Detection and Characterisation of Mutations Responsible for Allele-Specific Protein Thermostabilities at the Mn-Superoxide Dismutase Gene in the Deep-Sea Hydrothermal Vent Polychaete Alvinella pompejana

Alvinella pompejana (Polychaeta, Alvinellidae) is one of the most thermotolerant marine eukaryotes known to date. It inhabits chimney walls of deep-sea hydrothermal vents along the East Pacific Rise (EPR) and is exposed to various challenging conditions (e.g. high temperature, hypoxia and the presence of sulphides, heavy metals and radiations), which increase the production of dangerous reactive oxygen species (ROS). Two different allelic forms of a manganese-superoxide dismutase involved in ROS detoxification, ApMnSOD1 and ApMnSOD2, and differing only by two substitutions (M110L and A138G) were identified in an A. pompejana cDNA library. RFLP screening of 60 individuals from different localities along the EPR showed that ApMnSOD2 was rare (2 %) and only found in the heterozygous state. Dynamic light scattering measurements and residual enzymatic activity experiments showed that the most frequent form (ApMnSOD1) was the most resistant to temperature. Their half-lives were similarly long at 65 °C (>110 min) but exhibited a twofold difference at 80 °C (20.8 vs 9.8 min). Those properties are likely to be explained by the occurrence of an additional sulphur-containing hydrogen bond involving the M110 residue and the effect of the A138 residue on the backbone entropy. Our results confirm the thermophily of A. pompejana and suggest that this locus is a good model to study how the extreme thermal heterogeneity of the vent conditions may help to maintain old rare variants in those populations.     

Evolution of Cyclic Amidohydrolases: A Highly Diversified Superfamily

Dihydroorotases are universal proteins catalyzing the third step of pyrimidine biosynthesis. These zinc metalloenzymes belong to the superfamily of cyclic amidohydrolases, comprising also other enzymes that are involved in degradation of either purines (allantoinases), pyrimidines (dihydropyrimidinases) or hydantoins (hydantoinases). The evolutionary relationships between these mechanistically related enzymes were estimated after designing a method to build an accurate multiple sequence alignment. The amino acid sequences that have been crystallized were used to build a seed alignment. All the remaining homologues were progressively added by aligning their HMM profiles to the seed HMM profile, allowing to obtain a reliable phylogeny of the superfamily. This helped us to propose a new evolutionary classification of dihydroorotases into three major types, while at the same time disentangling an important part of the history of their complex structure–function relationships. Although differing in their substrate specificity, allantoinases, hydantoinases and dihydropyrimidinases are found to be phylogenetically closer to DHOase Type I than the proximity of the three DHOase types to each other. This suggests that the primordial cyclic amidohydrolase was a multifunctional, highly evolvable generalist, with high conformational diversity allowing for promiscuous activities. Then, successive gene duplications allowed resolving the primordial substrate ambiguity in various substrate specificities. The present-day superfamily of cyclic amidohydrolases is the result of the progressive divergence of these ancestral paralogous copies by descent with modification.     

Voltage-dependent anion channel (VDAC) participates in amyloid beta-induced toxicity and interacts with plasma membrane estrogen receptor α in septal and hippocampal neurons

Voltage-dependent anion channel (VDAC) is a porin known by its role in metabolite transport across mitochondria and participation in apoptotic processes. Although traditionally accepted to be located within mitochondrial outer membrane, some data has also reported its presence at the plasma membrane level where it seems to participate in regulation of normal redox homeostasis and apoptosis. Here, exposure of septal SN56 and hippocampal HT22 cells to specific anti-VDAC antibodies prior to amyloid beta (Aβ) peptide was observed to prevent neurotoxicity. In these cell lines, we identified a VDAC form associated with the plasma membrane that seems to be particularly abundant in caveolae. The two membrane-related isoforms of estrogen receptor α (mERα) (80 and 67 kDa), known in SN56 cells to participate in estrogen-induced neuroprotection against Aβ injury, were also observed to be present in caveolae. Interestingly, we demonstrated for the first time that both VDAC and mERα interact at the plasma membrane of these neurons as well as in microsomal fractions of the corresponding murine septal and hippocampal tissues. These proteins were also shown to associate with caveolin-1, thereby corroborating their presence in caveolar microdomains. Taken together, these results suggest that VDAC-mERα association at the plasma membrane level may participate in the modulation of Aβ-induced cell death.     

