Metal complexes as artificial proteases in proteomics: a palladium(II) complex cleaves various proteins in solutions containing detergents

Most popular agents for site-specific protein cleavage are proteolytic enzymes. Because they become denatured and inactivated by detergents, enzymes are inconvenient for proteomic analysis of hydrophobic proteins which require detergents as solubilizing agents. We used cis-[Pd(en)(H₂O)₂]²⁺ (in which en represents ethylenediamine) as an artificial protease to effect cleavage of three bovine proteins, namely ubiquitin, β-casein, and serum albumin, in separate experiments. Cleavage took place in aqueous solutions containing 1.0% wt./vol. of either 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or Zwittergent 3-14 at 2.5 < pH < 2.9 and 55-60 °C for 3-72 h. Digests were separated by HPLC and analyzed by tandem mass spectrometry. Peptides were identified by de novo sequencing and matched against the bovine genome. Because cleavage by Pd(II) complexes is rather selective and therefore infrequent, 72% of the identified peptides in the digests contained more than 10 amino acid. Palladium(II) complexes hold promise as cleavage agents in proteomics studies of membrane proteins.     

Mutational analysis and modeling reveal functionally critical residues in transmembrane segments 1 and 3 of the UapA transporter

Earlier, we identified mutations in the first transmembrane segment (TMS1) of UapA, a uric acid-xanthine transporter in Aspergillus nidulans, that affect its turnover and subcellular localization. Here, we use one of these mutations (H86D) and a novel mutation (I74D) as well as genetic suppressors of them, to show that TMS1 is a key domain for proper folding, trafficking and turnover. Kinetic analysis of mutants further revealed that partial misfolding and deficient trafficking of UapA does not affect its affinity for xanthine transport, but reduces that of uric acid and confers a degree of promiscuity towards the binding of other purines. This result strengthens the idea that subtle interactions among domains not directly involved in substrate binding refine the selectivity of UapA. Characterization of second-site suppressors of H86D revealed a genetic interaction of TMS1 with TMS3, the latter segment shown for the first time to be important for UapA function. Systematic mutational analysis of polar and conserved residues in TMS3 showed that Ser154 is crucial for UapA transport activity. Our results are in agreement with a topological model of UapA built on the recently published structure of UraA, a bacterial homolog of UapA.     

Impact of abscisic acid in overcoming the problem of albinism in horse chestnut androgenic embryos

Horse chestnut (Aesculus hyppocastanum L., Hyppocastanacea) is a relict species with a slow and complex reproductive cycle considered to have horticultural and medical importance. The cycle maybe circumvented via in vitro androgenesis. Androgenesis of horse chestnut was induced in microspores and anther culture on MS media. Some of the horse chestnut androgenic embryos were albinos. Addition of abscisic acid in media (in concentrations of 0.01, 0.1, 0.5, 1, 2, 5, 10, and 20 mg l^?1) with horse chestnut androgenic embryos has circumvented the reproduction cycle barriers. The best results were achieved on medium with the lowest abscisic acid concentration (0.01 mg l^?1) in microspore culture. The microspore culture proved to be a better model system for embryo production and albino embryo reduction than anther culture. Flow cytometry analysis after maturation treatments induced by ABA showed that 88 % of green embryos originating from microspore culture were haploid. However, 50 % of green embryos from anther culture were haploid. The remaining analyzed androgenic embryos, from both types of cultures were diploid.     

Soccer vs. running training effects in young adult men: which programme is more effective in improvement of body composition? Randomized controlled trial

