Fluorogenic and Affinity Derivatization Methods to Enable Proteomic Study of 3-Nitrotyrosine and 3,4-Dihydroxyphenylalanine as Markers of Oxidative Stress

Purpose: Oxidative post-translational modification of protein-bound tyrosine residues can have a significant impact on protein structure and function and thus may be important to physiological and pathological processes. Oxidative stress has been correlated with biological aging and many disease states, including diabetes, atherosclerosis, and neurodegeneration. Proteomic methods targeted to these modifications are important tools for determining which specific modifications may be significant in these conditions. Toward this end, a method designed to fluorogenically label the protein oxidation products 3-nitrotyrosine (3NY) and 3,4-dihydroxyphenylalanine (DOPA) using benzylamine-dependent chemistry is applied to model peptides and proteins, as well as cardiac tissue samples from a rat model for aging. Methods: Peptides or proteins are reacted with excess benzylamine (or a derivative thereof), in the presence of potassium ferricyanide, to fluorescently label DOPA residues by forming 2-phenylbenzoxazole derivatives. To label 3NY residues, the peptides or proteins are first reduced with sodium dithionite to give 3-aminotyrosine, which can undergo a similar reaction with benzylamine and oxidant to give the same products. Products are characterized by fluorescence spectroscopy, high-performance liquid chromatography (HPLC) with UV and fluorescence detection, mass spectrometry, and amino acid analysis. For enrichment by boronate-affinity HPLC, the benzylamine derivative (3R, 4S)-1-(4-(aminomethyl)phenylsulfonyl) pyrrolidine-3,4-diol (APPD) is used as the tagging reagent. Results: Cardiac proteins have been fluorescently labeled and separated, and some putative identifications have been made. A model protein, glycogen phosphorylase b (Ph-b), has been nitrated in vitro and labeled within a matrix of cardiac homogenate, and the products exhibit concentration-dependent fluorescence. The loss of 3NY from nitrated Ph-b upon mixing with cardiac homogenate has been observed and examined. Five model peptides have been labeled with APPD to determine the effect of primary structure on labeling efficiency, fluorescence quantum yield, and molar absorptivity. Conclusions: This method has great potential to aid identification of the protein oxidation products DOPA and 3NY in proteomic studies of tissue samples and can also be adapted for affinity enrichment and relative quantification of these low-abundance species

Prediction of 24-hour milk yield and composition in dairy cows from a single part-day yield and sample

peer-reviewedTeagasc PublicationIrish Journal of Agricultural and Food Research | Volume 58: Issue 1 Prediction of 24-hour milk yield and composition in dairy cows from a single part-day yield and sample S. McParlandemail , B. Coughlan , B. Enright , M. O’Keeffe , R. O’Connor , L. Feeney and D.P. Berry DOI: https://doi.org/10.2478/ijafr-2019-0007 | Published online: 09 Aug 2019 PDF Abstract Article PDF References Recommendations Abstract The objective was to evaluate the accuracy of predicting 24-hour milk yield and composition from a single morning (AM) or evening (PM) milk weight and composition. A calibration dataset of 37,481 test-day records with both AM and PM yields and composition was used to generate the prediction equations; equations were validated using 4,644 test-day records. Prediction models were developed within stage of lactation and parity while accounting for the inter-milking time interval. The mean correlation between the predicted 24-hour yields and composition of milk, fat and protein and the respective actual values was 0.97 when based on just an AM milk yield and composition with a mean correlation of 0.95 when based on just a PM milk yield and composition. The regression of predicted 24-hour yield and composition on the respective actual values varied from 0.97 to 1.01 with the exception of 24-hour fat percentage predicted from a PM sample (1.06). A single AM sample is useful to predict 24-hour milk yield and composition when the milking interval is known

Tyrosine Modifications in Aging

This is the publisher's version, also available electronically from http://online.liebertpub.com/doi/abs/10.1089/ars.2012.4595Significance: The understanding of physiological and pathological processes involving protein oxidation, particularly under conditions of aging and oxidative stress, can be aided by proteomic identification of proteins that accumulate oxidative post-translational modifications only if these detected modifications are connected to functional consequences. The modification of tyrosine (Tyr) residues can elicit significant changes in protein structure and function, which, in some cases, may contribute to biological aging and age-related pathologies, such as atherosclerosis, neurodegeneration, and cataracts. Recent Advances: Studies characterizing proteins in which Tyr has been modified to 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, 3,3′-dityrosine and other cross-links, or 3-chlorotyrosine are reviewed, with an emphasis on structural and functional consequences. Critical Issues: Distinguishing between inconsequential modifications and functionally significant ones requires careful biochemical and biophysical analysis of target proteins, as well as innovative methods for isolating the effects of the multiple modifications that often occur under oxidizing conditions. Future Directions: The labor-intensive task of isolating and characterizing individual modified proteins must continue, especially given the expanding list of known modifications. Emerging approaches, such as genetic and metabolic incorporation of unnatural amino acids, hold promise for additional focused studies of this kind. Antioxid. Redox Signal. 17, 1571–1579

