Chemical 'omics' approaches for understanding protein cysteine oxidation in biology

Oxidative cysteine modifications have emerged as a central mechanism for dynamic post-translational regulation of all major protein classes and correlate with many disease states. Elucidating the precise roles of cysteine oxidation in physiology and pathology presents a major challenge. This article reviews the current, targeted proteomic strategies that are available to detect and quantify cysteine oxidation. A number of indirect methods have been developed to monitor changes in the redox state of cysteines, with the majority relying on the loss of reactivity with thiol-modifying reagents or restoration of labeling by reducing agents. Recent advances in chemical biology allow for the direct detection of specific cysteine oxoforms based on their distinct chemical attributes. In addition, new chemical reporters of cysteine oxidation have enabled in situ detection of labile modifications and improved proteomic analysis of redox-regulated proteins. Progress in the field of redox proteomics should advance our knowledge of regulatory mechanisms that involve oxidation of cysteine residues and lead to a better understanding of oxidative biochemistry in health and disease.     

Elasticity and stress relaxation of a very small vocal fold

Across mammals many vocal sounds are produced by airflow induced vocal fold oscillation. We tested the hypothesis that stress-strain and stress-relaxation behavior of rat vocal folds can be used to predict the fundamental frequency range of the species' vocal repertoire. In a first approximation vocal fold oscillation has been modeled by the string model but it is not known whether this concept equally applies to large and small species. The shorter the vocal fold, the more the ideal string law may underestimate normal mode frequencies. To accommodate the very small size of the tissue specimen, a custom-built miniaturized tensile test apparatus was developed. Tissue properties of 6 male rat vocal folds were measured. Rat vocal folds demonstrated the typical linear stress-strain behavior in the low strain region and an exponential stress response at strains larger than about 40%. Approximating the rat's vocal fold oscillation with the string model suggests that fundamental frequencies up to about 6 kHz can be produced, which agrees with frequencies reported for audible rat vocalization. Individual differences and time-dependent changes in the tissue properties parallel findings in other species, and are interpreted as universal features of the laryngeal sound source.     

Alloimmune response results in expansion of autoreactive donor CD4+ T cells in transplants that can mediate chronic graft-versus-host disease

Chronic graft-versus-host disease (cGVHD) is considered an autoimmune-like disease mediated by donor CD4(+) T cells, but the origin of the autoreactive T cells is still controversial. In this article, we report that the transplantation of DBA/2 donor spleen cells into thymectomized MHC-matched allogeneic BALB/c recipients induced autoimmune-like cGVHD, although not in control syngeneic DBA/2 recipients. The donor-type CD4(+) T cells from the former but not the latter recipients induced autoimmune-like manifestations in secondary allogeneic BALB/c as well as syngeneic DBA/2 recipients. Transfer of donor-type CD4(+) T cells from secondary DBA/2 recipients with disease into syngeneic donor-type or allogeneic host-type tertiary recipients propagated autoimmune-like manifestations in both. Furthermore, TCR spectratyping revealed that the clonal expansion of the autoreactive CD4(+) T cells in cGVHD recipients was initiated by an alloimmune response. Finally, hybridoma CD4(+) T clones derived from DBA/2 recipients with disease proliferated similarly in response to stimulation by syngeneic donor-type or allogeneic host-type dendritic cells. These results demonstrate that the autoimmune-like manifestations in cGVHD can be mediated by a population of donor CD4(+) T cells in transplants that simultaneously recognize Ags presented by both donor and host APCs.     

Phytochrome control of its own accumulation and leaf expansion in tentoxin- and norflurazon-treated mung bean seedlings

Primary leaves of 4-day-old, dark-grown mung bean [ Vigna radiata (L.) Wilczek cv. Berken] seedlings were exposed to 24 h of white light (200 μmol m⁻² s⁻¹) which was terminated by a 15 min, phytochrome-saturating red or far-red light exposure. Phytochrome content (in vivo and in vitro) and leaf area were monitored during the subsequent dark period. Red light treatments resulted in lower phytochrome content and greater leaf expansion than did far-red treatments. Phytochrome accumulation and leaf expansion were less in norflurazon- (no carotenoids and very low Chl) than in tentoxin- (very low Chl) treated leaves. After 3 days of darkness, leaf expansion was about 25% greater and phytochrome content was about 50% less in red- than in far-red-treated leaves of all treatments. These effects generally took longer to develop in norflurazon- than in tentoxin-treated tissues. Norflurazon-treated tissues exposed to long white light periods apparently do not as accurately reflect phytochrome-controlled photomorphogenic events of green tissues as do tentoxin-treated tissues of mung bean seedlings.     

Primary human muscle precursor cells obtained from young and old donors produce similar proliferative,differentiation and senescent profiles in culture

The myogenic behaviour of primary human muscle precursor cells (MPCs) obtained from young (aged 20–25 years) and elderly people (aged 67–82 years) was studied in culture. Cells were compared in terms of proliferation, DNA damage, time course and extent of myogenic marker expression during differentiation, fusion, size of the formed myotubes, secretion of the myogenic regulatory cytokine TGF‐β1 and sensitivity to TGF‐β1 treatment. No differences were observed between cells obtained from the young and elderly people. The cell populations were expanded in culture until replicative senescence. Cultures that maintained their initial proportion of myogenic cells (desmin positive) with passaging (n = 5) were studied and compared with cells from the same individuals in the non‐senescent state. The senescent cells exhibited a greater number of cells with DNA damage (γ‐H2AX positive), showed impaired expression of markers of differentiation, fused less well, formed smaller myotubes and secreted more TGF‐β. The data strongly suggest that MPCs from young and elderly people have similar myogenic behaviour.     

