Spectroscopic characterization of Venus at the single molecule level

Venus is a recently developed, fast maturating, yellow fluorescent protein that has been used as a probe for in vivo applications. In the present work the photophysical characteristics of Venus were analyzed spectroscopically at the bulk and single molecule level. Through time-resolved single molecule measurements we found that single molecules of Venus display pronounced fluctuations in fluorescence emission, with clear fluorescence on- and off-times. These fluorescence intermittencies were found to occupy a broad range of time scales, ranging from milliseconds to several seconds. Such long off-times can complicate the analysis of single molecule counting experiments or single-molecule FRET experiments.     

QM/MM computational studies of substrate water binding to the oxygen-evolving centre of photosystem II

This paper reports computational studies of substrate water binding to the oxygen-evolving centre (OEC) of photosystem II (PSII), completely ligated by amino acid residues, water, hydroxide and chloride. The calculations are based on quantum mechanics/molecular mechanics hybrid models of the OEC of PSII, recently developed in conjunction with the X-ray crystal structure of PSII from the cyanobacterium Thermosynechococcus elongatus. The model OEC involves a cuboidal Mn3CaO4Mn metal cluster with three closely associated manganese ions linked to a single mu4-oxo-ligated Mn ion, often called the 'dangling manganese'. Two water molecules bound to calcium and the dangling manganese are postulated to be substrate molecules, responsible for dioxygen formation. It is found that the energy barriers for the Mn(4)-bound water agree nicely with those of model complexes. However, the barriers for Ca-bound waters are substantially larger. Water binding is not simply correlated to the formal oxidation states of the metal centres but rather to their corresponding electrostatic potential atomic charges as modulated by charge-transfer interactions. The calculations of structural rearrangements during water exchange provide support for the experimental finding that the exchange rates with bulk 18 O-labelled water should be smaller for water molecules coordinated to calcium than for water molecules attached to the dangling manganese. The models also predict that the S1-->S2 transition should produce opposite effects on the two water-exchange rates.     

QM/MM investigation of structure and spectroscopic properties of a vanadium-containing peroxidase

We present a comprehensive analysis of the most likely ground state configuration of the resting state of vanadium dependent chloroperoxidase (VCPO) based on quantum mechanics/molecular mechanics (QM/MM) evaluations of ground state properties, UV-vis spectra and NMR chemical shifts. Within the QM/MM framework, density functional theory (DFT) calculations are used to characterize the resting state of VCPO via time-dependent density functional theory (TD-DFT) calculations of electronic excitation energies and NMR chemical shifts. Comparison with available experimental data allows us to determine the most likely protonation state of VCPO, a state which results in a doubly protonated axial oxygen, a site largely stabilized by hydrogen bonds. We found that the bulk of the protein that is beyond the immediate layer surrounding the cofactor, has an important electrostatic effect on the absorption maximum. Through examination of frontier orbitals, we analyze the nature of two bound water molecules and the extent to which relevant residues in the active site influence the spectroscopy calculations.     

Microencapsulation of an anti-VE-cadherin antibody secreting 1B5 hybridoma cells.

Accumulating experimental evidence demonstrates that tumor growth and lethality are dependent on angiogenesis. Based on this concept, there is growing interest in the use of antiangiogenesis agents to inhibit tumor expansion. Compelling data implicate vascular endothelium (VE)-cadherin (an endothelium specific protein) as a key factor in the last step of angiogenesis, where the endothelial cells join one to each other and form microtubules (future blood vessels). We propose a novel approach to the inhibition of angiogenesis by immobilizing VE-cadherin-secreting hybridoma cells in alginate-agarose microcapsules. Hybridoma cells can be protected with biocompatible and semipermeable membranes that permit exit of anti-VE-cadherin monoclonal antibodies but not entry of cellular immune mediators. Stability studies were performed to select the suitable microcapsule for cell immobilization. Alginate and agarose solid beads coated with poly-L-lysine and alginate were chosen according to their stability and diffusional properties. 1B5 hybridoma cells were grown within the microcapsules and secreted anti-VE-cadherin antibodies during the 9 days of culture, reaching a cumulative concentration of 1.7 microg/mL. This antibody concentration inhibited microtubule formation (87%) in the in vitro angiogenesis Matrigel assay. Moreover, the antiangiogenic effect observed was antibody concentration related. These findings open a new alternative for the inhibition or prevention of angiogenesis and demonstrates the feasibility of using microencapsulated cells as a control-drug delivery system.     