The aims of this study were: 1) To determine the effects of a 12-week recreational soccer training programme and continuous endurance running on body composition of young adult men and 2) to determine which of these two programmes was more effective concerning body composition. Sixty-four participants completed the randomized controlled trial and were randomly assigned to one of three groups: a soccer training group (SOC; n=20), a running group (RUN; n=21) or a control group performing no physical training (CON; n=23). Training programmes for SOC and RUN lasted 12-week with 3 training sessions per week. Soccer sessions consisted of 60 min ordinary five-a-side, six-a-side or seven-a-side matches on a 30-45 m wide and 45-60 m long plastic grass pitch. Running sessions consisted of 60 min of continuous moderate intensity running at the same average heart rate as in SOC (~80% HR_max). All participants, regardless of group assignment, were tested for each of the following dependent variables: body weight, body height, body mass index, percent body fat, body fat mass, fat-free mass and total body water. In the SOC and RUN groups there was a significant decrease (p < 0.05) in body composition parameters from pre- to post-training values for all measures with the exception of fat-free mass and total body water. Body mass index, percent body fat and body fat mass did not differ between groups at baseline, but by week 12 were significantly lower (p < 0.05) in the SOC and RUN groups compared to CON. To conclude, recreational soccer training provides at least the same changes in body composition parameters as continuous running in young adult men when the training intensity is well matched.     

Near‐infrared human finger measurements based on self‐calibration point: Simulation and in vivo experiments

Near‐infrared light allows measuring tissue oxygenation. These measurements relay on oxygenation‐dependent absorption spectral changes. However, the tissue scattering, which is also spectral dependent, introduces an intrinsic error. Most methods focus on the volume reflectance from a semi‐infinite sample. We have proposed examining the full scattering profile (FSP), which is the angular intensity distribution. A point was found, that is, the iso‐path length (IPL) point, which is not dependent on the tissue scattering, and can serve for self‐calibration. This point is geometric dependent, hence in cylindrical tissues depends solely on the diameter. In this work, we examine an elliptic tissue cross section via Monte Carlo simulation. We have found that the IPL point of an elliptic tissue cross section is indifferent to the input illumination orientation. Furthermore, the IPL point is the same as in a circular cross section with a radius equal to the effective ellipse radius. This is despite the fact that the FSPs of the circular and elliptical cross sections are different. Hence, changing the orientation of the input illumination reveals the IPL point. In order to demonstrate this experimentally, the FSPs of a few female fingers were measured at 2 perpendicular orientations. The crossing point between these FSPs was found equivalent to the IPL point of a cylindrical phantom with a radius similar to the effective radius. The findings of this work will allow accurate pulse oximetry assessment of blood saturation.      

Behavioural and physiological plasticity of gypsy moth larvae to host plant switching

Larvae of the gypsy moth, Lymantria dispar L. (Lepidoptera: Lymantriidae), a generalist species, frequently encounter spatial and temporal variations in diet quality. Such variation favoured the evolution of high behavioural and physiological plasticity which, depending on forest stand composition, enables more or less successful exploitation of the environment. Even in mixed oak stands, a suitable habitat, interspecific and intraspecific host quality variation may provoke significant variation in gypsy moth performance and, consequently, defoliation severity. To elucidate the insufficiently explored relationship between gypsy moth and oaks (Fagaceae), we carried out reciprocal switches between Turkey oaks (Quercus cerris L.) and less nutritious Hungarian oaks (Quercus frainetto Ten.) (TH and HT groups), under controlled laboratory conditions, and compared larval performance between the switched larvae and larvae continuously fed on either Turkey oak (TT) or Hungarian oak (HH). We found that larval traits were most strongly affected by among‐tree variation in oak quality and identity of the host consumed during the fourth instar. Switching from Turkey to Hungarian oak (TH) led to a longer period of feeding, decrease of mass gain, growth, and consumption rate, lower efficiency of food use and nutrient conversion, and increase of protease and amylase activities. Larvae exposed to the reverse switch (HT) attained values of these traits characteristic for TT larvae. It appeared that the lower growth in the TH group than in the TT group was caused by both behavioural (consumption, pre‐ingestive) and metabolic (post‐digestive) effects from consuming oaks. Multivariate analyses of growth, consumption, and efficiency of food use revealed that early diet experience influenced the sensitivity of the most examined traits to less suitable Hungarian oaks, suggesting the development of behavioural and physiological adjustments. Our results indicate that lower risks of defoliation by gypsy moth might be expected in mixed stands with a higher proportion of Hungarian oak.     

