Kinetic model of phosphofructokinase-1 from Escherichia coli

This paper presents a kinetic model of phosphofructokinase-1 from Escherichia coli. A complete catalytic cycle has been reconstructed based on available information on the oligomeric structure of the enzyme and kinetic mechanism of its monomer. Applying the generalization of the Monod-Wyman-Changeux approach proposed by Popova and Sel'kov(35-37) to the reconstructed catalytic cycle rate equation has been derived. Dependence of the reaction rate on pH, magnesium, and effectors has been taken into account. Kinetic parameters have been estimated via fitting the rate equation against experimentally measured dependencies of initial rate on substrates, products, effectors, and pH available from the literature. The model of phosphofructokinase-1 predicts (1) cooperativity of binding both fructose-6-phosphate and ATPMg(2-), (2) significant inhibition of the enzyme resulting from an increase in total concentration of ATP under the condition of fixed concentration of Mg(2+) ions, and (3) dual effect of ADP consisting of allosteric activation and product inhibition of the enzyme. Moreover, the model developed can be used in the kinetic modeling of biochemical pathways containing phosphofructokinase-1.     

In-stent restenosis after revascularization of myocardium with drug-eluting stents is accompanied by elevated level of blood plasma eosinophil cationic protein

The aim of this study was to assess the involvement of eosinophil cationic protein, a marker of eosinophil activation, in the development of in-stent restenosis after drug-eluting stent implantation. Follow-up angiography at 6 to 12?months was performed in 32 patients who were treated with percutaneous coronary intervention and implantation of sirolimus-eluting stents. Blood plasma levels of eosinophil cationic protein (ECP) and total immunoglobulin E (IgE) were measured by enzyme-linked immunosorbent assay and the level of C-reactive protein (hs-CRP) by high-sensitivity nephelometry. According to angiography data, in-stent restenosis occurred in 13 patients, while 19 patients did not develop it. There were no differences between the hs-CRP and IgE levels in patients with or without restenosis. In contrast, ECP level was higher in patients with restenosis compared with that in patients without restenosis [17.7?ng/mL (11.2-24.0) vs. 9.0?ng/mL (6.4-12.9), p?= 0.017]. The incidence of in-stent restenoses was 63% in patients with ECP level higher than or equal to 11?ng/mL, and 19% in patients with an ECP level lower than 11?ng/mL (p?= 0.019). These findings suggest that elevated eosinophil activation may play an important role in the pathogenesis of in-stent restenosis after implantation of drug-eluting stents.     

Rapid production of functionalized recombinant proteins: marrying ligation independent cloning and in vitro protein ligation

Functional genomics and proteomics have been very active fields since the sequencing of several genomes was completed. To assign a physiological role to the newly discovered coding genes with unknown function, new generic methods for protein production, purification, and targeted functionalization are needed. This work presents a new vector, pCYSLIC, that allows rapid generation of Escherichia coli expression constructs via ligation-independent cloning (LIC). The vector is designed to facilitate protein purification by either Ni-NTA or GSH affinity chromatography. Subsequent proteolytic removal of affinity tags liberates an N-terminal cysteine residue that is then used for covalent modification of the target protein with different biophysical probes via protein ligation. The described system has been tested on 36 mammalian Rab GTPases, and it was demonstrated that recombinant GTPases produced with pCYSLIC could be efficiently modified with fluorescein or biotin in vitro. Finally, LIC was compared with the recently developed In-Fusion cloning method, and it was demonstrated that In-Fusion provides superior flexibility in choice of expression vector. By the application of In-Fusion cloning Cys-Rab6A GTPase with an N-terminal cysteine residue was generated employing unmodified pET30a vector and TVMV protease.     

Electroretinographic study of the magnetic compass in European robins

Migratory birds are known to be sensitive to external magnetic field (MF). Much indirect evidence suggests that the avian magnetic compass is localized in the retina. Previously, we showed that changes in the MF direction could modulate retinal responses in pigeons. In the present study, we performed similar experiments using the traditional model animal to study the magnetic compass, European robins. The photoresponses of isolated retina were recorded using ex vivo electroretinography (ERG). Blue- and red-light stimuli were applied under an MF with the natural intensity and two MF directions, when the angle between the plane of the retina and the field lines was 0° and 90°, respectively. The results were separately analysed for four quadrants of the retina. A comparison of the amplitudes of the a- and b-waves of the ERG responses to blue stimuli under the two MF directions revealed a small but significant difference in a- but not b-waves, and in only one (nasal) quadrant of the retina. The amplitudes of both the a- and b-waves of the ERG responses to red stimuli did not show significant effects of the MF direction. Thus, changes in the external MF modulate the European robin retinal responses to blue flashes, but not to red flashes. This result is in a good agreement with behavioural data showing the successful orientation of birds in an MF under blue, but not under red illumination.     

Optimal Schedules of Light Exposure for Rapidly Correcting Circadian Misalignment

Jet lag arises from a misalignment of circadian biological timing with the timing of human activity, and is caused by rapid transmeridian travel. Jet lag''s symptoms, such as depressed cognitive alertness, also arise from work and social schedules misaligned with the timing of the circadian clock. Using experimentally validated mathematical models, we develop a new methodology to find mathematically optimal schedules of light exposure and avoidance for rapidly re-entraining the human circadian system. In simulations, our schedules are found to significantly outperform other recently proposed schedules. Moreover, our schedules appear to be significantly more robust to both noise in light and to inter-individual variations in endogenous circadian period than other proposed schedules. By comparing the optimal schedules for thousands of different situations, and by using general mathematical arguments, we are also able to translate our findings into general principles of optimal circadian re-entrainment. These principles include: 1) a class of schedules where circadian amplitude is only slightly perturbed, optimal for dim light and for small shifts 2) another class of schedules where shifting occurs along the shortest path in phase-space, optimal for bright light and for large shifts 3) the determination that short light pulses are less effective than sustained light if the goal is to re-entrain quickly, and 4) the determination that length of daytime should be significantly shorter when delaying the clock than when advancing it.