Biophysical and structural investigation of bacterially expressed and engineered CCR5, a G protein-coupled receptor

The chemokine receptor CCR5 belongs to the class of G protein-coupled receptors. Besides its role in leukocyte trafficking, it is also the major HIV-1 coreceptor and hence a target for HIV-1 entry inhibitors. Here, we report Escherichia coli expression and a broad range of biophysical studies on E. coli-produced CCR5. After systematic screening and optimization, we obtained 10 mg of purified, detergent-solubilized, folded CCR5 from 1L culture in a triply isotope-labeled (²H/¹⁵N/¹³C) minimal medium. Thus the material is suitable for NMR spectroscopic studies. The expected α-helical secondary structure content is confirmed by circular dichroism spectroscopy. The solubilized CCR5 is monodisperse and homogeneous as judged by transmission electron microscopy. Interactions of CCR5 with its ligands, RANTES and MIP-1β were assessed by surface plasmon resonance yielding K_D values in the nanomolar range. Using size exclusion chromatography, stable monomeric CCR5 could be isolated. We show that cysteine residues affect both the yield and oligomer distribution of CCR5. HSQC spectra suggest that the transmembrane domains of CCR5 are in equilibrium between several conformations. In addition we present a model of CCR5 based on the crystal structure of CXCR4 as a starting point for protein engineering.     

Matching Pursuit with Asymmetric Functions for Signal Decomposition and Parameterization

The method of adaptive approximations by Matching Pursuit makes it possible to decompose signals into basic components (called atoms). The approach relies on fitting, in an iterative way, functions from a large predefined set (called dictionary) to an analyzed signal. Usually, symmetric functions coming from the Gabor family (sine modulated Gaussian) are used. However Gabor functions may not be optimal in describing waveforms present in physiological and medical signals. Many biomedical signals contain asymmetric components, usually with a steep rise and slower decay. For the decomposition of this kind of signal we introduce a dictionary of functions of various degrees of asymmetry – from symmetric Gabor atoms to highly asymmetric waveforms. The application of this enriched dictionary to Otoacoustic Emissions and Steady-State Visually Evoked Potentials demonstrated the advantages of the proposed method. The approach provides more sparse representation, allows for correct determination of the latencies of the components and removes the "energy leakage" effect generated by symmetric waveforms that do not sufficiently match the structures of the analyzed signal. Additionally, we introduced a time-frequency-amplitude distribution that is more adequate for representation of asymmetric atoms than the conventional time-frequency-energy distribution.     

Resonant modes and musical ratios in otoacoustic emissions

The ratios between frequency components of evoked otoacoustic emissions (OAE) were investigated for 100 ears. The signals were decomposed by means of an adaptive approximation method into basic waveforms coming from a very large and redundant dictionary of Gabor functions. The high time-frequency resolution of the method and the parametric representation of the waveforms allowed for an estimation of the frequency ratios of the basic components. A repetitive occurrence of the “fifths”, “fourths” and octaves connected with the Pythagorean temperament was found. The octaves containing “fifths” were identified. This kind of sequences in OAE tend to appear in the same form for tonal stimulations of different frequencies and for broadband stimuli. The significance of the results was confirmed by comparison to Monte Carlo simulations of the null hypothesis of random distribution of frequency modes. These findings support the resonance theory of hearing, which binds musical ratios with the geometrical spacing of outer hair cells in the cochlea.     

Use Reference Bands to Accurately Estimate ISCN Band Levels 400, 550, and 850

In their 2002 Guidelines for chromosome analysis of peripheral blood, the American College of Medical Genetics states that "The 550-band stage should be the goal of all constitutional studies..." The College of American Pathologists requires that the average case be analyzed at the 400-band level of resolution for routine work, and that the 550-band level be achieved in appropriate blood samples. The challenge is how to identify the 400, 550, and 850-band levels confidently and consistently. In this study, our objectives were to develop simple and reliable criteria to estimate band level, and to evaluate our laboratory's performance with respect to those criteria. Using the ISCN(1995) ideogram as a reference, candidate bands were selected for the three band levels: 400, 550 and 850. A pilot and two follow-up studies were conducted and a set of candidate bands were validated against the Vancouver method of evaluating band level so that band level scores were similar using either method. The final set of reference bands were the presence of 9q32 and 20q13.2 for the 400-band level; 5q33.2 and 10q22.2 for the 550-band level; and 3p26.1, 18q22.3 and 20q13.32 for the 850-band level. Cell selection improved after each technologist was provided a composite image of chromosomes with reference bands highlighted. The band level criteria presented here involve no band counting, appear to be objective, can help to improve quality and consistency among technologists, and can ensure compliance with regulatory agencies.     