Multinational study of oestrogen and progesterone receptor immunocytochemistry on breast carcinoma fine needle aspirates

?. P. Marin?ek, N. Nolde, I. Kardum‐Skelin, R. Nizzoli, B. Önal, T. Rezanko, E. Tani, K. T. Ostovi?, P. Vielh, F. Schmitt and G. Kocjan
Multinational study of oestrogen and progesterone receptor immunocytochemistry on breast carcinoma fine needle aspirates Objectives: To collect data on the variability of immunocytochemical (ICC) procedures used to detect oestrogen/progesterone receptors (ER/PR) on cytological material; to test the reproducibility of results; and to identify the crucial points in the ICC procedures that affect the result. Methods: Ten laboratories from eight countries participated in a two‐part study. In the first part, one of the participants (the coordinator) prepared and distributed cytospins from a fine needle aspirate of a primary breast carcinoma. Laboratories performed ICC staining for ER/PR according to their own methods on the test slides and in‐house positive controls. Slides were returned to the coordinator together with information on the preparation of positive control slides and the ICC methodology used. In the second part, obligatory methods of fixation and antigen retrieval were specified. Evaluation of results included grading the number of positive cells, staining intensity, background staining, cytoplasmic staining, sample condition and cellularity. Participants evaluated their own results, which were subsequently evaluated by the coordinator. Results: There was great variability in the preparation of slides for in‐house controls and ICC methodology. The outcome of ICC staining of in‐house control slides was excellent in two laboratories, adequate in three, sub‐optimal in four and inadequate in one. Only six obtained a positive reaction on the test slides and not all were of a high quality. Results of the second run were greatly improved in terms of cellularity of in‐house positive control slides, and scores for the percentage of stained cells and staining intensity of control and test slides. Cytospins and monolayer (ThinPrep^®) preparations were superior to direct smears; methods of fixation and antigen retrieval were the key points in the staining process. Conclusions: Our experience points to the need for guidelines for hormonal receptor determination and external quality control on cytological material, in order for cytological methods to be used in routine clinical practice with a suitable degree of confidence.     

Myotonic dystrophy protein kinase (DMPK) prevents ROS-induced cell death by assembling a hexokinase II-Src complex on the mitochondrial surface

The biological functions of myotonic dystrophy protein kinase (DMPK), a serine/threonine kinase whose gene mutations cause myotonic dystrophy type 1 (DM1), remain poorly understood. Several DMPK isoforms exist, and the long ones (DMPK-A/B/C/D) are associated with the mitochondria, where they exert unknown activities. We have studied the isoform A of DMPK, which we have found to be prevalently associated to the outer mitochondrial membrane. The kinase activity of mitochondrial DMPK protects cells from oxidative stress and from the ensuing opening of the mitochondrial permeability transition pore (PTP), which would otherwise irreversibly commit cells to death. We observe that DMPK (i) increases the mitochondrial localization of hexokinase II (HK II), (ii) forms a multimeric complex with HK II and with the active form of the tyrosine kinase Src, binding its SH3 domain and (iii) it is tyrosine-phosphorylated by Src. Both interaction among these proteins and tyrosine phosphorylation of DMPK are increased under oxidative stress, and Src inhibition selectively enhances death in DMPK-expressing cells after HK II detachment from the mitochondria. Down-modulation of DMPK abolishes the appearance of muscle markers in in vitro myogenesis, which is rescued by oxidant scavenging. Our data indicate that, together with HK II and Src, mitochondrial DMPK is part of a multimolecular complex endowed with antioxidant and pro-survival properties that could be relevant during the function and differentiation of muscle fibers.     

Geographically Related Variation in Epicuticular Wax Traits of Pinus nigra Populations from Southern Carpathians and Central Balkans – Taxonomic Considerations

The chemical composition of epicuticular waxes of nine populations from three Pinus nigra J. F. Arnold subspecies (namely subsp. nigra, subsp. banatica (Borbás ) Novák , and subsp. pallasiana (Lamb .) Holmboe ) from Southern Carpathians and central Balkan Peninsula were analyzed using GC/MS and GC/FID chromatography, and multivariate statistical techniques with respect to biogeography and taxonomy. In the needle waxes, four primary alcohols and 14 n‐alkanes ranging from C₂₁ to C₃₃ were identified, and the most abundant compounds were the four odd‐numbered n‐alkanes C₂₇, C₂₅, C₂₃, and C_29. Multivariate statistical analyses (CDA and CA) have shown existence of three P. nigra groups and suggested clinal differentiation as a mechanism of genetic variation across a geographic area: the first group consisted of the southernmost populations of subsp. pallasiana from Macedonia, the second consisted of the northernmost subsp. banatica populations from Romania, while all populations in Serbia described as three different subspecies (nigra, banatica, and pallasiana) formed the third group together with subsp. nigra population from Bosnia and Herzegovina. According to simple linear regression, geographic latitude and four bioclimatic parameters were moderately correlated with the contents of epicuticular wax compounds that are important in population discrimination, while stepwise multiple regression showed that latitude participated in most of the regression models for predicting the composition of the epicuticular waxes. These results agree with CDA and CA analysis, and confirmed the possibility of recognition of fine geographic differentiation of the analyzed P. nigra populations.     

