compared to the activity obtained on ammonium chloride. The levels of glutamine synthetase was regulated by the availability of nitrogen source like NH4 + , and further, the enzyme is repressed by increasing concentrations of NH4 +. In contrast to other micro-organisms, the Aspergillus niger enzyme was neither specifically inactivated by NH4+ or L-glutamine nor regulated by covalent modification.Glutamine synthetase from Aspergillus niger was purified to homogenity. The native enzyme is octameric with a molecular weight of 385,000±25,000. The enzyme also catalyses Mn2+ or Mg2+-dependent synthetase and Mn2+-dependent transferase activity.Aspergillus niger glutamine synthetase was completely inactivated by two mol of phenylglyoxal and one mol of N-ethylmaleimide with second order rate constants of 3·8 M–1 min–1 and 760 M–1 min–1 respectively. Ligands like Mg. ATP, Mg. ADP, Mg. AMP, L-glutamate NH4+, Mn2+ protected the enzyme against inactivation. The pattern of inactivation and protection afforded by different ligands against N-ethylamaleimide and phenylglyoxal was remarkably similar. These results suggest that metal ATP complex acts as a substrate and interacts with an arginine ressidue at the active site. Further, the metal ion and the free nucleotide probably interact at other sites on the enzyme affecting the catalytic activity.

A Comparative Study of Electrochemical Kinetic Parameters by the Potential Step Method. Electrode Reactions of CyDTA and EDTA Complexes of Cu(II) at DME

By using the same current-time (I-t) curves, electrochemical kinetic parameters are determined by two methods, (a) using the ratio of current at a given potential to the diffusion-controlled limiting current and (b) curve fitting method, for the reduction of Cu(II)–CyDTA complex. The analysis by the method (a) shows that the rate determining step involves only one electron although the overall reduction of the complex involves two electrons suggesting thereby the stepwise reduction of the complex. The nature of I-t curves suggests the adsorption of intermediate species at the electrode surface. Under these circumstances more reliable kinetic parameters can be obtained by the method (a) compared to that of (b). Similar observations are found in the case of reduction of Cu(II)–EDTA complex.

Kinetics of hydrogen evolution on a stainless steel electrode

The kinetic parameters for the hydrogen evolution reaction on a stainless steel substrate have been obtained from a study of the steady-state polarization curves as well as the galvanostatic transients. The high Tafel slope obtained in the steady-state polarization measurements was ascribed to the presence of an oxide film present on the surface of the stainless steel electrode.

Effect of amplifier imperfections on active networks

The deviation in the performance of active networks due to practical operational amplifiers (OA) is mainly because of the finite gain bandwidth productBand nonzero output resistanceR_0. The effect ofBandR_0on two OA impedances and single and multi-OA filters are discussed. In filters, the effect ofR_0is to add zeros to the transfer function often making it nonminimum phase. A simple method of analysis has been suggested for 3-OA biquad and coupled biquad circuits. A general method of noise minimization of the generalized impedance converter (GIC), while operating OA's within the prescribed voltage and current limits, is also discussed. The 3-OA biquadratic sections analyzed also exhibit noise behavior and signal handling capacity similar to the GIC. The GIC based structures are found to be better than other configurations both in biquadratic sections and direct realizations of higher order transfer functions.

Structure-Based Design of DevR Inhibitor Active against Nonreplicating Mycobacterium tuberculosis

Antitubercular treatment is directed against actively replicating organisms. There is an urgent need to develop drugs targeting persistent subpopulations of Mycobacterium tuberculosis. The DevR response regulator is believed to play a key role in bacterial dormancy adaptation during hypoxia. We developed a homology-based model of DevR and used it for the rational design of inhibitors. A phenylcoumarin derivative (compound 10) identified by in silico pharmacophore-based screening of 2.5 million compounds employing protocols with some novel features including a water-based pharmacophore query, was characterized further. Compound 10 inhibited DevR binding to target DNA, down-regulated dormancy genes transcription, and drastically reduced survival of hypoxic but not nutrient-starved dormant bacteria or actively growing organ ` isms. Our findings suggest that compound 10 ``locks'' DevR in an inactive conformation that is unable to bind cognate DNA and induce the dormancy regulon. These results provide proof-of-concept for DevR as a novel target to develop molecules with sterilizing activity against tubercle bacilli.