Stereoselective Covalent Binding of Anti-benzo(a)pyrene Diol Epoxide to DNA Conformation of Enantiomer Adducts

Abstract

The conformation of adducts derived from the reactions and covalent binding of the (+) and (-) enantiomers of 7β, 8α-dihydroxy-9α, 10α-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BaPDE) with double-stranded calf thymus DNA in vitro were investigated utilizing the electric linear dichroism technique. The linear dichroism and absorption spectra of the covalent DNA complexes are interpreted in terms of a superposition of two types of binding sites. One of these conformations (site I) is a complex in which the plane of the pyrene residue is close to parallel (within 30°) to the planes of the DNA bases (quasi-intercalation), while the other (site II) is an external binding site; this latter type of adduct is attributed to the covalent binding of anti-BaPDE to the exocyclic amino group of deoxyguanine (N2-dG), while site I adducts are attributed to the 06-deoxyguanine and N6-deoxyadenine adducts identified in the product analysis of P. Brookes and M.R. Osborne (Carcinogenesis (1982) 3, 1223–1226). Site II adducts are dominant (~90% in the covalent complexes derived from the (+) enantiomer), but account for only 50±5% of the adducts in the case of the (—)-enantiomer. The orientation of site II complexes is different by 20±10° in the adducts derived from the binding of the (+) and the (—) enantiomers to DNA, the long axis of the pyrene chromophore being oriented more parallel to the axis of the DNA helix in the case of the (+) enantiomer. These findings support the proposals by Brookes and Osborne that the difference in spatial orientation of the N2-dG adducts of (-)-anti-BaPDE together with their lower abundance may account for the lower biological activity of the (—) enantiomer. The external site II adducts, rather than site I adducts, appear to be correlated with the biological activity of these comoounds.     

Larval amphibian growth and development under varying density: are parasitized individuals poor competitors?

Population density and infection with parasites often are important factors affecting the growth and development of individuals. How these factors co-occur and interact in nature should have important consequences for individual fitness and higher-order phenomena, such as population dynamics of hosts and their interactions with other species. However, few studies have examined the joint effects of density and parasitism on host growth and development. We examined the co-influences of rearing density and parasitism, by the trematode Echinostoma trivolvis, on the growth and development of larval frogs, Rana (=Lithobates) pipiens. We also examined the potential role of parasite-mediated intraspecific competition by observing how unparasitized individuals performed when housed with other unparasitized tadpoles, versus housing with a combination of unparasitized and parasitized hosts. Mean mass and mean developmental stage were reduced under high rearing densities. The presence of parasitized conspecifics had no significant effect, but there was a significant interaction of density and parasitism presence on host mass, due to the fact that parasitized conspecifics grew poorly at high densities. Unparasitized individuals reared with parasitized and unparasitized conspecifics fared no better than unparasitized individuals reared only with one another. This result indicates that infected hosts compete as much as uninfected hosts for resources, even though infected individuals have reduced mass under high-density conditions. Resource acquisition and resource allocation are different processes, and parasitism, if it only affects the latter, might not have a discernible impact on competitive interactions.     

Teaching old enzymes new tricks: engineering and evolution of glycosidases and glycosyl transferases for improved glycoside synthesis.

The therapeutic potential of glycosides has made them an attractive target for drug development. The biological extraction and chemical synthesis of these molecules is often challenging and low yielding, thus alternative methods for the synthesis of polysaccharides are being pursued. A new class of enzymes, glycosynthases, which are nucleophile mutants of glycosidases, can perform the transglycosylation reaction without hydrolyzing the product, and thus provide a valuable resource for polysaccharide and glycan synthesis. Directed evolution of glycosynthases has expanded the repertoire of glycosidic linkages formed and the donors and acceptors (both sugar and nonsugar) that can be used by the glycosynthase. The application of new screening methods, such as FACS, to the directed evolution of glycosynthases will aid in the development of enzymes that are able to efficiently synthesize new, and therapeutically relevant glycosidic linkages.     

Chromosomal rearrangement inferred from comparisons of 12 Drosophila genomes

The availability of 12 complete genomes of various species of genus Drosophila provides a unique opportunity to analyze genome-scale chromosomal rearrangements among a group of closely related species. This article reports on the comparison of gene order between these 12 species and on the fixed rearrangement events that disrupt gene order. Three major themes are addressed: the conservation of syntenic blocks across species, the disruption of syntenic blocks (via chromosomal inversion events) and its relationship to the phylogenetic distribution of these species, and the rate of rearrangement events over evolutionary time. Comparison of syntenic blocks across this large genomic data set confirms that genetic elements are largely (95%) localized to the same Muller element across genus Drosophila species and paracentric inversions serve as the dominant mechanism for shuffling the order of genes along a chromosome. Gene-order scrambling between species is in accordance with the estimated evolutionary distances between them and we find it to approximate a linear process over time (linear to exponential with alternate divergence time estimates). We find the distribution of synteny segment sizes to be biased by a large number of small segments with comparatively fewer large segments. Our results provide estimated chromosomal evolution rates across this set of species on the basis of whole-genome synteny analysis, which are found to be higher than those previously reported. Identification of conserved syntenic blocks across these genomes suggests a large number of conserved blocks with varying levels of embryonic expression correlation in Drosophila melanogaster. On the other hand, an analysis of the disruption of syntenic blocks between species allowed the identification of fixed inversion breakpoints and estimates of breakpoint reuse and lineage-specific breakpoint event segregation.