Differential binding of ppGpp and pppGpp to E-coli RNA polymerase: photo-labeling and mass spectral studies

(p) ppGpp, a secondary messenger, is induced under stress and shows pleiotropic response. It binds to RNA polymerase and regulates transcription in Escherichia coli. More than 25 years have passed since the first discovery was made on the direct interaction of ppGpp with E. coli RNA polymerase. Several lines of evidence suggest different modes of ppGpp binding to the enzyme. Earlier cross-linking experiments suggested that the beta-subunit of RNA polymerase is the preferred site for ppGpp, whereas recent crystallographic studies pinpoint the interface of beta'/omega-subunits as the site of action. With an aim to validate the binding domain and to follow whether tetra-and pentaphosphate guanosines have different location on RNA polymerase, this work was initiated. RNA polymerase was photo-labeled with 8-azido-ppGpp/8-azido-pppGpp, and the product was digested with trypsin and subjected to mass spectrometry analysis. We observed three new peptides in the trypsin digest of the RNA polymerase labeled with 8-azido-ppGpp, of which two peptides correspond to the same pocket on beta'-subunit as predicted by X-ray structural analysis, whereas the third peptide was mapped on the beta-subunit. In the case of 8-azido-pppGpp-labeled RNA polymerase, we have found only one cross-linked peptide from the beta'-subunit. However, we were unable to identify any binding site of pppGpp on the beta-subunit. Interestingly, we observed that pppGpp at high concentration competes out ppGpp bound to RNA polymerase more efficiently, whereas ppGpp cannot titrate out pppGpp. The competition between tetraphosphate guanosine and pentaphosphate guanosine for E. coli RNA polymerase was followed by gel-based assay as well as by a new method known as DRaCALA assay.

Transient potassium channels augment degeneracy in hippocampal active dendritic spectral tuning

Hippocampal pyramidal neurons express an intraneuronal map of spectral tuning mediated by hyperpolarization-activated cyclic-nucleotide-gated nonspecific-cation channels. Modeling studies have predicted a critical regulatory role for A-type potassium (KA) channels towards augmenting functional robustness of this map. To test this, we performed patch-clamp recordings from soma and dendrites of rat hippocampal pyramidal neurons, and measured spectral tuning before and after blocking KA channels using two structurally distinct pharmacological agents. Consistent with computational predictions, we found that blocking KA channels resulted in a significant reduction in resonance frequency and significant increases in input resistance, impedance amplitude and action-potential firing frequency across the somato-apical trunk. Furthermore, across all measured locations, blocking KA channels enhanced temporal summation of postsynaptic potentials and critically altered the impedance phase profile, resulting in a significant reduction in total inductive phase. Finally, pair-wise correlations between intraneuronal percentage changes (after blocking KA channels) in different measurements were mostly weak, suggesting differential regulation of different physiological properties by KA channels. Our results unveil a pivotal role for fast transient channels in regulating theta-frequency spectral tuning and intrinsic phase response, and suggest that degeneracy with reference to several coexisting functional maps is mediated by cross-channel interactions across the active dendritic arbor.

Modified Simplified Rate Equation Model of Flowing Chemical Oxygen-Iodine Laser and its Application

A modified simplified rate equation (RE) model of flowing chemical oxygen-iodine laser (COIL), which is adapted to both the condition of homogeneous broadening and inhomogeneous broadening being of importance and the condition of inhomogeneous broadening being predominant, is presented for performance analyses of a COIL. By using the Voigt profile function and the gain-equal-loss approximation, a gain expression has been deduced from the rate equations of upper and lower level laser species. This gain expression is adapted to the conditions of very low gas pressure up to quite high pressure and can deal with the condition of lasing frequency being not equal to the central one of spectral profile. The expressions of output power and extraction efficiency in a flowing COIL can be obtained by solving the coupling equations of the deduced gain expression and the energy equation which expresses the complete transformation of the energy stored in singlet delta state oxygen into laser energy. By using these expressions, the RotoCOIL experiment is simulated, and obtained results agree well with experiment data. Effects of various adjustable parameters on the performances of COIL are also presented.