Buckling, stiffening, and negative dissipation in the dynamics of a biopolymer in an active medium

We present a generic theory for the dynamics of a stiff filament under tension, in an active medium with orientational correlations, such as a microtubule in contractile actin. In sharp contrast to the case of a passive medium, we find the filament can stiffen, and possibly oscillate or buckle, depending on both the contractile or tensile nature of the activity and the filament-medium anchoring interaction. We also demonstrate a strong violation of the fluctuation-dissipation (FD) relation in the effective dynamics of the filament, including a negative FD ratio. Our approach is also of relevance to the dynamics of axons, and our model equations bear a remarkable formal similarity to those in recent work [Martin P, Hudspeth AJ, Juelicher F (2001) Proc Natl Acad Sci USA 98: 14380-14385] on auditory hair cells. Detailed tests of our predictions can be made by using a single filament in actomyosin extracts or bacterial suspensions.

Semi-active vibration isolation using a near-zero-spring-rate device:steady state analysis of a single-degree-of-freedom model

A vibration isolator is described which incorporates a near-zero-spring-rate device within its operating range. The device is an assembly of a vertical spring in parallel with two inclined springs. A low spring rate is achieved by combining the equivalent stiffness in the vertical direction of the inclined springs with the stiffness of the vertical central spring. It is shown that there is a relation between the geometry and the stiffness of the individual springs that results in a low spring rate. Computer simulation studies of a single-degree-of-freedom model for harmonic base input show that the performance of the proposed scheme is superior to that of the passive schemes with linear springs and skyhook damping configuration. The response curves show that, for small to large amplitudes of base disturbance, the system goes into resonance at low frequencies of excitation. Thus, it is possible to achieve very good isolation over a wide low-frequency band. Also, the damper force requirements for the proposed scheme are much lower than for the damper force of a skyhook configuration or a conventional linear spring with a semi-active damper.

Mutational analysis of active-site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp(122) and Asp(193) are crucial to the double-stranded DNA cleavage activity whereas Asp(218) is not

Mycobacterium leprae recA harbors an in-frame insertion sequence that encodes an intein homing endonuclease (PI-MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their ctive center. A common feature of LAGLIDADG-type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI-MleI is distinctive from other members of the family of LAGLIDADG-type HEases for its modular structure with functionally separable domains for DNA-binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI-MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active-site residues essential for DNA target site recognition and double-stranded DNA cleavage, we performed site-directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild-type PI-MleI and its variants disclosed that the two amino acid residues, Asp(122) (in Block C) and Asp(193) (in functional Block E), are crucial to the double-stranded DNA endonuclease activity, whereas Asp(218) (in pseudo-Block E) is not. However, despite the reduced catalytic activity, the PI-MleI variants, like the wild-type PI-MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA-binding affinities, but abolished the double-stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double-stranded DNA cleavage activity, compared with the wild-type PI-MleI. These results provide compelling evidence that Asp(122) and Asp(193) in DOD motif I and II, respectively, are bona fide active-site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.

Isolation and visualization of active presynaptic filaments of recA protein and single-stranded DNA

In the presence of ATP, recA protein forms a presynaptic complex with single-stranded DNA that is an obligatory intermediate in homologous pairing. Presynaptic complexes of recA protein and circular single strands that are active in forming joint molecules can be isolated by gel filtration. These isolated active complexes are nucleoprotein filaments with the following characteristics: (i) a contour length that is at least 1.5 times that of the corresponding duplex DNA molecule, (ii) an ordered structure visualized by negative staining as a striated filament with a repeat distance of 9.0 nm and a width of 9.3 nm, (iii) approximately 8 molecules of recA protein and 20 nucleotide residues per striation. The widened spacing between bases in the nucleoprotein filament means that the initial matching of complementary sequences must involve intertwining of the filament and duplex DNA, unwinding of the latter, or some combination of both to equalize the spacing between nascent base pairs. These experiments support the concept that recA protein first forms a filament with single-stranded DNA, which in turn binds to duplex DNA to mediate both homologous pairing and subsequent strand exchange.

Active nucleoprotein filaments of single-stranded binding protein and recA protein on single-stranded DNA have a regular repeating structure

When E. coli single-stranded DNA binding protein (SSB) coats single-stranded DNA (ssDNA) in the presence of 1 mM MgCl2 it inhibits the subsequent binding of recA protein, whereas SSB binding to ssDNA in 12 mM MgCl2 promotes the binding of recA protein. These two conditions correspond respectively to those which produce 'smooth' and 'beaded' forms of ssDNA-SSB filaments. By gel filtration and immunoprecipitation we observed active nucleoprotein filaments of recA protein and SSB on ssDNA that contained on average 1 monomer of recA protein per 4 nucleotides and 1 monomer of SSB per 20-22 nucleotides. Filaments in such a mixture, when digested with micrococcal nuclease produced a regular repeating pattern, approximately every 70-80 nucleotides, that differed from the pattern observed when only recA protein was bound to the ssDNA. We conclude that the beaded ssDNA-SSB nucleoprotein filament readily binds recA protein and forms an intermediate that is active in the formation of joint molecules and can retain substantially all of the SSB that was originally bound.

