Consequences of DNA damage for gene transcription : direct effects of modified nucleobases and the role of base excision repair = Folgen von DNA-Sch��den f��r die Gentranskription

The presence of damaged nucleobases in DNA can negatively influence transcription of genes. One of the mechanisms by which DNA damage interferes with reading of genetic information is a direct blockage of the elongating RNA polymerase complexes ��� an effect well described for bulky adducts induced by several chemical substances and UV-irradiation. However, other mechanisms must exist as well because many of the endogenously occurring non-bulky DNA base modifications have transcription-inhibitory properties in cells, whilstrnnot constituting a roadblock for RNA polymerases under cell free conditions. The inhibition of transcription by non-blocking DNA damage was investigated in this work by employing the reporter gene-based assays. Comparison between various types of DNA damage (UV-induced pyrimidine photoproducts, oxidative purine modifications induced by photosensitisation, defined synthetic modified bases such as 8-oxoguanine and uracil, and sequence-specific single-strand breaks) showed that distinct mechanisms of inhibition of transcription can be engaged, and that DNA repair can influence transcription of the affectedrngenes in several different ways.rnQuantitative expression analyses of reporter genes damaged either by the exposure of cells to UV or delivered into cells by transient transfection supported the earlier evidence that transcription arrest at the damage sites is the major mechanism for the inhibition of transcription by this kind of DNA lesions and that recovery of transcription requires a functional nucleotide excision repair gene Csb (ERCC6) in mouse cells. In contrast, oxidisedrnpurines generated by photosensitisation do not cause transcriptional blockage by a direct mechanism, but rather lead to transcriptional repression of the damaged gene which is associated with altered histone acetylation in the promoter region. The whole chain of events leading to transcriptional silencing in response to DNA damage remains to be uncovered. Yet, the data presented here identify repair-induced single-strand breaks ��� which arise from excision of damaged bases by the DNA repair glycosylases or endonucleases ��� as arnputative initiatory factor in this process. Such an indirect mechanism was supported by requirement of the 8-oxoguanine DNA glycosylase (OGG1) for the inhibition of transcription by synthetic 8-oxodG incorporated into a reporter gene and by the delays observed for the inhibition of transcription caused by structurally unrelated base modifications (8-oxoguanine and uracil). It is thereby hypothesized that excision of the modified bases could be a generalrnmechanism for inhibition of transcription by DNA damage which is processed by the base excision repair (BER) pathway. Further gene expression analyses of plasmids containing single-strand breaks or abasic sites in the transcribed sequences revealed strong transcription inhibitory potentials of these lesions, in agreement with the presumption that BER intermediates are largely responsible for the observed effects. Experiments with synthetic base modifications positioned within the defined DNA sequences showed thatrninhibition of transcription did not require the localisation of the lesion in the transcribed DNA strand; therefore the damage sensing mechanism has to be different from the direct encounters of transcribing RNA polymerase complexes with DNA damage.rnAltogether, this work provides new evidence that processing of various DNA basernmodifications by BER can perturb transcription of damaged genes by triggering a gene silencing mechanism. As gene expression can be influenced even by a single DNA damage event, this mechanism could have relevance for the endogenous DNA damage induced in cells under normal physiological conditions, with a possible link to gene silencing in general.

Numerical and semi-analytical models of sliding masses: application to the 1783 Scilla tsunamigenic landslide

