Influence of the acetylcholinesterase active site protonation on omega loop and active site dynamics

Existence of alternative entrances in acetylcholinesterase (AChE) could explain the contrast between the very high AChE catalytic efficiency and the narrow and long access path to the active site revealed by X-ray crystallography. Alternative entrances could facilitate diffusion of the reaction products or at least water and ions from the active site. Previous molecular dynamics simulations identified side door and back door as the most probable alternative entrances. The simulations of non-inhibited AChE suggested that the back door opening events occur only rarely (0.8% of the time in the 10ns trajectory). Here we present a molecular dynamics simulation of non-inhibited AChE, where the back door opening appears much more often (14% of the time in the 12ns trajectory) and where the side door opening was observed quite frequently (78% of trajectory time). We also present molecular dynamics, where the back door does not open at all, or where large conformational changes of the AChE omega loop occur together with alternative passage opening events. All these differences in AChE dynamical behavior are caused by different protonation states of two glutamate residues located on bottom of the active site gorge (Glu202 and G450 in Mus musculus AChE). Our results confirm the results of previous molecular dynamics simulations, expand the view and suggest the probable reasons for the overall conformational behavior of AChE omega loop.     

Novel antimicrobial peptides from the venom of the eusocial bee Halictus sexcinctus (Hymenoptera: Halictidae) and their analogs

Two novel antimicrobial peptides, named halictines, were isolated from the venom of the eusocial bee Halictus sexcinctus. Their primary sequences were established by ESI-QTOF mass spectrometry, Edman degradation and enzymatic digestion as Gly-Met-Trp-Ser-Lys-Ile-Leu-Gly-His-Leu-Ile-Arg-NH₂ (HAL-1), and Gly-Lys-Trp-Met-Ser-Leu-Leu-Lys–His-Ile-Leu-Lys-NH₂ (HAL-2). Both peptides exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria but also noticeable hemolytic activity. The CD spectra of HAL-1 and HAL-2 measured in the presence of trifluoroethanol or SDS showed ability to form an amphipathic α-helical secondary structure in an anisotropic environment such as bacterial cell membrane. NMR spectra of HAL-1 and HAL-2 measured in trifluoroethanol/water confirmed formation of helical conformation in both peptides with a slightly higher helical propensity in HAL-1. Altogether, we prepared 51 of HAL-1 and HAL-2 analogs to study the effect of such structural parameters as cationicity, hydrophobicity, α-helicity, amphipathicity, and truncation on antimicrobial and hemolytic activities. The potentially most promising analogs in both series are those with increased net positive charge, in which the suitable amino acid residues were replaced by Lys. This improvement basically relates to the increase of antimicrobial activity against pathogenic Pseudomonas aeruginosa and to the mitigation of hemolytic activity.     

Structural diversity of nucleoside phosphonic acids as a key factor in the discovery of potent inhibitors of rat T-cell lymphoma thymidine phosphorylase

Structurally diverse, sugar-modified, thymine-containing nucleoside phosphonic acids were evaluated for their ability to inhibit thymidine phosphorylase (TP, EC 2.4.2.4) purified from spontaneous T-cell lymphomas of an inbred Sprague-Dawley rat strain. From a large set of tested compounds, among them a number of pyrrolidine-based derivatives, 10 nucleotide analogues with IC₅₀ values below 1 μM were selected. Out of them, four compounds strongly inhibited the enzyme with IC₅₀ values lying in a range of 11–45 nM. These most potent compounds might be bi-substrate analogues.     

Cyanide inhibition and pyruvate-induced recovery of cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase

The mechanism of cyanide’s inhibitory effect on the mitochondrial cytochrome c oxidase (COX) as well as the conditions for its recovery have not yet been fully explained. We investigated three parameters of COX function, namely electron transport (oxygen consumption), proton transport (mitochondrial membrane potential Δψ _m) and the enzyme affinity to oxygen (p ₅₀ value) with regard to the inhibition by KCN and its reversal by pyruvate. 250 μM KCN completely inhibited both the electron and proton transport function of COX. The inhibition was reversible as demonstrated by washing of mitochondria. The addition of 60 mM pyruvate induced the maximal recovery of both parameters to 60–80% of the original values. When using low KCN concentrations of up to 5 μM, we observed a profound, 30-fold decrease of COX affinity for oxygen. Again, this decrease was completely reversed by washing mitochondria while pyruvate induced only a partial, yet significant recovery of oxygen affinity. Our results demonstrate that the inhibition of COX by cyanide is reversible and that the potential of pyruvate as a cyanide poisoning antidote is limited. Importantly, we also showed that the COX affinity for oxygen is the most sensitive indicator of cyanide toxic effects.     

