Pure manganese(III) 5,10,15,20-tetrakis(4-benzoic acid)porphyrin (MnTBAP) is not a superoxide dismutase mimic in aqueous systems: a case of structure–activity relationship as a watchdog mechanism in experimental therapeutics and biology

Superoxide is involved in a plethora of pathological and physiological processes via oxidative stress and/or signal transduction pathways. Superoxide dismutase (SOD) mimics have, thus, been actively sought for clinical and mechanistic purposes. Manganese(III) 5,10,15,20-tetrakis(4-benzoic acid)porphyrin (MnTBAP) is one of the most intensely explored "SOD mimics" in biology and medicine. However, we show here that this claimed SOD activity of MnTBAP in aqueous media is not corroborated by comprehensive structure-activity relationship studies for a wide set of Mn porphyrins and that MnTBAP from usual commercial sources contains different amounts of noninnocent trace impurities (Mn clusters), which inhibited xanthine oxidase and had SOD activity in their own right. In addition, the preparation and thorough characterization of a high-purity MnTBAP is presented for the first time and confirmed that pure MnTBAP has no SOD activity in aqueous medium. These findings call for an assessment of the relevance and suitability of using MnTBAP (or its impurities) as a mechanistic probe and antioxidant therapeutic; conclusions on the physiological and pathological role of superoxide derived from studies using MnTBAP of uncertain purity should be examined judiciously. An unequivocal distinction between the biological effects due to MnTBAP and that of its impurities can only be unambiguously made if a pure sample is/was used. This work also illustrates the contribution of fundamental structure-activity relationship studies not only for drug design and optimization, but also as a "watchdog" mechanism for checking/spotting eventual incongruence of drug activity in chemical and biological settings.     

Mass spectrometry-based glycoproteomic approach involving lysine derivatization for structural characterization of recombinant human erythropoietin

Lysine-containing peptides comprising glycosylation sites derived from recombinant human erythropoietin (rHuEPO) by trypsin or Lys-C and PNGase F dual digestion were derivatized with 2-methoxy-4,5-dihydro-1H-imidazole and its deuterated analogues. In the same reaction, under reducing conditions (beta-mercaptoethanol), cysteines were converted into methyl-cysteines and lysines into Lys-4,5-dihydro-1H-imidazole. Both modifications on cysteines and lysines simplified the CID-MS/MS spectra, while preserving the structural information by yielding y-series ions and improved the mass spectral signal intensity up to 25 times. Moreover, by this approach, the N-glycan occupation sites were unambiguously determined. O-Glycosylation sites as well as O-glycan structures were determined by a LC-MS/MS experiment carried out on dually digested rHuEPO. N-Glycan mixture purified on a graphitized carbon column using a newly developed method that extracted only sialylated carbohydrates was analyzed first using MALDI-TOF in negative linear ion mode with low mass accuracy but without interferences and metastabile ions and then a reflectron with high mass accuracy. After defining the precursor ions, we performed the nanoESI QTOF MS/MS analysis on N-glycans, mainly targeting the distinction between carbohydrates with sialylated antennae and those lacking sialic acid moieties.     

Probabilistic kinetic model of slow oxidation of low-density lipoprotein: II. Experiments

Theoretical probabilistic kinetic model has been applied to describe the measurements of several oxidation markers as a function of time, during slow oxidation of low-density lipoprotein (LDL). It has been demonstrated that such a process could be described as tocopherol-mediated peroxidation (TMP), initiated and sustained by the action of copper ions, present in LDL in trace amounts. In that process concentration of alpha-tocopherol remains essentially unaltered. Tocopherol and copper ions act as catalysts, oscillating between the oxidized and reduced states. The fitting of the theoretical model to the experimental data resulted in determination of the numerical values for the kinetic parameters. It has been found that the parameter values used for the fitting of the data collected for a number of samples from various donors differ rather little. The kinetic chain length of 1.3 (in presence of co-antioxidants) and 2.9 (in the absence of co-antioxidants) is shorter than found by others. The difference probably comes from the much lower concentration of copper ions in our systems (about 0.1 ion per LDL particle).     