The Scilla rock avalanche occurred on 6 February 1783 along the coast of the Calabria region (southern Italy), close to the Messina Strait. It was triggered by a mainshock of the Terremoto delle Calabrie seismic sequence, and it induced a tsunami wave responsible for more than 1500 casualties along the neighboring Marina Grande beach. The main goal of this work is the application of semi-analtycal and numerical models to simulate this event. The first one is a MATLAB code expressly created for this work that solves the equations of motion for sliding particles on a two-dimensional surface through a fourth-order Runge-Kutta method. The second one is a code developed by the Tsunami Research Team of the Department of Physics and Astronomy (DIFA) of the Bologna University that describes a slide as a chain of blocks able to interact while sliding down over a slope and adopts a Lagrangian point of view. A wide description of landslide phenomena and in particular of landslides induced by earthquakes and with tsunamigenic potential is proposed in the first part of the work. Subsequently, the physical and mathematical background is presented; in particular, a detailed study on derivatives discratization is provided. Later on, a description of the dynamics of a point-mass sliding on a surface is proposed together with several applications of numerical and analytical models over ideal topographies. In the last part, the dynamics of points sliding on a surface and interacting with each other is proposed. Similarly, different application on an ideal topography are shown. Finally, the applications on the 1783 Scilla event are shown and discussed.

Cleavage of IgG1 and IgG3 by gingipain K from Porphyromonas gingivalis may compromise host defense in progressive periodontitis

Degradation of immunoglobulins is an effective strategy of bacteria to evade the immune system. We have tested whether human IgG is a substrate for gingipain K of Porphyromonas gingivalis and found that the enzyme can hydrolyze subclass 1 and 3 of human IgG. The heavy chain of IgG(1) was cleaved at a single site within the hinge region, generating Fab and Fc fragments. IgG(3) was also cleaved within the heavy chain, but at several sites around the CH2 region. Investigation of the enzyme kinetics of IgG proteolysis by gingipain K, using FPLC- and isothermal titration calorimetry-based assays followed by Hill plots, revealed non-Michaelis-Menten kinetics involving a mechanism of positive cooperativity. In ex vivo studies, it was shown that gingipain K retained its IgG hydrolyzing activity in human plasma despite the high content of natural protease inhibitors; that IgG(1) cleavage products were detected in gingival crevicular fluid samples from patients with severe periodontitis; and that gingipain K treatment of serum samples from patients with high antibody titers against P. gingivalis significantly hindered opsonin-dependent phagocytosis of clinical isolates of P. gingivalis by neutrophils. Altogether, these findings underline a biological function of gingipain K as an IgG protease of pathophysiological importance.

Characterization of Molecular Determinants of the Conformational Stability of Macrophage Migration Inhibitory Factor: Leucine 46 Hydrophobic Pocket

Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the ��-strand ��3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF���s trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state.

Total synthesis of (-)-zampanolide and structure-activity relationship studies on (-)-dactylolide derivatives

A new total synthesis of the marine macrolide (-)-zampanolide (1) and the structurally and stereochemically related non-natural levorotatory enantiomer of (+)-dactylolide (2), that is, ent-2, has been developed. The synthesis features a high-yielding, selective intramolecular Horner-Wadsworth-Emmons (HWE) reaction to close the 20-membered macrolactone ring of 1 and ent-2. The ��-keto phosphonate/aldehyde precursor for the ring-closure reaction was obtained by esterification of a ��-diethylphosphono carboxylic acid fragment and a secondary alcohol fragment incorporating the THP ring that is embedded in the macrocyclic core structure of 1 and ent-2. THP ring formation was accomplished through a segment coupling Prins-type cyclization. Employing the same overall strategy, 13-desmethylene-ent-2 as well as the monocyclic desTHP derivatives of 1 and ent-2 were prepared. Synthetic 1 inhibited human cancer cell growth in vitro with nM IC(50) values, while ent-2, which lacks the diene-containing hemiaminal-linked side chain of 1, is 25- to 260-fold less active. 13-Desmethylene-ent-2 as well as the reduced versions of ent-2 and 13-desmethylene-ent-2 all showed similar cellular activity as ent-2 itself. The same activity level was attained by the monocyclic desTHP derivative of 1. Oxidation of the aldehyde functionality of ent-2 gave a carboxylic acid that was converted into the corresponding N-hexyl amide. The latter showed only ��M antiproliferative activity, thus being several hundred-fold less potent than 1.

