Pore formation by equinatoxin, a eukaryotic pore-forming toxin, requires a flexible N-terminal region and a stable beta-sandwich

Actinoporins are eukaryotic pore-forming proteins that create 2-nm pores in natural and model lipid membranes by the self-association of four monomers. The regions that undergo conformational change and form part of the transmembrane pore are currently being defined. It was shown recently that the N-terminal region (residues 10-28) of equinatoxin, an actinoporin from Actinia equina, participates in building of the final pore wall. Assuming that the pore is formed solely by a polypeptide chain, other parts of the toxin should constitute the conductive channel and here we searched for these regions by disulfide scanning mutagenesis. Only double cysteine mutants where the N-terminal segment 1-30 was attached to the beta-sandwich exhibited reduced hemolytic activity upon disulfide formation, showing that other parts of equinatoxin, particularly the beta-sandwich and importantly the C-terminal alpha-helix, do not undergo large conformational rearrangements during the pore formation. The role of the beta-sandwich stability was independently assessed via destabilization of a part of its hydrophobic core by mutations of the buried Trp117. These mutants were considerably less stable than the wild-type but exhibited similar or slightly lower permeabilizing activity. Collectively these results show that a flexible N-terminal region and stable beta-sandwich are pre-requisite for proper pore formation by the actinoporin family.     

Cholinesterases in Single Nerve Cells Isolated from the Locus Ceruleus and from Nucleus of the Facial Nerve of the Rat: A Microgasometric Study

Cholinesterase activity in single nerve cell bodies isolated from the locus ceruleus and nucleus of the facial nerve of the rat was analyzed by the microgasometric method. Acetylcholinesterase activity is about the same in both types of cells. Nonspecific cholinesterase is present in noradrenergic cells of the locus ceruleus but not in the cholinergic cells of the nucleus of the facial nerve. The total activity of cholinesterases and the activity of acetylcholinesterase in nerve cell bodies isolated from the locus ceruleus remains practically unchanged from the tenth postnatal day until the age of 24 months. Depletion of noradrenaline by a high dose of reserpine does not influence the total activity of cholinesterases in nerve cell bodies of locus ceruleus.     

A theoretical study on effect of the initial redox state of cytochrome b559 on maximal chlorophyll fluorescence level (F(M)): implications for photoinhibition of photosystem II

In this work, we extended the reversible radical pair model which describes energy utilization and electron transfer up to the first quinone electron acceptor (Q(A)) in photosystem II (PSII), by redox reactions involving cytochrome (cyt) b559. In the model, cyt b559 accepts electrons from the reduced primary electron acceptor in PSII, pheophytin, and donates electrons to the oxidized primary electron donor in PSII (P680+). Theoretical simulations of chlorophyll fluorescence rise based on the model show that the maximal fluorescence, F(M), increases with an increasing amount of initially reduced cyt b559. In this work we applied, the first to our knowledge, metabolic control analysis (MCA) to a model of reactions in PSII. The MCA was used to determine to what extent the reactions occurring in the model control the F(M) level and how this control depends on the initial redox state of cyt b559. The simulations also revealed that increasing the amount of initially reduced cyt b559 could protect PSII against photoinhibition. Also experimental data, which might be used to validate our theory, are presented and discussed.     

Alpha-lipoic acid affects the oxidative stress in various brain structures in mice with methionine and choline deficiency

Deficiency in methionine or choline can induce oxidative stress in various organs such as liver, kidney, heart, and brain. This study was to examine the effects of alpha-lipoic acid (LA) on oxidative stress induced by methionine and choline deficiency (MCD) in several brain structures. Male mice C57BL/6 (n = 28) were divided into four groups: (1) control – continuously fed with standard chow; (2) LA – fed with standard chow and receiving LA; (3) MCD2 – fed with MCD diet for two weeks, and (4) MCD2+LA – fed with MCD diet for two weeks and receiving LA (100 mg/kg/day intraperitonealy [i.p.]). Brain tissue (cortex, hypothalamus, striatum and hippocampus) was taken for determination of oxidative stress parameters. MCD diet induced a significant increase in malondialdehyde and NOx concentration in all brain regions, while LA restored their content to normal values. Similar to this, in MCD2 group, activity of total SOD, MnSOD, and Cu/ZnSOD was reduced by MCD diet, while LA treatment improved their activities in all brain structures. Besides, in MCD2 group a decrease in catalase activity in cortex and GSH content in hypothalamus was evident, while LA treatment induced an increase in catalase activity in cortex and striatum and GSH content in hypothalamus. LA treatment can significantly reduce lipid peroxidation and nitrosative stress, caused by MCD diet, in all brain regions by restoring antioxidant enzymes activities, predominantly total SOD, MnSOD, and Cu/ZnSOD, and to a lesser extent by modulating catalase activity and GSH content. LA supplementation may be used in order to prevent brain oxidative injury induced by methionine and choline deficiency.     

