The Role of Structural Elements of the 5'-Terminal Region of p53 mRNA in Translation under Stress Conditions Assayed by the Antisense Oligonucleotide Approach

The p53 protein is one of the major factors responsible for cell cycle regulation and stress response. In the 5’-terminal region of p53 mRNA, an IRES element has been found which takes part in the translational regulation of p53 expression. Two characteristic hairpin motifs are present in this mRNA region: G56-C169, with the first AUG codon, and U180-A218, which interacts with the Hdm2 protein (human homolog of mouse double minute 2 protein). 2′-OMe modified antisense oligomers hybridizing to the 5''-terminal region of p53 mRNA were applied to assess the role of these structural elements in translation initiation under conditions of cellular stress. Structural changes in the RNA target occurring upon oligomers’ binding were monitored by the Pb²⁺-induced cleavage method. The impact of antisense oligomers on the synthesis of two proteins, the full-length p53 and its isoform Δ40p53, was analysed in HT-29, MCF-7 and HepG2 cells, under normal conditions and under stress, as well as in vitro conditions. The results revealed that the hairpin U180-A218 and adjacent single-stranded region A219-A228 were predominantly responsible for high efficacy of IRES-mediated translation in the presence of stress factors. These motifs play a role of cis-acting elements which are able to modulate IRES activity, likely via interactions with protein factors.     

Structural and Functional Analysis of Human HtrA3 Protease and Its Subdomains

Human HtrA3 protease, which induces mitochondria-mediated apoptosis, can be a tumor suppressor and a potential therapeutic target in the treatment of cancer. However, there is little information about its structure and biochemical properties. HtrA3 is composed of an N-terminal domain not required for proteolytic activity, a central serine protease domain and a C-terminal PDZ domain. HtrA3S, its short natural isoform, lacks the PDZ domain which is substituted by a stretch of 7 C-terminal amino acid residues, unique for this isoform. This paper presents the crystal structure of the HtrA3 protease domain together with the PDZ domain (ΔN-HtrA3), showing that the protein forms a trimer whose protease domains are similar to those of human HtrA1 and HtrA2. The ΔN-HtrA3 PDZ domains are placed in a position intermediate between that in the flat saucer-like HtrA1 SAXS structure and the compact pyramidal HtrA2 X-ray structure. The PDZ domain interacts closely with the LB loop of the protease domain in a way not found in other human HtrAs. ΔN-HtrA3 with the PDZ removed (ΔN-HtrA3-ΔPDZ) and an N-terminally truncated HtrA3S (ΔN-HtrA3S) were fully active at a wide range of temperatures and their substrate affinity was not impaired. This indicates that the PDZ domain is dispensable for HtrA3 activity. As determined by size exclusion chromatography, ΔN-HtrA3 formed stable trimers while both ΔN-HtrA3-ΔPDZ and ΔN-HtrA3S were monomeric. This suggests that the presence of the PDZ domain, unlike in HtrA1 and HtrA2, influences HtrA3 trimer formation. The unique C-terminal sequence of ΔN-HtrA3S appeared to have little effect on activity and oligomerization. Additionally, we examined the cleavage specificity of ΔN-HtrA3. Results reported in this paper provide new insights into the structure and function of ΔN-HtrA3, which seems to have a unique combination of features among human HtrA proteases.     

Relative Brain and Brain Part Sizes Provide Only Limited Evidence that Machiavellian Behaviour in Cleaner Wrasse Is Cognitively Demanding

It is currently widely accepted that the complexity of a species’ social life is a major determinant of its brain complexity, as predicted by the social brain hypothesis. However, it remains a challenge to explain what social complexity exactly is and what the best corresponding measures of brain anatomy are. Absolute and relative size of the brain and of the neocortex have often been used as a proxy to predict cognitive performance. Here, we apply the logic of the social brain hypothesis to marine cleaning mutualism involving the genus Labroides. These wrasses remove ectoparasites from ‘client’ reef fish. Conflict occurs as wrasse prefer client mucus over ectoparasites, where mucus feeding constitutes cheating. As a result of this conflict, cleaner wrasse show remarkable Machiavellian-like behaviour. Using own data as well as available data from the literature, we investigated whether the general brain anatomy of Labroides provides any indication that their Machiavellian behaviour is associated with a more complex brain. Neither data set provided evidence for an increased encephalisation index compared to other wrasse species. Published data on relative sizes of brain parts in 25 species of the order Perciformes suggests that only the diencephalon is relatively enlarged in Labroides dimidiatus. This part contains various nuclei of the social decision making network. In conclusion, gross brain anatomy yields little evidence for the hypothesis that strategic behaviour in cleaning selects for larger brains, while future research should focus on more detailed aspects like the sizes of specific nuclei as well as their cryoarchitectonic structure and connectivity.     