An eDNA/eRNA-based approach to investigate the life cycle of non-cultivable shellfish micro-parasites: the case of Bonamia ostreae,a parasite of the European flat oyster Ostrea edulis

Environmental DNA approaches are increasingly used to detect microorganisms in environmental compartments, including water. They show considerable advantages to study non-cultivable microorganisms like Bonamia ostreae, a protozoan parasite inducing significant mortality in populations of flat oyster Ostrea edulis. Although B. ostreae development within the host has been well described, questions remain about its behaviour in the environment. As B. ostreae transmission is direct, seawater appears as an interesting target to develop early detection tools and improve our understanding of disease transmission mechanisms. In this context, we have developed an eDNA/eRNA approach allowing detecting and quantifying B. ostreae 18S rDNA/rRNA as well as monitoring its presence in seawater by real-time PCR. B. ostreae DNA could be detected up to 4 days while RNA could be detected up to 30 days, suggesting a higher sensitivity of the eRNA-based tool. Additionally, more than 90% of shed parasites were no longer detected after 2 days outside the oysters. By allowing B. ostreae detection in seawater, this approach would not only be useful to monitor the presence of the parasite in oyster production areas but also to evaluate the effect of changing environmental factors on parasite survival and transmission.     

Doubled lifespan and patient‐like pathologies in progeria mice fed high‐fat diet

Hutchinson‐Gilford Progeria Syndrome (HGPS) is a devastating premature aging disease. Mouse models have been instrumental for understanding HGPS mechanisms and for testing therapies, which to date have had only marginal benefits in mice and patients. Barriers to developing effective therapies include the unknown etiology of progeria mice early death, seemingly unrelated to the reported atherosclerosis contributing to HGPS patient mortality, and mice not recapitulating the severity of human disease. Here, we show that progeria mice die from starvation and cachexia. Switching progeria mice approaching death from regular diet to high‐fat diet (HFD) rescues early lethality and ameliorates morbidity. Critically, feeding the mice only HFD delays aging and nearly doubles lifespan, which is the greatest lifespan extension recorded in progeria mice. The extended lifespan allows for progeria mice to develop degenerative aging pathologies of a severity that emulates the human disease. We propose that starvation and cachexia greatly influence progeria phenotypes and that nutritional/nutraceutical strategies might help modulate disease progression. Importantly, progeria mice on HFD provide a more clinically relevant animal model to study mechanisms of HGPS pathology and to test therapies.     

Age-related changes in glutathione synthesis in the eye lens.

Eye lenses from young rats or mice synthesize GSH from methionine or N-acetylcysteine. However, lenses from old animals do not synthesize GSH from methionine. This is due to the absence of cystathionase activity in old lenses. GSH monoethyl ester, but not free GSH, increases GSH content and protects the lens against experimental oxidative stress. The importance of these results in the prevention of cataractogenesis is discussed.     

Differential functional behavior of viral phi29, Nf and GA-1 SSB proteins

DNA replication of 29 and related phages takes place via a strand displacement mechanism, a process that generates large amounts of single-stranded DNA (ssDNA). Consequently, phage-encoded ssDNA-binding proteins (SSBs) are essential proteins during phage 29-like DNA replication. In the present work we analyze the helix-destabilizing activity of the SSBs of 29 and the related phages Nf and GA-1, their ability to eliminate non-productive binding of 29 DNA polymerase to ssDNA and their stimulatory effect on replication by 29 DNA polymerase in primed M13 ssDNA replication, a situation that resembles type II replicative intermediates that occur during 29-like DNA replication. Significant differences have been appreciated in the functional behavior of the three SSBs. First, the GA-1 SSB is able to display helix-destabilizing activity and to stimulate dNTP incorporation by 29 DNA polymerase in the M13 DNA replication assay, even at SSB concentrations at which the 29 and Nf SSBs do not show any effect. On the other hand, the 29 SSB is the only one of the three SSBs able to increase the replication rate of 29 DNA polymerase in primed M13 ssDNA replication. From the fact that the 29 SSB, but not the Nf SSB, stimulates the replication rate of Nf DNA polymerase we conclude that the different behaviors of the SSBs on stimulation of the replication rate of 29 and Nf DNA polymerases is most likely due to formation of different nucleoprotein complexes of the SSBs with the ssDNA rather than to a specific interaction between the SSB and the corresponding DNA polymerase. A model that correlates the thermodynamic parameters that define SSB–ssDNA nucleoprotein complex formation with the functional stimulatory effect of the SSB on 29-like DNA replication has been proposed.