M. Cristina Vega

On the reliability of MINDO/3, MNDO and AM1 radical geometries for the computation of INDO electron spin resonance hyperfine coupling constants

Abstract Electron spin resonance hyperfine coupling constants (hfcs) for H and C nuclei of 21 radicals have been calculated using the INDO method from MINDO/3, MNDO and AM1 optimized geometries. In all cases, the correlation coefficients are better than those in the literature using standard geometries, although a dependence of hfc values on the geometry is observed. MINDO/3 geometries provide the best results for the H nucleus, whereas MNDO and AM1 geometries give very accurate INDO hfc values for the C nucleus.

A MNDO and AM1 quantum chemical study of the reaction mechanism of the McLafferty type rearrangement in the butanal radical cation

Abstract The stepwise and concerted reaction mechanisms of the McLafferty type rearrangement in the butanal radical cation have been studied by both MNDO and AM1 (Dewars semi-empirical methods). The calculations indicate a favored concerted reaction mechanism. Results obtained from the study of this reaction at semiempirical level were consistent with both ab initio and experimental data.

Nucleases of Metallo-Beta-Lactamase and Protein Phosphatase Families in DNA Repair

All living organisms must struggle to maintain genomic integrity and long-term stability in the face of the lesions that are constantly inflicted upon the genome by environmental factors, e.g., genotoxic chemicals, UV light, ionizing radiation (IR), and endogenous factors, e.g., during DNA replication. These various DNA lesions (or injuries) encompass a bewildering array of chemical and physical modifications to the DNA structure that must be repaired to preserve the faithful maintenance of the genome. A prevalent class of DNA lesion consists of a break across both DNA strands, termed double strand break (DSB) (Fig. 1 and Table 1). Only of endogenous origin, about 50 DSBs have been calculated to occur per human cell division (Vilenchik and Knudson 2003). Many of these DSBs are generated by IR, reactive oxygen species, and DNA replication across a nick (Ma, J.L. et al. 2003). If left unrepaired, DSBs can cause dire effects such as gene loss during cell division, chromosomal translocations, increased mutation rates, and carcinogenesis (Khanna and Jackson 2001). The various cellular mechanisms that are collectively referred to as DNA repair include DNA damage detection (or sensing), binding and recruitment of specialized protein complex machinery to the site of damage, signaling, initiation of repair, repair, and resolution of the lesion (Fig. 1). Central to all DNA repair processes are nucleases, enzymes and enzyme complexes that can cleave DNA either in a sugar specific fashion (e.g., DNA and RNA nucleases) or in a sugar unspecific fashion (Marti and Fleck 2004). Nucleases can be further divided into exonucleases, which remove nucleotides from a free 5’ or 3’ end, and endonucleases, which hydrolyze internal phosphodiester bonds without the requirement for a free end. DNA nucleases, which can cleave single stranded (ss) or double stranded (ds) DNA, cleave a phosphodiester bond between a deoxyribose and a phosphate group, thus producing one cleavage product with a 5’ terminal phosphate group and another product with a 3’ terminal hydroxyl group. Two kinds of DNA lesions, double strand breaks (DSBs) and interstrand crosslinks (ICLs) (Fig. 1), are significantly dependent on the timely action of DNA nucleases, since the initiating step in the repair pathways of DSBs and ICLs often consists of an exonucleolytic or endonucleolytic cleavage that exposes the substrate for the next DNA repair activity. Without the action of a nuclease, the DNA lesion would stay unrepaired because of chemically inaccessible or sterically blocked DNA intermediates. Therefore, nucleases are an integral part of the cellular mechanisms that have evolved to handle DNA damage. Indeed,