Adaptive radiations in butterflies: evolutionary history of the genus Erebia (Nymphalidae: Satyrinae)

We studied the speciose butterfly genus Erebia by reconstructing its phylogenetic relationships using parsimony and Bayesian approaches. We estimated times and rates of diversification for its lineages and employed a biogeographical analysis in order to reconstruct its evolutionary history. DNA sequence data from one mitochondrial gene and three nuclear genes were analyzed for a total of 74 species in Erebia. The estimated dates of origin and diversification for clades, in combination with a biogeographical analysis, suggest that the genus originated in Asian Russia and started its diversification process around 23 Myr. An important event was the dispersal of a lineage from Asia to Western Europe between 23 and 17 Myr, which allowed the radiation of most of species in the genus. The diversification pattern is consistent with a model of diversity limited by clade richness, which implies an early rapid diversification followed by deceleration due to a decrease in speciation. We argue that these characteristics of the evolutionary history of Erebia are consistent with a density‐dependent scenario, with species radiation limited by filling of niche space and reduced resources. We found that the Boeberia parmenio appears strongly supported in the genus Erebia and therefore we place Boeberia Prout, 1901 as a junior synonym of Erebia Dalman, 1816 ( syn. nov. ).     

Stage-specific requirement for Isa1 and Isa2 proteins in the mitochondrion of Trypanosoma brucei and heterologous rescue by human and Blastocystis orthologues

IscA/Isa proteins function as alternative scaffolds for the assembly of Fe-S clusters and/or provide iron for their assembly in prokaryotes and eukaryotes. Isa are usually non-essential and in most organisms are confined to the mitochondrion. We have studied the function of TbIsa1 and TbIsa2 in Trypanosoma brucei, where the requirement for both of them to sustain cell growth depends on the life cycle stage. The TbIsa proteins are abundant in the procyclic form, which contains an active organelle. Both proteins are indispensable for growth, as they are required for the assembly of Fe-S clusters in mitochondrial aconitase, fumarase and succinate dehydrogenase. Reactive oxygen species but not iron accumulate in the procyclic mitochondrion upon ablation of the TbIsa proteins, but their depletion does not influence the assembly of Fe-S clusters in cytosolic proteins. In the bloodstream form, which has a downregulated mitochondrion, the TbIsa proteins are non-essential. The Isa2 orthologue of the anaerobic protist Blastocystis partially rescued the growth and enzymatic activities of TbIsa1/2 knock-down. Rescues of single knock-downs as well as heterologous rescues with human Isa orthologues partially recovered the activities of aconitase and fumarase. These results show that the Isa1 and Isa2 proteins of diverse eukaryotes have overlapping functions.     

Uranium,thorium and rare earth elements in macrofungi: what are the genuine concentrations?

Concentrations of uranium, thorium and rare earth elements (REE) in 36 species of ectomycorrhizal (26 samples) and saprobic (25 samples) macrofungi from unpolluted sites with differing bedrock geochemistry were analyzed by inductively coupled plasma mass spectrometry (ICP-MS). Analytical results are supported by use of certified reference materials (BCR-670, BCR-667, NIST-1575a) and the reliability of the determination of uranium was verified by epithermal neutron activation analysis (ENAA). It appears that data recently published on these elements are erroneous, in part because of use of an inappropriate analytical method; and in part because of apparent contamination by soil particles resulting in elevated levels of thorium and REE. Macrofungi from unpolluted areas, in general, did not accumulate high levels of the investigated metals. Concentrations of uranium and thorium were generally below 30 and 125 μg kg⁻¹ (dry weight), respectively. Concentrations of REE in macrofungi did not exceed 360 μg kg⁻¹ (dry weight) and their distribution more or less followed the trend observed in post-Archean shales and loess.     