A fluorescently labeled dendronized polymer-enzyme conjugate carrying multiple copies of two different types of active enzymes

A hybrid structure of a synthetic dendronized polymer, two different types of enzymes (superoxide dismutase and horseradish peroxidase), and a fluorescent dye (fluorescein) was synthesized. Thereby, a single polymer chain carried multiple copies of the two enzymes and the fluorescein. The entire attachment chemistry is based on UV/vis-quantifiable bis-aryl hydrazone bond formation that allows direct quantification of bound molecules: 60 superoxide dismutase, 120 horseradish peroxidase, and 20 fluorescein molecules on an average polymer chain of 2000 repeating units. To obtain other enzyme ratios the experimental conditions were altered accordingly. Moreover, it could be shown that both enzymes remained fully active and catalyzed a two-step cascade reaction.

Molecular characterization of the equine collagen, type IX, alpha 2 (COL9A2) gene on horse chromosome 2p16-->p15

The mammalian collagen, type IX, alpha 2 gene (COL9A2) encodes the alpha-2 chain of type IX collagen and is located on horse chromosome 2p16-->p14 harbouring a quantitative trait locus for osteochondrosis. We isolated a bacterial artificial chromosome (BAC) clone containing the equine COL9A2 gene and determined the complete genomic sequence of this gene. Cloning and characterization of equine COL9A2 revealed that the equine gene consists of 32 exons spanning approximately 15 kb. The COL9A2 transcript encodes a single protein of 688 amino acids. Thirty two single nucleotide polymorphisms (SNPs) equally distributed in the gene were detected in a mutation scan of eight unrelated Hanoverian warmblood stallions, including one SNP that affects the amino acid sequence of COL9A2. Comparative analyses between horse, human, mouse and rat indicate that the chromosomal location of equine COL9A2 is in agreement with known chromosomal synteny relationships. The comparison of the gene structure and transcript revealed a high degree of conservation towards the other mammalian COL9A2 genes. We chose three informative SNPs for association and linkage disequilibrium tests in three to five paternal half-sib families of Hanoverian warmblood horses consisting of 44 to 75 genotyped animals. The test statistics did not reach the significance threshold of 5% and so we could not show an association of COL9A2 with equine osteochondrosis.

Molecular characterization of the porcine DNAL4 gene

The DNAL4 (dynein, axonemal, light polypeptide 4) gene encodes a light chain of dynein. Dyneins are motor proteins that contribute to axonal transport. Cloning and characterization of the porcine DNAL4 revealed a conserved organization with respect to the human ortholog. The porcine DNAL4 gene consists of 4 exons and codes for a peptide of 105 amino acids. The porcine DNAL4 gene is located on SSC5p15. Analysis of the naturally occurring variation of the DNAL4 gene in pigs from the Pi��train und Duroc breeds revealed five SNPs in non-coding regions of the gene.

Contact system activation in human sepsis - 47kD HK, a marker of sepsis severity?

AIM: This pilot study seeks to determine whether contact system activation (CSA) occurs in human sepsis patients and to characterise blood levels of the 47kD light chain of high-molecular weight kininogen (47kD HK). METHODS: Six consecutive patients with clinical suspicion of sepsis were evaluated on days 1, 2, 3 and 6-8 for 47kD HK blood levels expressed in U/ml of whole blood and as percent of total HK. 47kD HK was measured in whole blood by quantitative immunoblot analysis. RESULTS: On study day 1 or 2, analysis of 47kD HK in U/ml of whole blood identified CSA in 3/6 patients.When 47kD HK levels were expressed as percent of total HK, 4/6 patients were identified with CSA before day 3. The degree of CSA as assayed by the presence of 47kD HK correlated with the severity of the systemic inflammatory syndrome (SIRS), i.e. mean CSA increased progressively from basal levels in healthy controls (0.08 U/ml or 10.4%) to patients without SIRS (0.10 U/ml or 15.1%), to patients with sepsis (0.12 U/ml or 15.0%), and finally to patients in a combined category of severe sepsis and septic shock (0.13 U/ml or 17.4%). CONCLUSION: CSA, defined by increased 47kD HK, occurred early on in the course of sepsis in a subset of sepsis patients. 47kD HK levels, an indicator of bradykinin release, correlated with sepsis severity. Future larger studies will need to evaluate the role of 47kD HK as a biomarker for both prognosis and treatment response in human sepsis..