Monte Carlo filtering and smoothing with application to time-varying spectral estimation

We develop methods for performing filtering and smoothing in non-linear non-Gaussian dynamical models. The methods rely on a particle cloud representation of the filtering distribution which evolves through time using importance sampling and resampling ideas. In particular, novel techniques are presented for generation of random realisations from the joint smoothing distribution and for MAP estimation of the state sequence. Realisations of the smoothing distribution are generated in a forward-backward procedure, while the MAP estimation procedure can be performed in a single forward pass of the Viterbi algorithm applied to a discretised version of the state space. An application to spectral estimation for time-varying autoregressions is described.

Towards a perceptually optimal spectral amplitude estimator for audio signal enhancement

We present a statistical model-based approach to signal enhancement in the case of additive broadband noise. Because broadband noise is localised in neither time nor frequency, its removal is one of the most pervasive and difficult signal enhancement tasks. In order to improve perceived signal quality, we take advantage of human perception and define a best estimate of the original signal in terms of a cost function incorporating perceptual optimality criteria. We derive the resultant signal estimator and implement it in a short-time spectral attenuation framework. Audio examples, references, and further information may be found at http://www-sigproc.eng.cam.ac.uk/~pjw47.

Effects of Translational Nonequilibrium on Performance of Flowing Chemical Oxygen-Iodine Laser

The effect of the translational nonequilibrium on performance modeling of flowing chemical oxygen-iodine lasers (COIL) is emphasized in this paper. The spectral line broadening (SLB) model is a basic factor for predicting the performances of flowing COIL. The Voigt profile function is a well-known SLB model and is usually utilized. In the case of gas pressure in laser cavity less than 5 torr, a low pressure limit expression of the Voigt profile function is used. These two SLB models imply that ail lasing particles can interact with monochromatic laser radiation. Basically, the inhomogeneous broadening effects are not considered in these two SLB models and they cannot predict the spectral content. The latter requires consideration of finite translational relaxation rate. Unfortunately, it is rather difficult to solve simultaneously the Navier-Stokes (NS) equations and the conservation equations of the number of lasing particles per unit volume and per unit frequency interval. In the operating condition of flowing COIL, it is possible to obtain a perturbational solution of the conservational equations for lasing particles and deduce a new relation between the gain and the optical intensity, i.e., a new gain-saturation relation. By coupling the gain-saturation relation with other governing equations (such as the NS equations, chemical reaction equations and the optical model of gain-equal-loss), We have numerically calculated the performances of flowing COIL. The present results are compared with those obtained by the common rate-equation (RE) model, in which the Voigt profile function and its low pressure limit expression are used. The difference of different model's results is great. For instance, in the case of lasing frequency coinciding with the central frequency of line profile and very low gas pressure, the gain-saturation relation of the present model is quite different with that of the RE model.

Semi-gas kinetics model for performance modeling of flowing chemical oxygen-iodine lasers (COIL)

A semi-gas kinetics (SGK) model for performance analyses of flowing chemical oxygen-iodine laser (COIL) is presented. In this model, the oxygen-iodine reaction gas flow is treated as a continuous medium, and the effect of thermal motions of particles of different laser energy levels on the performances of the COIL is included and the velocity distribution function equations are solved by using the double-parameter perturbational method. For a premixed flow, effects of different chemical reaction systems, different gain saturation models and temperature, pressure, yield of excited oxygen, iodine concentration and frequency-shift on the performances of the COIL are computed, and the calculated output power agrees well with the experimental data. The results indicate that the power extraction of the SGK model considering 21 reactions is close to those when only the reversible pumping reaction is considered, while different gain saturation models and adjustable parameters greatly affect the output power, the optimal threshold gain range, and the length of power extraction.