Locomotor activity of the Holarctic molluscs Radix auricularia (from Lake Baikal) in various light pollution conditions

Light pollution is a modern environmental problem. The scale of light pollution is increasing yearly and is negatively affecting the functioning of terrestrial and aquatic ecosystems. Of the aquatic ecosystems, marine ecosystems are the most studied, while there is very little information on the effect of artificial lighting on freshwater ecosystems. Among freshwater aquatic organisms, there are relatively little data on the effect of artificial light on crustaceans and fish, while we could find no meaningful data on the effect of artificial light on molluscs are practically absent. Here we test whether different types of artificial lighting, differing in their spectra, affect the activity of the Holarctic mollusc Radix auricularia. For this, we used two light sources (with warm and cold light) and a 1-m long aquarium. We found that both light sources affect individuals of this species, but the effects of this exposure are different. Artificial lighting (depending on the spectral characteristics) can increase the activity of molluscs of this species or reduce it. In the long term, the impact on the ecosystem will depend on the type of water body where light pollution is present, where individuals of this species live, and the type of light sources

Effect of Artificial Light on Physiological and Hematological Parameters of Individuals of Phoxinus phoxinus (Linnaeus, 1758)

In the natural environment, the life activity of organisms takes place under conditions of stable daily, lunar and annual light cycles. However, human activities aimed at creating comfortable conditions for people have resulted in light becoming one of the factors of anthropogenic environmental pollution. Artificial lighting at night can cause physiological and behavioural changes and disturbances in aquatic organisms, affecting their vital functions. Fish are one of the groups of aquatic organisms that are most susceptible to the influence of light at night, largely due to the anatomical structure of their eyes. The aim of this work was to test whether keeping Phoxinus phoxinus (Linnaeus, 1758) under constant light exposure promotes the growth of inflammatory processes in them, and also to study whether lighting at night affects oxygen consumption. Experiments showed that the oxygen consumption of P. phoxinus increased statistically significantly (p = 0.04303) at night with light compared to night without light. In an experiment in which blood cell counts were performed, the results showed a statistically significant increase in leukocytes (p = 0.01506) in the third experimental group of four kept under constant artificial light for 17 days. Based on the results of our study, it has been confirmed that keeping fish under abnormal light conditions, i.e. using different sources of artificial light at night near water bodies, can lead to physiological changes that can have a negative impact on the life of organisms. In fish, the level of oxygen consumption increases, indicating an increase in the level of metabolism, which in turn affects the growth and formation of organisms, causing a decrease in the intensity of various physiological processes such as feeding and reproduction. There is also an increase in the level of leukocytes, which indicates an increase in inflammatory processes in the organisms, which can lead to a decrease in immune function and, as a result, the susceptibility of fish to various diseases

Rate Limiting Factors For Protein Folding.

Abstract. This thesis describes factors that are rate limiting for the folding of two small proteins, U1A and CI2 which fold without accumulating intermediates. The [GdnHCl] dependencies of the unfolding- and refolding kinetics of U1A display downward curvatures. However, as the curvatures are precisely matched and no indications of formation of partially structured intermediates are seen, the folding behaviour is still consistent with a two-state model. Instead, the curvatures seem related to Hammond behaviour, i.e. movements of the transition state on the reaction coordinate. This implies that the free energy barrier is broad and rather flat. Consequently the search for productive interactions in the folding protein does not take place at ground state level but at high energy. Analysis of mutants of CI2 shows, that to a larger extent than previously thought, these mutants also display transition state movements. Possibly, the activation barrier for folding is generally broad but with a more or less rugged surface. When the ruggedness involves localised features that are much higher than the surrounding barrier, the protein will appear to have a localised transition state. Mutations that lower these localised features will then set free movements of the transition state. One way to monitor diffusive events in protein folding is to measure the rate of folding as a function of solvent viscosity. Unfortunately the viscogens (osmolytes) added in these studies also tend to stabilise the protein and thus have dual effects on the folding rate. When CI2 is refolded in the presence of several different osmolytes we find no viscosity dependence on the refolding kinetics. However, we find that the osmolytes, in addition to their other effects also induce a collapse of the denatured protein. The collapse increases the reconfiguration time of the protein and thereby retards folding. At [protein]>1mM, U1A aggregates early in kinetic refolding experiments. This leads to a retardation of folding at low [denaturant], which resembles the accumulation of an intermediate. The retardation results from kinetic competition: at high [protein] the fraction of protein that aggregates increases and eventually dominates the refolding amplitude. As the rate of folding after aggregation is slower than direct folding, this results in an apparent decrease of the folding rate. Similar experiments with CI2 show that also this protein undergoes transient aggregation. Furthermore, as both U1A and CI2 fold without accumulating intermediates the protein aggregates likely form directly from the coil

