Structure of a hydroxyl radical induced cross-link of thymine and tyrosine

DNA-protein cross-links are formed when living cells or isolated chromatin is exposed to ionizing radiation. Little is known about the actual cross-linked products of DNA and proteins. In this work, a novel hydroxyl radical induced cross-link of thymine and tyrosine has been isolated along with a tyrosine dimer by high-performance liquid chromatography of aqueous mixtures of tyrosine and thymine that had been exposed to hydroxyl radicals generated by ionizing radiation. The isolated compounds have been examined by gas chromatography-mass spectrometry, high-resolution mass spectrometry, and 1H and 13C nuclear magnetic resonance spectroscopy. The structure of the thymine-tyrosine cross-link has been identified as the product from the formation of a covalent bond between the methyl group of the thymine and carbon 3 of the tyrosine ring. In addition, the 3,3' tyrosine dimer was isolated and characterized. The mechanism of the formation of these compounds is discussed. This work presents the first complete chemical characterization of a hydroxyl radical induced DNA base-amino acid cross-link.     

The influence of limb alignment and transfemoral amputation technique on muscle capacity during gait

Many factors influence successful outcomes following transfemoral amputation. One factor is surgical technique. In this study, the influence of limb alignment and surgical technique on a muscle’s capacity to generate force was examined using musculoskeletal modeling. Non-amputee and transfemoral amputee models were analyzed while hip adduction, femur length, and reattached muscle wrap position, tension and stabilization technique were systematically varied. With muscle tension preserved, wrap position and femur length had little influence on muscle capacity. However, limb alignment, muscle tension and stabilization technique notably influenced muscle capacity. Overall, myodesis stabilization provided greater muscle balance and function than myoplasty stabilization.     

Pollen–plant–climate relationships in sub-Saharan Africa

Aim  To demonstrate that incorporating the bioclimatic range of possible contributor plants leads to improved accuracy in interpreting the palaeoclimatic record of taxonomically complex pollen types.
Location  North Tropical Africa.
Methods  The geographical ranges of selected African plants were extracted from the literature and geo-referenced. These plant ranges were compared with the pollen percentages obtained from a network of surface sediments. Climate-response surfaces were graphed for each pollen taxon and each corresponding plant species.
Results  Several patterns can be identified, including taxa for which the pollen and plant distributions coincide, and others where the range limits diverge. Some pollen types display a reduced climate range compared with that of the corresponding plant species, due to low pollen production and/or dispersal. For other taxa, corresponding to high pollen producers such as pioneer taxa, pollen types display a larger climatic envelope than that of the corresponding plants. The number of species contained in a pollen taxon is an important factor, as the botanical species included in a taxon may have different geographical and climate distributions.
Main conclusions  The comparison between pollen and plant distributions is an essential step towards more precise vegetation and climate reconstructions in Africa, as it identifies taxa that have a high correspondence between pollen and plant distribution patterns. Our method is a useful tool to reassess biome reconstructions in Africa and to characterize accurately the vegetation and climate conditions at a regional scale, from pollen data.     

Interleukin 2 and concanavalin A stimulate interferon-gamma production in a murine cytolytic T cell clone by different pathways

We identified a variant murine cytolytic T lymphocyte (CTL) clone which, in contrast to the parent clone and all other murine T cell populations tested, was found to have acquired spontaneously the ability to produce interferon-gamma (IFN-gamma) in response to recombinant interleukin 2 (rIL-2). IFN-gamma production in response to concanavalin A (Con A), which was characteristic of all T cell populations tested, was preserved in this variant. The IFN produced by the variant in response to either stimulus was active in both a macrophage-activating factor assay and an anti-viral assay. Both activities induced by either stimulus could be blocked by monoclonal anti-IFN-gamma antibodies. Upon Northern blot analysis using an IFN-gamma-specific cDNA probe, the IFN-gamma RNA isolated from variant cells stimulated with Con A or IL-2 were found to migrate equivalently. The unusual pattern of responsiveness in this variant CTL was exploited to compare the mechanisms involved in induction of IFN-gamma production by Con A or IL-2. Striking differences were observed. Unlike IFN-gamma production induced by Con A, IFN-gamma production induced by IL-2 was not accompanied by an elevation of intracellular Ca2+ levels, did not require physiologic extracellular Ca2+ levels, and was not inhibited by the immunosuppressive agent cyclosporin A. Thus, in this variant CTL clone, conditions that have ordinarily been associated in an obligate manner with lymphokine gene expression were found instead to be related to the specific mode of stimulation.     