High efficiency protocol of DNA extraction from Micromys minutus mandibles from owl pellets: a tool for molecular research of cryptic mammal species

Owl pellets have high potential as a source of DNA. However, this noninvasive method of collecting DNA is rarely used, and its methodological aspects are poorly understood. We investigated the methodology for DNA extraction and amplification from owl pellets containing the smallest European rodent—the Harvest mouse Micromys minutus—as an example. We used mandibles identified in owl pellets for mitochondrial and nuclear DNA amplification. For DNA extraction, we tested two commercial protocols and utilized a protocol being a combination of two commercial kits which ensured high efficiency of DNA extraction. Additionally, we recorded that the amount of DNA was five times higher in extracts from teeth as compared to DNA extracts from jawbones derived from the same mandible. The quantity of DNA was significantly positively correlated with biological sample weight; however, the age of the pellet remains had an impact on the level of inhibition. We recorded inhibition in 40 % of mtDNA extracts derived from pellets older than 150 months, whereas in DNA extracts from pellets younger than 80 months, we did not observe a negative impact of inhibition on PCR efficiency. The amplification success rate was 89.9 % for the mitochondrial fragment and 39.4 % in the case of the nuclear fragment. We observed partial degradation of DNA evidenced by the fact that the longest fragments that we were able to amplify in the case of mtDNA were 450 and 200 bp for nuDNA. The study shows that pellets can be considered as a source of DNA and have high potential for molecular research in the case of threatened species and species that are difficult to study using standard field techniques.     

Difficulties in diagnosis and treatment of acromegaly in a patient with a McCune-Albright syndrome. A case report and a review of literature

We describe a female patient aged 43, who at the age of five was diagnosed with polyostotic fibrous dysplasia (FD). The patient was intermittently treated in our department since the age 33, for approximately 10 years, with intravenous bisphosphonates. At the age of 42 acromegaly was diagnosed incidentally, since clinical manifestations were poor, and, if present earlier, they had been related to FD. Only retrospectively, having biochemical confirmation of GH excess, we could relate them to acromegaly. Because of the involvement of the base of the skull there was no possibility of transphenoidal surgery. Long-acting somatostatin analogues were started, but no response was observed, with IGF-1 and GH being even higher during than before treatment. After the 37-year-history of FD, the occurrence of additional endocrine disorder enabled to make diagnosis of McCune-Albright syndrome (MAS) even in the absence of two out of three classical manifestations such as café-au-lait skin pigmentation and peripheral precocious puberty in the past medical history.     

Ca2+-modulated membrane guanylate cyclase in the testes

To date, the calcium-regulated membrane guanylate cyclase Rod Outer Segment Guanylate Cyclase type 1 (ROS-GC1) transduction system in addition to photoreceptors is known to be expressed in three other types of neuronal cells: in the pinealocytes, mitral cells of the olfactory bulb and the gustatory epithelium of tongue. Very recent studies from our laboratory show that expression of ROS-GC1 is not restricted to the neuronal cells; the male gonads and the spermatozoa also express ROS-GC1. In this presentation, the authors review the existing information on the localization and function of guanylate cyclase with special emphasis on Ca²⁺-modulated membrane guanylate cyclase, ROS-GC1, in the testes. The role of ROS-GC1 and its Ca²⁺-sensing modulators in the processes of spermatogenesis and fertilization are discussed.     