Semiotic Analysis in the Study of Social Work

Research and professional practices have the joint aim of re-structuring the preconceived notions of reality. They both want to gain the understanding about social reality. Social workers use their professional competence in order to grasp the reality of their clients, while researchers��� pursuit is to open the secrecies of the research material. Development and research are now so intertwined and inherent in almost all professional practices that making distinctions between practising, developing and researching has become difficult and in many aspects irrelevant. Moving towards research-based practices is possible and it is easily applied within the framework of the qualitative research approach (Dominelli 2005, 235; Humphries 2005, 280). Social work can be understood as acts and speech acts crisscrossing between social workers and clients. When trying to catch the verbal and non-verbal hints of each others��� behaviour, the actors have to do a lot of interpretations in a more or less uncertain mental landscape. Our point of departure is the idea that the study of social work practices requires tools which effectively reveal the internal complexity of social work (see, for example, Adams & Dominelli & Payne 2005, 294 ��� 295). The boom of qualitative research methodologies in recent decades is associated with much profound the rupture in humanities, which is called the linguistic turn (Rorty 1967). The idea that language is not transparently mediating our perceptions and thoughts about reality, but on the contrary it constitutes it was new and even confusing to many social scientists. Nowadays we have got used to read research reports which have applied different branches of discursive analyses or narratologic or semiotic approaches. Although differences are sophisticated between those orientations they share the idea of the predominance of language. Despite the lively research work of today���s social work and the research-minded atmosphere of social work practice, semiotics has rarely applied in social work research. However, social work as a communicative practice concerns symbols, metaphors and all kinds of the representative structures of language. Those items are at the core of semiotics, the science of signs, and the science which examines people using signs in their mutual interaction and their endeavours to make the sense of the world they live in, their semiosis. When thinking of the practice of social work and doing the research of it, a number of interpretational levels ought to be passed before reaching the research phase in social work. First of all, social workers have to interpret their clients��� situations, which will be recorded in the files. In some very rare cases those past situations will be reflected in discussions or perhaps interviews or put under the scrutiny of some researcher in the future. Each and every new observation adds its own flavour to the mixture of meanings. Social workers have combined their observations with previous experience and professional knowledge, furthermore, the situation on hand also influences the reactions. In addition, the interpretations made by social workers over the course of their daily working routines are never limited to being part of the personal process of the social worker, but are also always inherently cultural. The work aiming at social change is defined by the presence of an initial situation, a specific goal, and the means and ways of achieving it, which are ��� or which should be ��� agreed upon by the social worker and the client in situation which is unique and at the same time socially-driven. Because of the inherent plot-based nature of social work, the practices related to it can be analysed as stories (see Dominelli 2005, 234), given, of course, that they are signifying and told by someone. The research of the practices is concentrating on impressions, perceptions, judgements, accounts, documents etc. All these multifarious elements can be scrutinized as textual corpora, but not whatever textual material. In semiotic analysis, the material studied is characterised as verbal or textual and loaded with meanings. We present a contribution of research methodology, semiotic analysis, which has to our mind at least implicitly references to the social work practices. Our examples of semiotic interpretation have been picked up from our dissertations (Laine 2005; Saurama 2002). The data are official documents from the archives of a child welfare agency and transcriptions of the interviews of shelter employees. These data can be defined as stories told by the social workers of what they have seen and felt. The official documents present only fragmentations and they are often written in passive form. (Saurama 2002, 70.) The interviews carried out in the shelters can be described as stories where the narrators are more familiar and known. The material is characterised by the interaction between the interviewer and interviewee. The levels of the story and the telling of the story become apparent when interviews or documents are examined with the use of semiotic tools. The roots of semiotic interpretation can be found in three different branches; the American pragmatism, Saussurean linguistics in Paris and the so called formalism in Moscow and Tartu; however in this paper we are engaged with the so called Parisian School of semiology which prominent figure was A. J. Greimas. The Finnish sociologists Pekka Sulkunen and Jukka T��rr��nen (1997a; 1997b) have further developed the ideas of Greimas in their studies on socio-semiotics, and we lean on their ideas. In semiotics social reality is conceived as a relationship between subjects, observations, and interpretations and it is seen mediated by natural language which is the most common sign system among human beings (Mounin 1985; de Saussure 2006; Sebeok 1986). Signification is an act of associating an abstract context (signified) to some physical instrument (signifier). These two elements together form the basic concept, the ���sign���, which never constitutes any kind of meaning alone. The meaning will be comprised in a distinction process where signs are being related to other signs. In this chain of signs, the meaning becomes diverged from reality. (Greimas 1980, 28; Potter 1996, 70; de Saussure 2006, 46-48.) One interpretative tool is to think of speech as a surface under which deep structures ��� i.e. values and norms ��� exist (Greimas & Courtes 1982; Greimas 1987). To our mind semiotics is very much about playing with two different levels of text: the syntagmatic surface which is more or less faithful to the grammar, and the paradigmatic, semantic structure of values and norms hidden in the deeper meanings of interpretations. Semiotic analysis deals precisely with the level of meaning which exists under the surface, but the only way to reach those meanings is through the textual level, the written or spoken text. That is why the tools are needed. In our studies, we have used the semiotic square and the actant analysis. The former is based on the distinctions and the categorisations of meanings, and the latter on opening the plotting of narratives in order to reach the value structures.