P2 nucleotide receptors on C2C12 satellite cells

In developing muscle cells environmental stimuli transmitted by purines binding to the specific receptors are crucial proliferation regulators. C2C12 myoblasts express numerous purinergic receptors representing both main classes: P2X and P2Y. Among P2Y receptors we have found the expression of P2Y(1), P2Y(2), P2Y(4), P2Y(6) and P2Y(12) family members while among P2X receptors P2X(4), P2X(5) and P2X(7) were discovered. We have been able to show that activation of those receptors is responsible for ERK class kinase activity, responsible for regulation of cell proliferation pathway. We have also demonstrated that this activity is calcium dependent suggesting Ca(2+) ions as secondary messenger between receptor and kinase regulatory system. More specifically, we do suspect that in C2C12 myoblasts calcium channels of P2X receptors, particularly P2X(5) play the main role in proliferation regulation. In further development of myoblasts into myotubes, when proliferation is gradually inhibited, the pattern of P2 receptors is changed. This phenomenon is followed by diminishing of the P2Y(2)-dependent Ca(2+) signaling, while the mRNA expression of P2Y(2) receptor reminds still on the high level. Moreover, P2X(2) receptor mRNA, absent in myoblasts appears in myotubes. These data show that differentiation of C2C12 cell line satellite myoblasts is accompanied by changes in P2 receptors expression pattern.     

Effect of 6-week endurance training on hemodynamic and neurohormonal responses to lower body negative pressure (LBNP) in healthy young men.

Endurance training is considered as a factor impairing orthostatic tolerance although an improvement and lack of effect have been also reported. The mechanisms of the changes and their relation to initial tolerance of orthostasis are not clear. In the present study, effect of moderate running training on hemodynamic and neurohormonal changes during LBNP, a laboratory test simulating orthostasis, was investigated in subjects with high (HT) and low (LT) tolerance of LBNP. Twenty four male, healthy subjects were submitted to graded LBNP (-15, -30 and -50 mmHg) before and after training. During each test heart rate (HR), stroke volume (SV) and blood pressure, plasma catecholamines, ACTH, adrenomedullin, atrial natriuretic peptide, and renin activity were determined. Basing on initial test, 13 subjects who withstood LBNP at -50 mmHg for 10 min were allocated into HT group and 11 subjects who earlier showed presyncopal symptoms to LT group. Training improved LBNP tolerance in six LT subjects. This was associated with attenuated rate of HR increase and SV decline (before training, at -30 mmHg deltaHR was 21 +/- 4 beats/min and deltaSV - -36+/- 8 ml while after training the respective values were 8 +/- 4 beats/min and -11+/- 6 ml). No differences in hemodynamic response were found in HT subjects and those from LT group whose LBNP tolerance was unchanged. In neither group training affected neurohormonal changes except inhibition of plasma ACTH rise in subjects with improvement of LBNP tolerance. It is concluded that some subjects with low orthostatic tolerance may benefit from moderate training due to improvement of cardiac function regulation.     

The molecular and biochemical characteristics of proline iminopeptidase from rye seedlings (Secale cereale L.)

A proline iminopeptidase (EC. 3.4.11.5) was isolated from shoots of 3 day old seedlings. The purification procedure consisted of 5 steps: acid precipitation, gel filtration on Sephadex G-200, ion-exchange chromatography on Sepharose CL 6B, twice repeated hydrophoic chromatography on Phenyl-Sepharose HP. The enzyme was purified 404.8-fold, with the specific activity of 8.5 units mg⁻¹ of protein with recovery yield of 3%. The purified enzyme had a molecular mass of 225 kDa estimated by gel filtration and 55.4 kDa by SDS PAGE. This indicates that native enzyme is composed of four subunits. The enzyme was specific for proline β-naphtylamide among various amino acid β-naphtylamides. An optimal activity was observed at 37 °C at pH 7.75. The enzyme was thermostable up to 37 °C for 30 min. The enzyme was strongly inhibited by pHMB, E-64, heavy metal ions and partially by PMSF, DFP. The results suggest that cysteine and serine residues may participate in the enzyme activity.