Predictions of Experimentally Observed Stochastic Ground Vibrations Induced by Blasting

In the present paper, we investigate the blast induced ground motion recorded at the limestone quarry “Suva Vrela” near Kosjerić, which is located in the western part of Serbia. We examine the recorded signals by means of surrogate data methods and a determinism test, in order to determine whether the recorded ground velocity is stochastic or deterministic in nature. Longitudinal, transversal and the vertical ground motion component are analyzed at three monitoring points that are located at different distances from the blasting source. The analysis reveals that the recordings belong to a class of stationary linear stochastic processes with Gaussian inputs, which could be distorted by a monotonic, instantaneous, time-independent nonlinear function. Low determinism factors obtained with the determinism test further confirm the stochastic nature of the recordings. Guided by the outcome of time series analysis, we propose an improved prediction model for the peak particle velocity based on a neural network. We show that, while conventional predictors fail to provide acceptable prediction accuracy, the neural network model with four main blast parameters as input, namely total charge, maximum charge per delay, distance from the blasting source to the measuring point, and hole depth, delivers significantly more accurate predictions that may be applicable on site. We also perform a sensitivity analysis, which reveals that the distance from the blasting source has the strongest influence on the final value of the peak particle velocity. This is in full agreement with previous observations and theory, thus additionally validating our methodology and main conclusions.     

Oxidoreduction reactions involving the electrostatic and the covalent complex of cytochrome c and plastocyanin: importance of the protein rearrangement for the intracomplex electron-transfer reaction

Horse heart cytochrome c and French bean plastocyanin are cross-linked one-to-one by a carbodiimide [Geren, L. M., Stonehuerner, J., Davis, D. J., & Millett, F. (1983) Biochim. Biophys. Acta 724, 62] in the same general orientation in which they associate electrostatically [King, G. C., Binstead, R. A., & Wright, P. E. (1985) Biochim. Biophys. Acta 806, 262]. The reduction potentials of the Fe and Cu atoms in the covalent diprotein complex are respectively 245 and 385 mV vs NHE; the EPR spectra of the two metals are not perturbed by cross-linking. Four isomers of the covalent diprotein complex, which probably differ slightly from one another in the manner of cross-linking, are separated efficiently by cation-exchange chromatography. Stopped-flow spectrophotometric experiments with the covalent diprotein complex show that the presence of plastocyanin somewhat inhibits oxidation of ferrocytochrome c by [Fe(CN)6]3- and somewhat promotes oxidation of this protein by [Fe(C5H5)2]+. These changes in reactivity are explained in terms of electrostatic and steric effects. Pulse-radiolysis experiments with the electrostatic diprotein complex yield association constants of greater than or equal to 5 X 10(6) and 1 X 10(5) M-1 at ionic strengths of 1 and 40 mM, respectively, and the rate constant of 1.05 X 10(3) s-1, regardless of the ionic strength, for the intracomplex electron-transfer reaction. Analogous pulse-radiolysis experiments with each of the four isomers of the covalent diprotein complex, at ionic strengths of both 2 and 200 mM, show an absence of the intracomplex electron-transfer reaction. A rearrangement of the proteins for this reaction seems to be possible (or unnecessary) in the electrostatic complex but impossible in the covalent complex.     

The cytokine midkine and its receptor RPTPζ regulate B cell survival in a pathway induced by CD74

Lasting B cell persistence depends on survival signals that are transduced by cell surface receptors. In this study, we describe a novel biological mechanism essential for survival and homeostasis of normal peripheral mature B cells and chronic lymphocytic leukemia cells, regulated by the heparin-binding cytokine, midkine (MK), and its proteoglycan receptor, the receptor-type tyrosine phosphatase ζ (RPTPζ). We demonstrate that MK initiates a signaling cascade leading to B cell survival by binding to RPTPζ. In mice lacking PTPRZ, the proportion and number of the mature B cell population are reduced. Our results emphasize a unique and critical function for MK signaling in the previously described MIF/CD74-induced survival pathway. Stimulation of CD74 with MIF leads to c-Met activation, resulting in elevation of MK expression in both normal mouse splenic B and chronic lymphocytic leukemia cells. Our results indicate that MK and RPTPζ are important regulators of the B cell repertoire. These findings could pave the way toward understanding the mechanisms shaping B cell survival and suggest novel therapeutic strategies based on the blockade of the MK/RPTPζ-dependent survival pathway.