Distinguishing Antimicrobial Models with Different Resistance Mechanisms via Population Pharmacodynamic Modeling

Semi-mechanistic pharmacokinetic-pharmacodynamic (PK-PD) modeling is increasingly used for antimicrobial drug development and optimization of dosage regimens, but systematic simulation-estimation studies to distinguish between competing PD models are lacking. This study compared the ability of static and dynamic in vitro infection models to distinguish between models with different resistance mechanisms and support accurate and precise parameter estimation. Monte Carlo simulations (MCS) were performed for models with one susceptible bacterial population without (M1) or with a resting stage (M2), a one population model with adaptive resistance (M5), models with pre-existing susceptible and resistant populations without (M3) or with (M4) inter-conversion, and a model with two pre-existing populations with adaptive resistance (M6). For each model, 200 datasets of the total bacterial population were simulated over 24h using static antibiotic concentrations (256-fold concentration range) or over 48h under dynamic conditions (dosing every 12h; elimination half-life: 1h). Twelve-hundred random datasets (each containing 20 curves for static or four curves for dynamic conditions) were generated by bootstrapping. Each dataset was estimated by all six models via population PD modeling to compare bias and precision. For M1 and M3, most parameter estimates were unbiased (<10%) and had good imprecision (<30%). However, parameters for adaptive resistance and inter-conversion for M2, M4, M5 and M6 had poor bias and large imprecision under static and dynamic conditions. For datasets that only contained viable counts of the total population, common statistical criteria and diagnostic plots did not support sound identification of the true resistance mechanism. Therefore, it seems advisable to quantify resistant bacteria and characterize their MICs and resistance mechanisms to support extended simulations and translate from in vitro experiments to animal infection models and ultimately patients.     

Effects of MDMA Injections on the Behavior of Socially-Housed Long-Tailed Macaques (Macaca fascicularis)

3,4-methylenedioxy-N-methyl amphetamine (MDMA) is one of the few known molecules to increase human and rodent prosocial behaviors. However, this effect has never been assessed on the social behavior of non-human primates. In our study, we subcutaneously injected three different doses of MDMA (1.0, 1.5 or 2.0mg/kg) to a group of three, socially housed, young male long-tailed macaques. More than 200 hours of behavioral data were recorded, during 68 behavioral sessions, by an automatic color-based video device that tracked the 3D positions of each animal and of a toy. This data was then categorized into 5 exclusive behaviors (resting, locomotion, foraging, social contact and object play). In addition, received and given social grooming was manually scored. Results show several significant dose-dependent behavioral effects. At 1.5mg/kg only, MDMA induces a significant increase in social grooming behavior, thus confirming the prosocial effect of MDMA in macaques. Additionally, at 1.5 and 2.0 mg/kg MDMA injection substantially decreases foraging behavior, which is consistent with the known anorexigenic effect of this compound. Furthermore, at 2.0 mg/kg MDMA injection induces an increase in locomotor behavior, which is also in accordance with its known stimulant property. Interestingly, MDMA injected at 1.0mg/kg increases the rate of object play, which might be interpreted as a decrease of the inhibition to manipulate a unique object in presence of others, or, as an increase of the intrinsic motivation to manipulate this object. Together, our results support the effectiveness of MDMA to study the complex neurobiology of primates’ social behaviors.     

Influence of Obesity and Metabolic Disease on Carotid Atherosclerosis in Patients with Coronary Artery Disease (CordioPrev Study)

BackgroundRecent data suggest that the presence of associated metabolic abnormalities may be important modifiers of the association of obesity with a poorer prognosis in coronary heart disease. We determined the influence of isolated overweight and obesity on carotid intima media thickness (IMT-CC), and also assessed whether this influence was determined by the presence of metabolic abnormalities.Methods1002 participants from the CordioPrev study were studied at entry. We determined their metabolic phenotypes and performed carotid ultrasound assessment. We evaluated the influence of obesity, overweight and metabolic phenotypes on the IMT-CC.ResultsMetabolically sick participants (defined by the presence of two or more metabolic abnormalities) showed a greater IMT-CC than metabolically healthy individuals (p = 4 * 10⁻⁶). Overweight and normal weight patients who were metabolically healthy showed a lower IMT-CC than the metabolically abnormal groups (all p<0.05). When we evaluated only body weight (without considering metabolic phenotypes), overweight or obese patients did not differ significantly from normal-weight patients in their IMT-CC (p = 0.077). However, obesity was a determinant of IMT-CC when compared to the composite group of normal weight and overweight patients (all not obese).ConclusionsIn coronary patients, a metabolically abnormal phenotype is associated with a greater IMT-CC, and may be linked to a higher risk of suffering new cardiovascular events. The protection conferred in the IMT-CC by the absence of metabolic abnormality may be blunted by the presence of obesity.

Trial Registration

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