High-energy channeling in protein folding

Recent controversy about the role of populated intermediates in protein folding emphasizes the need to better characterize other events on the folding pathway. A complication is that these involve high-energy states which are difficult to target experimentally since they do not accumulate kinetically. Here, we explore the energetics of high-energy states and map out the shape of the free-energy profile for folding of the two-state protein U1A. The analysis is based on nonlinearities in the GdnHCl dependence of the activation energy for unfolding, which we interpret in terms of structural changes of the protein-folding transition state. The result suggests that U1A folds by high-energy channeling where most of the conformational search takes place isoenergetically at transition-state level. This is manifested in a very broad and flat activation barrier, the top of which covers more than 60% of the reaction coordinate. The interpretation favors a folding mechanism where the pathway leading to the native protein is determined by the sequence's ability to stabilize productive transitio

Transient aggregates in protein folding are easily mistaken for folding intermediates

It has been questioned recently whether populated intermediates are important for the protein folding process or are artefacts trapped in nonproductive pathways. We report here that the rapidly formed intermediate of the spliceosomal protein U1A is an off-pathway artefact caused by transient aggregation of denatured protein under native conditions. Transient aggregates are easily mistaken for structured monomers and could be a general problem in time-resolved folding studies

Transmembrane topology of the Acr3 family arsenite transporter from Bacillus subtilis.

The transmembrane topology of the Acr3 family arsenite transporter Acr3 from Bacillus subtilis was analysed experimentally using translational fusions with alkaline phosphatase and green fluorescent protein and in silico by topology modelling. Initial topology prediction resulted in two models with 9 and 10 TM helices respectively. 32 fusion constructs were made between truncated forms of acr3 and the reporter genes at 17 different sites throughout the acr3 sequence to discriminate between these models. Nine strong reporter protein signals provided information about the majority of the locations of the cytoplasmic and extracellular loops of Acr3 and showed that both the N- and the C-termini are located in the cytoplasm. Two ambiguous data points indicated the possibility of an alternative 8 helix topology. This possibility was investigated using another 10 fusion variants, but no experimental support for the 8 TM topology was obtained. We therefore conclude that Acr3 has 10 transmembrane helices. Overall, the loops which connect the membrane spanning segments are short, with cytoplasmic loops being somewhat longer than the extracellular loops. The study provides the first ever experimentally derived structural information on a protein of the Acr3 family which constitutes one of the largest classes of arsenite transporters

Miniature biofuel cell as a potential power source for glucose-​sensing contact lenses

A microscale membrane-​less biofuel cell, capable of generating elec. energy from human lachrymal liq., was developed by using the ascorbate and oxygen naturally present in tears as fuel and oxidant. The biodevice is based on three-​dimensional nanostructured gold electrodes covered with abiotic (conductive org. complex) and biol. (redox enzyme) materials functioning as efficient anodic and cathodic catalysts, resp. Three-​dimensional nanostructured electrodes were fabricated by modifying 100 μm gold wires with 17 nm gold nanoparticles, which were further modified with tetrathiafulvalene-​tetracyanoquinodimethane conducting complex to create the anode and with Myrothecium verrucaria bilirubin oxidase to create the biocathode. When operated in human tears, the biodevice exhibited the following characteristics: an open circuit voltage of 0.54 V, a maximal power d. of 3.1 μW cm-​2 at 0.25 V and 0.72 μW cm-​2 at 0.4 V, with a stable c.d. output of over 0.55 μA cm-​2 at 0.4 V for 6 h of continuous operation. These findings support the authors' proposition that an ascorbate​/oxygen biofuel cell could be a suitable power source for glucose-​sensing contact lenses to be used for continuous health monitoring by diabetes patients

The Changing Nature of the Protein Folding Transition State: Implications for the Shape of the Free-energy Profile for Folding

According to landscape theory proteins do not fold by localised pathways, but find their native conformation by a progressive organisation of an ensemble of partly folded structures down a folding funnel. Here, we use kinetics and protein engineering to investigate the shape of the free-energy profile for two-state folding, which is the macroscopic view of the funnel process for small and rapidly folding proteins. Our experiments are based mainly on structural changes of the transition state of chymotrypsin inhibitor 2 (CI2) upon destabilisation with temperature and GdnHCl. The transition state ensemble of CI2 is a localised feature in the free-energy profile that is sharply higher than the other parts of the activation barrier. The relatively fixed position of the CI2 transition state on the reaction coordinate makes it easy to characterise but contributes also to overshadow the rest of the free-energy profile, the shape of which is inaccessible for analysis. Results from mutants of CI2 and comparison with other two-state proteins, however, point at the possibility that the barrier for folding is generally broad and that localised transition states result from minor ripples in the free-energy profile. Accordingly, variabilities in the folding kinetics may not indicate different folding mechanisms, but could be accounted for by various degrees of ruggedness on top of very broad activation barriers for folding. The concept is attractive since it summarises a wide range of folding data which have previously seemed unrelated. It is also supported by theory. Consistent with experiment, broad barriers predict that new transition state ensembles are exposed upon extreme destabilisation or radical mutations