Dementia and legal competency

The legal competency or capability to exercise rights is level of judgment and decision-making ability needed to manage one's own affairs and to sign official documents. With some exceptions, the person entitles this right in age of majority. It is acquired without legal procedures, however the annulment of legal capacity requires a juristic process. This resolution may not be final and could be revoked thorough the procedure of reverting legal capacity - fully or partially. Given the increasing number of persons with dementia, they are often subjects of legal expertise concerning their legal capacity. On the other part, emphasis on the civil rights of mentally ill also demands their maximal protection. Therefore such distinctive issue is approached with particular attention. The approach in determination of legal competency is more focused on gradation of it's particular aspects instead of existing dual concept: legally capable - legally incapable. The main assumption represents how person with dementia is legally capable and should enjoy all the rights, privileges and obligations as other citizens do. The aspects of legal competency for which person with dementia is going to be deprived, due to protection of one's rights and interests, are determined in legal procedure and then passed over to the guardian decided by court. Partial annulment of legal competency is measure applied when there is even one existing aspect of preserved legal capability (pension disposition, salary or pension disposition, ability of concluding contract, making testament, concluding marriage, divorce, choosing whereabouts, independent living, right to vote, right to decide course of treatment ect.). This measure is most often in favour of the patient and rarely for protection of other persons and their interests. Physicians are expected to precisely describe early dementia symptoms which may influence assessment of specific aspects involved in legal capacity (memory loss, impaired task execution, language difficulties, loosing perception of time and space, changes in mood and behaviour, personality alterations, loss of interests and initiative). Towards more accurate determination of legal competency the psychometric tests are being used. The appliance of these tests must be guided with basic question during evaluation: "For what is or is not he/she capable?" In prediction of possible dementia development, the modern diagnostic procedures are used as help for potentially demented individuals in order to plan own affairs and by oneself determine future guardian. This ensures the maximal respect and protection of rights among persons with dementia in order to independently manage life one step ahead of progressive illness. Finally, it is to be distinguished medical concept of legal capacity which is universal and judicial concept which is restricted by rules of national legal system differing from country to country.     

Electrostatic contribution to the thermodynamic and kinetic stability of the homotrimeric coiled coil Lpp-56: A computational study

The protein moiety of the Braun's E. coli outer membrane lipoprotein (Lpp-56) is an attractive object of biophysical investigation in several aspects. It is a homotrimeric, parallel coiled coil, a class of coiled coils whose stability and folding have been studied only occasionally. Lpp-56 possesses unique structural properties and exhibits extremely low rates of folding and unfolding. It is natural to ask how the specificity of the structure determines the extraordinary physical chemical properties of this protein. Recently, a seemingly controversial data on the stability and unfolding rate of Lpp-56 have been published (Dragan et al., Biochemistry 2004;43: 14891-14900; Bjelic et al., Biochemistry 2006;45:8931-8939). The unfolding rate constant measured using GdmCl as the denaturing agent, though extremely low, was substantially higher than that obtained on the basis of thermal unfolding. If this large difference arises from the effect of screening of electrostatic interactions induced by GdmCl, electrostatic interactions would appear to be an important factor determining the unusual properties of Lpp-56. We present here a computational analysis of the electrostatic properties of Lpp-56 combining molecular dynamics simulations and continuum pK calculations. The pH-dependence of the unfolding free energy is predicted in good agreement with the experimental data: the change in DeltaG between pH 3 and pH 7 is approximately 60 kJ mol(-1). The results suggest that the difference in the stability of the protein observed using different experimental methods is mainly because of the effect of the reduction of electrostatic interactions when the salt (GdmCl) concentration increases. We also find that the occupancy of the interhelical salt bridges is unusually high. We hypothesize that electrostatic interactions, and the interhelical salt bridges in particular, are an important factor determining the low unfolding rate of Lpp-56.     

New concepts and new words--how do languages cope with the problem of neology?

It is a well-known fact that languages react differently when foreign words denoting new concepts have to be integrated into the native system. The procedure mostly depends on the degree of purism present in a linguistic community: some languages are rather open to foreign influences and do not demonstrate any special hostility towards new words which are easily accepted and adapted to the phonological and morphological systems of the receiving language. Languages, which have a strong puristic tradition, usually channel their borrowings into the loan translation field using internal word formation resources as a means of creating neologisms. Regardless of whether they are built of native elements or appear as loans, neologisms are necessarily the result of linguistic changes.     