Codon usage in the vertebrate hemoglobins and its implications

A study of codon usage in vertebrate hemoglobins revealed an evolutionary trend toward elevated numbers of CpG codon boundary pairs in mammalian hemoglobin alpha genes. Selection for CpG codon boundaries countering the generally observed CpG suppression is strongly suggested by these data. These observations parallel recently published experimental results that indicate that constitutive expression of the human alpha-globin gene appears to be determined by regulatory information encoded within the structural gene. The possibility is raised that, in the absence of selection, CpG decay can be used to date the evolutionary origin of a mammalian alpha pseudogene from its active alpha gene.      

Evidence implicating utilization of different T cell receptor-associated signaling pathways by TH1 and TH2 clones

We have reported recently that high concentrations of anti-CD3 mAb inhibited IL-2-dependent proliferation of TH1 but not TH2 clones. The selective inhibitory effect on TH1 clones suggested that the two helper T lymphocyte subsets might utilize different TCR-associated signal transduction mechanisms. In the present study, we demonstrate that this distinction was not due to a gross difference in the level of TCR expression by TH1 and TH2 clones. Inhibition of TH1 proliferation by anti-CD3 mAb appeared to depend on calcium for maximal effect, suggesting that a substantial elevation of intracellular free calcium concentration ([Ca2+]i) might not occur after ligation of the TCR complex of TH2 clones. Calcium ionophore inhibited IL-2-dependent proliferation of both subsets, suggesting that receptor/ligand systems which stimulate elevated [Ca2+]i would be expected to inhibit proliferation. Although elevated [Ca2+]i and generation of inositol phosphates were readily detected in TH1 clones, these second messengers were not detected following stimulation of TH2 clones via the TCR complex. In addition, lymphokine production by TH1 clones was more sensitive to inhibition by cholera toxin, 8-bromoadenosine 3':5'-cyclic monophosphate, and cyclosporin A than was lymphokine production by TH2 clones. Collectively, these results suggest that TH1 and TH2 clones utilize distinct TCR-associated signal transduction mechanisms for lymphokine gene expression. The difference in signaling mechanisms suggests a potential pharmacologic target for intervention in situations where inappropriate activation of TH1 or TH2 cells occurs in vivo.     

Damage to the bases in DNA induced by hydrogen peroxide and ferric ion chelates

Incubation of a number of ferric ion chelates with H2O2 at pH 7.4 generated a reactive species able to produce chemical modifications of the bases in DNA that are very similar to those produced in DNA by the hypoxanthine/xanthine oxidase system (Aruoma, O.I., Halliwell, B., and Dizdaroglu, M. (1989) J. Biol. Chem. 264, 13024-13028). Products were identified and quantitated by the use of gas chromatography-mass spectrometry with selected-ion monitoring. Compared with other complexes used, ferric ion-nitrilotriacetic acid produced by far the largest amount of the base products. Typical hydroxyl radical scavengers and superoxide dismutase provided significant decreases in the yields of the products. On this basis, it is proposed that ferric ion complexes react with H2O2 to produce hydroxyl radical; this was also shown using the deoxyribose assay. Inhibition of product formation by superoxide dismutase suggests the involvement of superoxide radical in this reaction. It is likely that hydroxyl radical generated by reaction of the ferric ion-nitrilotriacetic acid complex with H2O2 contributes to the carcinogenicity and nephrotoxicity associated with this chelating agent.     