Predicting urban outdoor thermal comfort by the Universal Thermal Climate Index UTCI—a case study in Southern Brazil

Recognising that modifications to the physical attributes of urban space are able to promote improved thermal outdoor conditions and thus positively influence the use of open spaces, a survey to define optimal thermal comfort ranges for passers-by in pedestrian streets was conducted in Curitiba, Brazil. We applied general additive models to study the impact of temperature, humidity, and wind, as well as long-wave and short-wave radiant heat fluxes as summarised by the recently developed Universal Thermal Climate Index (UTCI) on the choice of clothing insulation by fitting LOESS smoothers to observations from 944 males and 710 females aged from 13 to 91 years. We further analysed votes of thermal sensation compared to predictions of UTCI. The results showed that females chose less insulating clothing in warm conditions compared to males and that observed values of clothing insulation depended on temperature, but also on season and potentially on solar radiation. The overall pattern of clothing choice was well reflected by UTCI, which also provided for good predictions of thermal sensation votes depending on the meteorological conditions. Analysing subgroups indicated that the goodness-of-fit of the UTCI was independent of gender and age, and with only limited influence of season and body composition as assessed by body mass index. This suggests that UTCI can serve as a suitable planning tool for urban thermal comfort in sub-tropical regions.     

Deriving the operational procedure for the Universal Thermal Climate Index (UTCI)

The Universal Thermal Climate Index (UTCI) aimed for a one-dimensional quantity adequately reflecting the human physiological reaction to the multi-dimensionally defined actual outdoor thermal environment. The human reaction was simulated by the UTCI-Fiala multi-node model of human thermoregulation, which was integrated with an adaptive clothing model. Following the concept of an equivalent temperature, UTCI for a given combination of wind speed, radiation, humidity and air temperature was defined as the air temperature of the reference environment, which according to the model produces an equivalent dynamic physiological response. Operationalising this concept involved (1) the definition of a reference environment with 50% relative humidity (but vapour pressure capped at 20 hPa), with calm air and radiant temperature equalling air temperature and (2) the development of a one-dimensional representation of the multivariate model output at different exposure times. The latter was achieved by principal component analyses showing that the linear combination of 7 parameters of thermophysiological strain (core, mean and facial skin temperatures, sweat production, skin wettedness, skin blood flow, shivering) after 30 and 120 min exposure time accounted for two-thirds of the total variation in the multi-dimensional dynamic physiological response. The operational procedure was completed by a scale categorising UTCI equivalent temperature values in terms of thermal stress, and by providing simplified routines for fast but sufficiently accurate calculation, which included look-up tables of pre-calculated UTCI values for a grid of all relevant combinations of climate parameters and polynomial regression equations predicting UTCI over the same grid. The analyses of the sensitivity of UTCI to humidity, radiation and wind speed showed plausible reactions in the heat as well as in the cold, and indicate that UTCI may in this regard be universally useable in the major areas of research and application in human biometeorology.     

Characterization of the bifunctional aminoglycoside-modifying enzyme ANT(3')-Ii/AAC(6')-IId from Serratia marcescens

A newly discovered bifunctional antibiotic resistance enzyme from Serratia marcescens catalyzes adenylation and acetylation of aminoglycoside antibiotics. The structure assignment of the enzymic products indicated that acetylation takes place on the 6'-amine of kanamycin A and the adenylation on 3'- and 9-hydroxyl groups of streptomycin and spectinomycin, respectively. The adenyltransferase domain appears to be highly specific to spectinomycin and streptomycin, while the acetyltransferase domain shows a broad substrate profile. Initial velocity patterns indicate that both domains follow a sequential kinetic mechanism. The use of dead-end and product inhibition, the solvent isotope effect, and the solvent viscosity effect reveals that the adenyltransferase domain catalyzes the reaction by a Theorell-Chance kinetic mechanism, where ATP binds to the enzyme prior to the aminoglycoside and the modified antibiotic is the last product to be released. The acetyltransferase domain follows an ordered bi-bi kinetic mechanism, in which the antibiotic is the first substrate that binds to the active site and CoASH is released prior to the modified aminoglycoside. The merging of two genes to create bifunctional resistance enzymes with expanded profiles has now been documented in four instances, including the subject of study in this report, which suggests a new trend in the emergence of resistance to aminoglycoside antibiotics among pathogens.     

Polyglutamine repeats and β‐helix structure: Molecular dynamics study

Neurodegenerative diseases are often associated with the formation of highly insoluble aggregates. Despite the efforts devoted to the characterization of these aggregates, their structure remains elusive. Several neurodegenerative diseases are characterized by the expansion of CAG repeats, which code for Gln. Among the structural models proposed for the aggregates observed in polyQ-linked diseases, the nanotube beta-helix model proposed by Perutz and colleagues Proc Natl Acad Sci U S A 2002;99:5591-5595 has been influential. In the present study, the stability of this beta-helix model has been investigated by performing molecular dynamics simulations on polyQ fragments of different lengths. The results indicate that models shorter than two full beta-helix turns are unstable and collapse toward irregular structures. On the other hand, longer beta-helix models, containing more than 40 residues, achieve a dynamic regular structure. This finding is in line with the observed threshold of Gln repeats (approximately 40) correlated with the insurgence of the disease. Notably, the structure of the final state of the models longer than 40 residues strictly depends on their size. A compact stable ellipsoidal structure is formed by the model made of two full helical turns (41 residues), whereas water filled tubular structures emerge from simulation on longer polypeptides. These results have been interpreted taking into account the experimental data on polyQ aggregates. A structural interpretation of the literature data has been proposed by assuming that different beta-helical models are involved in the different stages of the aggregation process.