A theoretical model for the magnetic helicity of solar active regions

Active regions on the solar surface are known to possess magnetic helicity, which is predominantly negative in the northern hemisphere and positive in the southern hemisphere. Choudhuri et al. [Choudhuri, A.R. On the connection between mean field dynamo theory and flux tubes. Solar Phys. 215, 31–55, 2003] proposed that the magnetic helicity arises due to the wrapping up of the poloidal field of the convection zone around rising flux tubes which form active regions. Choudhuri [Choudhuri, A.R., Chatterjee, P., Nandy, D. Helicity of solar active regions from a dynamo model. ApJ 615, L57–L60, 2004] used this idea to calculate magnetic helicity from their solar dynamo model. Apart from getting broad agreements with observational data, they also predict that the hemispheric helicity rule may be violated at the beginning of a solar cycle. Chatterjee et al. [Chatterjee, P., Choudhuri, A.R., Petrovay, K. Development of twist in an emerging magnetic flux tube by poloidal field accretion. A&A 449, 781–789, 2006] study the penetration of the wrapped poloidal field into the rising flux tube due to turbulent diffusion using a simple 1-d model. They find that the extent of penetration of the wrapped field will depend on how weak the magnetic field inside the rising flux tube becomes before its emergence. They conclude that more detailed observational data will throw light on the physical conditions of flux tubes just before their emergence to the photosphere.

Comparison of performance parameters of poly(3,4 ethylenedioxythiophene) (PEDOT) based electrochromic device on glass with and without counter electrode

Conjugated polymers are promising materials for electrochromic device technology. Aqueous dispersions of poly(3,4-ethylenedioxythiophene)-(PEDOT) were spin coated onto transparent conducting oxide (TCO) coated glass substrates. A seven-layer electrochromic device was fabricated with the following configuration: glass/transparent conducting oxide (TCO)/PEDOT (main electrochromic layer)/gel electrolyte/prussian blue (counter electrode)/TCO/glass. The device fabricated with counter electrode (Prussian blue) showed a contrast of 18% and without counter electrode showed visible contrast of 5% at 632 nm at a voltage of 1.9 V. The comparison of the device is done in terms of the colouration efficiency of the devices with and without counter electrode.

A dynamic renormalization group study of active nematics

We carry out a systematic construction of the coarse-grained dynamical equation of motion for the orientational order parameter for a two-dimensional active nematic, that is a nonequilibrium steady state with uniaxial, apolar orientational order. Using the dynamical renormalization group, we show that the leading nonlinearities in this equation are marginally irrelevant. We discover a special limit of parameters in which the equation of motion for the angle field bears a close relation to the 2d stochastic Burgers equation. We find nevertheless that, unlike for the Burgers problem, the nonlinearity is marginally irrelevant even in this special limit, as a result of a hidden fluctuation-dissipation relation. 2d active nematics therefore have quasi-long-range order, just like their equilibrium counterparts.

Experimental Demonstration of H{infty} Control based Active Vibration Suppression in Composite Fin-tip of Aircraft using Optimally Placed Piezoelectric Patch Actuators

The goal of this study is the multi-mode structural vibration control in the composite fin-tip of an aircraft. Structural model of the composite fin-tip with surface bonded piezoelectric actuators is developed using the finite element method. The finite element model is updated experimentally to reflect the natural frequencies and mode shapes accurately. A model order reduction technique is employed for reducing the finite element structural matrices before developing the controller. Particle swarm based evolutionary optimization technique is used for optimal placement of piezoelectric patch actuators and accelerometer sensors to suppress vibration. H{infty} based active vibration controllers are designed directly in the discrete domain and implemented using dSpace® (DS-1005) electronic signal processing boards. Significant vibration suppression in the multiple bending modes of interest is experimentally demonstrated for sinusoidal and band limited white noise forcing functions.

High-level expression of biologically active glycoprotein hormones in Pichia pastoris strains—selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated 15N-labeled phCG

The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active 15N-labeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerol-methanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using NH4Cl/glucose–glycerol–methanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man8–15GlcNAc2). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type N-glycans (Manup-to-30GlcNAc2). The latter finding made the X-33 strain not very suitable for generating 15N-labeled material. Therefore, 15N-phCG was expressed in the GS115 strain using the new optimized protocol. The 15N-enrichment was evaluated by 15N-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that 15N-phCG/GS115 had the same folding as urinary hCG. Furthermore, 15N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor assays, and in vitro bioassays.