Immune response of horses to vaccination with the recombinant Hc domain of botulinum neurotoxin types C and D

Botulinum neurotoxins, predominantly serotypes C and D, cause equine botulism through forage poisoning. The C-terminal part of the heavy chain of botulinum neurotoxin types C and D (HcBoNT/C and D) was expressed in Escherichia coli and evaluated as a recombinant mono- and bivalent vaccine in twelve horses in comparison to a commercially available toxoid vaccine. A three-dose subcutaneous immunization of adult horses elicited robust serum antibody response in an ELISA using the immunogen as a capture antigen. Immune sera showed dose-dependent high potency in neutralizing specifically the active BoNT/C and D in the mouse protection assay. The aluminium hydroxide based mono- and bivalent recombinant HcBoNT/C and D vaccines were characterized by good compatibility and the ability to elicit protective antibody titers similar or superior to the commercially available toxoid vaccine.

Effects of oligotrophication on primary production in peri-alpine lakes

During the second half of the 20th century untreated sewage released from housing and industry into natural waters led to a degradation of many freshwater lakes and reservoirs worldwide. In order to mitigate eutrophication, wastewater treatment plants, including Fe-induced phosphorus precipitation, were implemented throughout the industrialized world, leading to reoligotrophication in many freshwater lakes. To understand and assess the effects of reoligotrophication on primary productivity, we analyzed 28 years of 14C assimilation rates, as well as other biotic and abiotic parameters, such as global radiation, nutrient concentrations and plankton densities in peri-alpine Lake Lucerne, Switzerland. Using a simple productivity-light relationship, we estimated continuous primary production and discussed the relation between productivity and observed limnological parameters. Furthermore, we assessed the uncertainty of our modeling approach based on monthly 14C assimilation measurements using Monte Carlo simulations. Results confirm that monthly sampling of productivity is sufficient for identifying long-term trends in productivity and that conservation management has successfully improved water quality during the past three decades via reducing nutrients and primary production in the lake. However, even though nutrient concentrations have remained constant in recent years, annual primary production varies significantly from year to year. Despite the fact that nutrient concentrations have decreased by more than an order of magnitude, primary production has decreased only slightly. These results suggest that primary production correlates well to nutrients availability but meteorological conditions lead to interannual variability regardless of the trophic status of the lake. Accordingly, in oligotrophic freshwaters meteorological forcing may reduce productivity impacting on the entire food chain of the ecosystem.