Complement in leucocyte development and function

The complement system is a critical part of vertebrate innate immunity and, as has become increasingly clear over the last decade, also of adaptive immunity. Complement comprises about 20 soluble components, many of which are initially produced as inactive proteases, and several membrane components that prevent self-damage or potentiate complement function. Microbial polysaccharides not present in mammals, immune complexes and apoptotic cell debris can all activate complement by starting a cascade-like process, with each protease activating the next one in the pathway. As a result of complement activation, pathogens may be completely covered by complement-activated fragments (a process termed opsonization), stimulating their phagocytosis by macrophages and neutrophils. Opsonization can also trigger direct pathogen lysis by complement-mediated insertion of the membrane-attack complex. At the same time, complement activation releases small peptide fragments, or anaphylatoxins, that recruit macrophages and neutrophils to sites of infection through direct chemotaxis or indirectly, by activation of local mast cells. Thus, the three-pronged action of complement (enhancing phagocytosis, lysing pathogens and recruiting leucocytes while maintaining innate self-tolerance) is vital for survival. Complement deficiency or dysfunction can give rise to infections (immunodeficiency) or self-damage (autoimmunity, inflammation) in susceptible tissues such as red blood cells, kidney, brain or retina. Research in complement-targeted therapies is an expanding field that has already improved the prognosis of severe diseases such as atypical Hemolytic Uremic Syndrome (aHUS) or Paroxysmal Nocturnal Hemoglobinuria (PNH). A thorough account of the most important methods used in the clinic to assess the complement system of healthy and diseased individuals is central to understand the potential role of complement in diagnostics and therapeutics [1]. The distinct pathogen-fighting functions of complement provide a formidable barrier for most pathogens, which have evolved numerous immunoevasion mechanisms in an attempt to hide and escape from or subvert the complement system [2]. Complement activation also results in cross-talk with the adaptive immune system. Indeed, it is currently appreciated that a full-fledged cellular immune response de

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N 6 -threonylcarbamoyladenosine TcdA Function

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of tRNA(UUU) to TcdA, an N6-threonylcarbamoyladenosine (t6A) dehydratase, which cyclizes the threonylcarbamoyl side chain attached to A37 in the anticodon stem loop (ASL) of tRNAs to cyclic t6A (ct6A). Transcription of the synthetic gene encoding tRNA(UUU) is induced in E. coli with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and the cells containing tRNA are harvested 24 h post-induction. The RNA fraction is purified using the acid phenol extraction method. Pure tRNA is obtained by a gel filtration chromatography that efficiently separates the small-sized tRNA molecules from larger intact or fragmented nucleic acids. To analyze TcdA binding to tRNA(UUU), TcdA is mixed with tRNA(UUU) and separated on a native agarose gel at 4 °C. The free tRNA(UUU) migrates faster, while the TcdA-tRNA(UUU) complexes undergo a mobility retardation that can be observed upon staining of the gel. We demonstrate that TcdA is a tRNA(UUU)-binding enzyme. This gel retardation assay can be used to study TcdA mutants and the effects of additives and other proteins on binding.

Mechanism of deoxygenation of butyraldehyde by atomic carbon

Abstract The reaction mechanism of the deoxygenation of butyraldehyde by atomic carbon has been studied using the AM1 method. The “end-on” mechanism is favoured over the “sideways” one. However, an alternative mechanism which is a mixing of the “end-on” and “sideways” mechanisms is preferred from an energetic point of view. This new mechanism is consistent with the experimental evidence reported in the literature.

A comparative study of the performance of Dewar's semiempirical methods to compute electron spin resonance hyperfine coupling constants

Abstract The performance of Dewars semiempirical methods MINDO/3, MNDO and AM1 in computing electron spin resonance spectroscopy hyperfine coupling constants (hfcs) is assessed. A set of 32 molecules including neutral, cation and anion radicals were studied and the H, N and C nuclei were investigated. The linear relationship between hfcs and electronic spin densities was analysed and the different correlation and dispersion coefficients used to assess the performance of the methods. Using the optimized geometries within each method, only MINDO/3 provides reliable results for the three nuclei investigated. AM1 and MNDO provide reasonable results only for N and C. However, when experimental geometries are used, reasonable hfcs are obtained with the three methods for all the nuclei investigated.