Naturally occurring compounds in differentiation based therapy of cancer

Differentiation of cancer cells entails the reversion of phenotype from malignant to the original. The conversion to cell type characteristic for another tissue is named transdifferentiation. Differentiation/transdifferentiation of malignant cells in high grade tumor mass could serve as a nonaggressive approach that potentially limits tumor progression and augments chemosensitivity. While this therapeutic strategy is already being used for treatment of hematological cancers, its feasibility for solid malignancies is still debated. We will presently discuss the natural compounds that show these properties, with focus on anthraquinones from Aloe vera, Senna, Rheum sp. and hop derived prenylflavonoids.     

Do captive conditions favor shedding of parasites in the reared Atlantic bluefin tuna (Thunnus thynnus)?

Tuna (Thunnus spp.) has been characterized by long distance migrations, highly predatory behavior and longevity, all of which in turn, enable infections with a wide spectrum of different parasitic groups, reflecting in a remarkable diversity of tuna parasite communities. Since 2003, we have been monitoring parasite communities of Atlantic bluefin tuna (Thunnus thynnus) that are caught from the wild and transferred into cages during spring-summer months, as well as assemblages in fish that exit rearing cycle during the winter harvest period after 1.5 years. Interestingly in reared tuna, parasitic populations exhibit a significant decreasing trend at the end of the rearing cycle, rarely observed in other intensive productions that represent a suitable environment for the emergence, establishment and transmission of pathogens. In order to assess epizootiological behavior of tuna parasites assemblages at the beginning (B group) and at the end (A group) of 1.5 year rearing cycle, we examined data on parasite prevalence and abundance over 4 years. The aim was to evaluate parasite diversity indices and emerging differences between newly caught and harvested fish, as well as community compositions and their nestedness in respect to the event in the rearing cycle (capture or harvest time). In order to be able to predict classification of tuna in two categories (newly caught or heavily infected and harvested or less infected fish), based on empirical didymozoids abundances and year of sampling, we built a decision tree model. Results suggest that specificities of parasite assemblages and their dynamics in tuna before and after farming have no similar precedents in aquaculture. A trend of parasitic pauperization repeating in each rearing cycle over four-years time, in once diverse and species rich parasite communities is observed, however, structures of both B and A group rearing assemblages remain nested, with the same species being core parasites (Didymosulcus katsuwonicola and Koellikerioides intestinalis). The B group exhibited significantly higher total parasite richness and mean parasites abundance, as well as the heteroxenous species richness and abundance in comparison to A group, where monoxenous species were not recorded at all. Eleven parasite species out of 26 taxa were selected as important in discriminating between B and A groups' parasites assemblages, while significantly the most abundant in B group were D. katsuwonicola, Platocystis alalongae, K. intestinalis, Koellikerioides internogastricus, Didymocystis abdominalis and Anisakis sp. It is hard to postulate the combination of factors affecting these parasite populations, but environmental, anthropogenic or host intrinsic influence has to be taken into account for further investigation.     

Possible Influence of B Chromosomes on Genes Included in Immune Response and Parasite Burden in Apodemus flavicollis

Genetic background underlying wild populations immune response to different parasites is still not well understood. We studied immune response to multiple infections and to competition between different parasite species at different developmental stages in population of yellow-necked mouse, Apodemus flavicollis. Quantitative real-time PCR was used to investigate associations of MHC II-DRB, IL-10 and Tgf-β genes expressions with presence of intestinal parasites at different developmental stages. Furthermore, we were interested whether the host related characteristics (sex, age, body condition, presence of B chromosomes or expression of other genes) or characteristics of present parasites (number of adult parasites of each identified species, egg count of each parasite genus, total number of nematode individuals) affect differential expression of the studied genes. A significant invert association between the expression of MHC II-DRB and Tgf-β gene was found, which together with absence of IL-10 association confirmed modified Th2 as the main type of immune response to nematode infections. Effect of recorded parasites and parasite life-cycle stage on expression levels of MHC II-DRB gene was detected only through interactions with host-related characteristics such as sex, age, and the presence of B chromosomes. The presence of B chromosomes is associated with lower expression level of Tgf-β gene. Although the influence of host genetic background on parasite infection has already been well documented, this is the first study in mammals that gave presence of B chromosomes on immune response full consideration.