Alarm systems and catastrophes from a diverse point of view

Using a chain of urns, we build a Bayesian nonparametric alarm system to predict catastrophic events, such as epidemics, black outs, etc. Differently from other alarm systems in the literature, our model is constantly updated on the basis of the available information, according to the Bayesian paradigm. The papers contains both theoretical and empirical results. In particular, we test our alarm system on a well-known time series of sunspots.

Using state variables to model the response of tumour cells to radiation and heat: a novel multi-hit-repair approach

In order to overcome the limitations of the linear-quadratic model and include synergistic effects of heat and radiation, a novel radiobiological model is proposed. The model is based on a chain of cell populations which are characterized by the number of radiation induced damages (hits). Cells can shift downward along the chain by collecting hits and upward by a repair process. The repair process is governed by a repair probability which depends upon state variables used for a simplistic description of the impact of heat and radiation upon repair proteins. Based on the parameters used, populations up to 4-5 hits are relevant for the calculation of the survival. The model describes intuitively the mathematical behaviour of apoptotic and nonapoptotic cell death. Linear-quadratic-linear behaviour of the logarithmic cell survival, fractionation, and (with one exception) the dose rate dependencies are described correctly. The model covers the time gap dependence of the synergistic cell killing due to combined application of heat and radiation, but further validation of the proposed approach based on experimental data is needed. However, the model offers a work bench for testing different biological concepts of damage induction, repair, and statistical approaches for calculating the variables of state.

Pharmacological characterization of nipecotic acid ethyl ester: A novel cholinergic muscarinic agonist

The aim of this dissertation was to examine the hypothesis that (R)-nipecotic acid ethyl ester ((R)-NAEE) is a cholinergic agonist that is selective for a particular subclass (M$\sb1$ or M$\sb2$) of muscarinic receptors.^ Ligand binding studies indicated that like cholinergic agonists (R)-NAEE selectively interacts with rat heart (M$\sb2$) and brain (M$\sb1$) muscarinic binding sites. Physiological studies revealed that unlike cholinergic agonists (R)-NAEE stimulated only those responses coupled to M$\sb2$ muscarinic receptors (acid secretion, negative inotropic response, smooth muscle contraction). Moreover, in rat brain (R)-NAEE differentiated between M$\sb2$ receptors negatively coupled to adenylate cyclase activity and M$\sb1$ receptors mediating PI turnover, being a weak competitive antagonist at these latter sites. In isolated rat gastric mucosal cells (R)-NAEE also differentiated between two M$\sb2$ coupled responses where it potentiated acid secretion but could not stimulate PI turnover. Atropine, a selective antimuscarinic agent, competitively antagonized all agonist effects of (R)-NAEE.^ Unlike (R)-NAEE, the muscarinic agonist arecoline, which is structurally similar to (R)-NAEE, stimulates both M$\sb1$ and M$\sb2$ receptors. Structure activity studies revealed that saturation of the piperidine ring and the length of the ester side chain of (R)-NAEE are the most important determinants for both M$\sb2$ efficacy and selectivity.^ The results of this dissertation establish that (R)-NAEE is a cholinergic muscarinic receptor agonist that displays greater efficacy at M$\sb2$ than at M$\sb1$ receptors, being a weak antagonist at the M$\sb1$ site. With such selectivity, (R)-NAEE may be regarded as a prototype for a unique class of cholinergic muscarinic M$\sb2$ receptor agonists. Because of these unique properties, (R)-NAEE should be useful in the further characterization of muscarinic receptors, and could lead to the development of a new class of therapeutic agents. ^