Electron attachment-induced DNA single-strand breaks at the pyrimidine sites

To elucidate the contribution of pyrimidine in DNA strand breaks caused by low-energy electrons (LEEs), theoretical investigations of the LEE attachment-induced C_3′–O_3′, and C_5′–O_5′ σ bond as well as N-glycosidic bond breaking of 2′-deoxycytidine-3′,5′-diphosphate and 2′-deoxythymidine-3′,5′-diphosphate were performed using the B3LYP/DZP++ approach. The base-centered radical anions are electronically stable enough to assure that either the C–O or glycosidic bond breaking processes might compete with the electron detachment and yield corresponding radical fragments and anions. In the gas phase, the computed glycosidic bond breaking activation energy (24.1 kcal/mol) excludes the base release pathway. The low-energy barrier for the C_3′–O_3′ σ bond cleavage process (∼6.0 kcal/mol for both cytidine and thymidine) suggests that this reaction pathway is the most favorable one as compared to other possible pathways. On the other hand, the relatively low activation energy barrier (∼14 kcal/mol) for the C_5′–O_5′ σ bond cleavage process indicates that this bond breaking pathway could be possible, especially when the incident electrons have relatively high energy (a few electronvolts). The presence of the polarizable medium greatly increases the activation energies of either C–O σ bond cleavage processes or the N-glycosidic bond breaking process. The only possible pathway that dominates the LEE-induced DNA single strands in the presence of the polarizable surroundings (such as in an aqueous solution) is the C_3′–O_3′ σ bond cleavage (the relatively low activation energy barrier, ∼13.4 kcal/mol, has been predicted through a polarizable continuum model investigation). The qualitative agreement between the ratio for the bond breaks of C_5′–O_5′, C_3′–O_3′ and N-glycosidic bonds observed in the experiment of oligonucleotide tetramer CGAT and the theoretical sequence of the bond breaking reaction pathways have been found. This consistency between the theoretical predictions and the experimental observations provides strong supportive evidences for the base-centered radical anion mechanism of the LEE-induced single-strand bond breaking around the pyrimidine sites of the DNA single strands.     

Evidence for Light Wavelength-Specific Photoelectrophysiological Signaling and Memory of Excess Light Episodes in Arabidopsis

Although light is essential for photosynthesis, excess light can damage the photosynthetic apparatus and deregulate other cellular processes. Thus, protective integrated regulatory responses that can dissipate excess of absorbed light energy and simultaneously optimize photosynthesis and other cellular processes under variable light conditions can prove highly adaptive. Here, we show that the local and systemic responses to an excess light episode are associated with photoelectrophysiological signaling (PEPS) as well as with changes in nonphotochemical quenching and reactive oxygen species levels. During an excess light incident, PEPS is induced by quantum redox changes in photosystem II and in its proximity and/or by changes in glutathione metabolism in chloroplasts. PEPS is transduced, at least in part, by bundle sheath cells and is light wavelength specific. PEPS systemic propagation speed and action potential are dependent on ASCORBATE PEROXIDASE2 function. Excess light episodes are physiologically memorized in leaves, and the cellular light memory effect is specific for an excess of blue (450 nm) and red (650 nm) light of similar energy. It is concluded that plants possess a complex and dynamic light training and memory system that involves quantum redox, reactive oxygen species, hormonal, and PEPS signaling and is used to optimize light acclimation and immune defenses.     

Purification of extracellular laccase from Cerrena unicolor

Cerrena unicolor was found to produce large amounts of extracellular laccase when grown aerobically on the optimized Lindenberg and Holm medium in fermenter culture with an automatic pH control. The laccase from this source was purified to homogeneity by a rapid procedure, using ion-exchange chromatography, affinity chromatography, and chromatofocusing. The enzymes isoforms were recovered with a 65- to 92-fold increase in specific activity and a yield for Ia1?=?6.7%; Ia2?=?27.5%; Ib?=?9.7%; and IIa1?=?21%. The molecular mass of the purified enzymes proved to be 45, 47, 54, and 62?kD, respectively, as determined by size-exclusion high-performance liquid chromatography (HPLC). The isoelectric points were in the range of 4.7 to 4.2, and the carbohydrate content in the purified enzymes was between 1.6 and 3.5%.