Neutrophils-induced increase of adenosine triphosphate depletion in rat neonatal cardiac myocytes with impaired energy metabolism.

Isolated, cultured rat neonatal cardiac myocytes were placed in medium supplemented with mitochondrial respiratory inhibitor potassium cyanide which caused a rapid adenosine triphosphate (ATP) depletion. These myocytes with the impaired energy metabolism ("hypoxia-like state") were exposed to unstimulated human neutrophils. Effect of human neutrophils on the myocytes in the "hypoxia-like state" was quantified as a total change in the amount of ATP in cardiac cells. After 5 hours of incubation of neutrophils with the myocytes in the "hypoxia-like state" an additional decrease (of 50 per cent) in ATP content was observed. Since catalase (which destroys hydrogen peroxide) prevented the further decline in ATP level in the myocytes with impaired energy metabolism, it seem that hydrogen peroxide and possibly their products are responsible for this effect. These results suggest that unstimulated human neutrophils after activation by the contact with injured cardiac cells caused further decrease of ATP level in target cells.     

Mechanistic studies of ionizing radiation and oxidative mutagenesis: genetic effects of a single 8-hydroxyguanine (7-hydro-8-oxoguanine) residue inserted at a unique site in a viral genome

T4 RNA ligase was used to construct a deoxypentanucleotide containing a single 8-hydroxyguanine (7-hydro-8-oxoguanine; G8-OH) residue, which is one of the putatively mutagenic DNA adducts produced by oxidants and ionizing radiation. The pentamer d(GCTAG8-OH)p was prepared by the ligation of a chemically synthesized acceptor molecule, d(GCTA), to an adducted donor, 8-hydroxy-2'-deoxyguanosine 5',3'-bisphosphate. The acceptor was efficiently converted to the reaction product (greater than 95%), and the final product yield was 50%. Following 3'-dephosphorylation, the pentamer was characterized by UV spectroscopy, by high-pressure liquid chromatography, and by gas chromatography-mass spectrometry of the nucleosides released by enzymatic hydrolysis. Both d(GCTAG8-OH) and an unmodified control were 5'-phosphorylated by using [gamma -32P]ATP and incorporated covalently by DNA ligase into a five-base gap at a unique NheI restriction site in the otherwise duplex genome of an M13mp19 derivative. The ligation product contained G8-OH at the 3' residue of an in-frame amber codon (5'-TAG-3') (genome position 6276) of the phage lacZ alpha gene. The adduct was part of a nonsense codon in a unique restriction site in order to facilitate the identification and selection of mutants generated by the replication of the modified genome in Escherichia coli. Both control and adducted pentamers ligated into the genome at 50% of the maximum theoretical efficiency, and nearly all (approximately 90%) of the site-specifically adducted products possessed pentanucleotides that were covalently linked at both 5' and 3' termini. The G8-OH lesion in the NheI site inhibited the cleavage of the site by a 200-fold excess of NheI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Medial Temporal Lobe Roles in Human Path Integration

Path integration is a process in which observers derive their location by integrating self-motion signals along their locomotion trajectory. Although the medial temporal lobe (MTL) is thought to take part in path integration, the scope of its role for path integration remains unclear. To address this issue, we administered a variety of tasks involving path integration and other related processes to a group of neurosurgical patients whose MTL was unilaterally resected as therapy for epilepsy. These patients were unimpaired relative to neurologically intact controls in many tasks that required integration of various kinds of sensory self-motion information. However, the same patients (especially those who had lesions in the right hemisphere) walked farther than the controls when attempting to walk without vision to a previewed target. Importantly, this task was unique in our test battery in that it allowed participants to form a mental representation of the target location and anticipate their upcoming walking trajectory before they began moving. Thus, these results put forth a new idea that the role of MTL structures for human path integration may stem from their participation in predicting the consequences of one''s locomotor actions. The strengths of this new theoretical viewpoint are discussed.