Coordination conjugates of therapeutic proteins with drug carriers: a new approach for versatile advanced drug delivery

We present here a general system for the coordination attachment of therapeutic proteins to a drug delivery system and its application in combined therapy. Proof of concept is demonstrated by the synthesis and testing of the targeted drug delivery system for cytostatics, which is based on a combination of the drug carrier Zn-porphyrin-cyclodextrin conjugates and their supramolecular coordination complexes with immunoglobulins. This system can be as readily used for a variety of therapeutic and targeting proteins including PAs, MAs, lectins, and HSA. Moreover, it allows combined photodynamic therapy, cell targeted chemotherapy and immunotherapy. When tested in a mouse model with human C32 carcinoma, the therapeutic superiority of the coordination assembly nanosystem was shown in comparison with the efficacy of building blocks used for the construction of the system.     

Fatal attraction phenomenon in humans: cat odour attractiveness increased for toxoplasma-infected men while decreased for infected women

Background

Latent toxoplasmosis, a lifelong infection with the protozoan Toxoplasma gondii, has cumulative effects on the behaviour of hosts, including humans. The most impressive effect of toxoplasmosis is the “fatal attraction phenomenon,” the conversion of innate fear of cat odour into attraction to cat odour in infected rodents. While most behavioural effects of toxoplasmosis were confirmed also in humans, neither the fatal attraction phenomenon nor any toxoplasmosis-associated changes in olfactory functions have been searched for in them.

Principal Findings

Thirty-four Toxoplasma-infected and 134 noninfected students rated the odour of urine samples from cat, horse, tiger, brown hyena and dog for intensity and pleasantness. The raters were blind to their infection status and identity of the samples. No signs of changed sensitivity of olfaction were observed. However, we found a strong, gender dependent effect of toxoplasmosis on the pleasantness attributed to cat urine odour (p = 0.0025). Infected men rated this odour as more pleasant than did the noninfected men, while infected women rated the same odour as less pleasant than did noninfected women. Toxoplasmosis did not affect how subjects rated the pleasantness of any other animal species'' urine odour; however, a non-significant trend in the same directions was observed for hyena urine.

Conclusions

The absence of the effects of toxoplasmosis on the odour pleasantness score attributed to large cats would suggest that the amino acid felinine could be responsible for the fatal attraction phenomenon. Our results also raise the possibility that the odour-specific threshold deficits observed in schizophrenia patients could be caused by increased prevalence of Toxoplasma-infected subjects in this population rather than by schizophrenia itself. The trend observed with the hyena urine sample suggests that this carnivore, and other representatives of the Feliformia suborder, should be studied for their possible role as definitive hosts in the life cycle of Toxoplasma.     

Modulator of intracellular Ca(2+), thapsigargin, interferes with in vitro secretion of cytokines and nitric oxide

Interference of thapsigargin (TG), an inhibitor of endoplasmic reticulum Ca(2+) ATPase, with immune reactivity of murine macrophages was investigated under conditions in vitro. The activation of cells with lipopolysaccharide (LPS), interferon-(gamma) (IFN-(gamma)), and with acyclic nucleoside phosphonate N(6)-isobutyl-9-[2-(phosphonomethoxy)ethyl]- 2,6-diaminopurine (N(6)-isobutyl-PMEDAP) resulted in enhanced production of cytokines TNF-alpha, IL-10, chemokines RANTES/CCL5 and MIP-1alpha/CCL3, as well as in substantially augmented production of nitric oxide (NO) triggered by IFN-(gamma). The effects were in a dual mode of action influenced by TG (1 microM). While TG upregulated secretion of TNF-alpha, it inhibited secretion of IL-10 and RANTES. The immune-stimulated secretion of MIP-1alpha remained virtually unaffected, though TG on its own activated expression of MIP-1alpha in macrophages. The high-output NO production induced by IFN-(gamma), high concentrations of LPS, or by combination of IFN-(gamma) plus LPS or N(6)-isobutyl-PMEDAP was inhibited by TG. On the other hand, production of NO which was marginally activated by low concentration of LPS was upregulated by TG.