The combined use of selective deuteration and double resonance experiments in assigning the 1H resonances of valine and tyrosine residues of dihydrofolate reductase

AbstractSelective deuteration is a general solution to the resolution problem which limits the application of double resonance experiments to the assignment of the 1H NMR spectra of proteins. Spin-decoupling and NOE experiments have been carried out on Lactobacillus casei dihydrofolate reductase and on selectively deuterated derivatives of the enzyme containing either [γ-2H6]Val or (α,δ2,ϵ1-2H3]His, [α,δ1,δ2,ϵ1,ϵ2,ζ-2H6]Phe, [α,δ1,ϵ3,ζ2,ζ3,η2-2H6]Trp and [α,ϵ1,ϵ2-2H3]Tyr. When combined with ring-current shift calculations based on the crystal structure of the enzyme, these experiments allow us to assign 1H resonances of Val 61, Val 115, Tyr 46 and Tyr 68

Raising argument strength using negative evidence: A constraint on models of induction

Both intuitively, and according to similarity-based theories of induction, relevant evidence raises argument strength when it is positive and lowers it when it is negative. In three experiments, we tested the hypothesis that argument strength can actually increase when negative evidence is introduced. Two kinds of argument were compared through forced choice or sequential evaluation: single positive arguments (e.g., “Shostakovich’s music causes alpha waves in the brain; therefore, Bach’s music causes alpha waves in the brain”) and double mixed arguments (e.g., “Shostakovich’s music causes alpha waves in the brain, X’s music DOES NOT; therefore, Bach’s music causes alpha waves in the brain”). Negative evidence in the second premise lowered credence when it applied to an item X from the same subcategory (e.g., Haydn) and raised it when it applied to a different subcategory (e.g., AC/DC). The results constitute a new constraint on models of induction

Patterns of dairy food intake, body composition and markers of metabolic health in Ireland:results from the National Adult Nutrition Survey

Background: Studies examining the association between dairy consumption and metabolic health have shown mixed results. This may be due, in part, to the use of different definitions of dairy, and to single types of dairy foods examined in isolation. Objective: The objective of the study was to examine associations between dairy food intake and metabolic health, identify patterns of dairy food consumption and determine whether dairy dietary patterns are associated with outcomes of metabolic health, in a cross-sectional survey. Design: A 4-day food diary was used to assess food and beverage consumption, including dairy (defined as milk, cheese, yogurt, cream and butter) in free-living, healthy Irish adults aged 18–90 years (n=1500). Fasting blood samples (n=897) were collected, and anthropometric measurements taken. Differences in metabolic health markers across patterns and tertiles of dairy consumption were tested via analysis of covariance. Patterns of dairy food consumption, of different fat contents, were identified using cluster analysis. Results: Higher (total) dairy was associated with lower body mass index, %body fat, waist circumference and waist-to-hip ratio (P<0.001), and lower systolic (P=0.02) and diastolic (P<0.001) blood pressure. Similar trends were observed when milk and yogurt intakes were considered separately. Higher cheese consumption was associated with higher C-peptide (P<0.001). Dietary pattern analysis identified three patterns (clusters) of dairy consumption; 'Whole milk', 'Reduced fat milks and yogurt' and 'Butter and cream'. The 'Reduced fat milks and yogurt' cluster had the highest scores on a Healthy Eating Index, and lower-fat and saturated fat intakes, but greater triglyceride levels (P=0.028) and total cholesterol (P=0.015). conclusion: Overall, these results suggest that while milk and yogurt consumption is associated with a favourable body phenotype, the blood lipid profiles are less favourable when eaten as part of a low-fat high-carbohydrate dietary pattern. More research is needed to better understand this association. Conclusion: Overall, these results suggest that although milk and yogurt consumption is associated with a favourable body phenotype, the blood lipid profiles are less favourable when eaten as part of a low-fat high-carbohydrate dietary pattern. More research is needed to better understand this association

A viral CTL escape mutation leading to immunoglobulin-like transcript 4-mediated functional inhibition of myelomonocytic cells

Viral mutational escape can reduce or abrogate recognition by the T cell receptor (TCR) of virus-specific CD8+ T cells. However, very little is known about the impact of cytotoxic T lymphocyte (CTL) epitope mutations on interactions between peptide–major histocompatibility complex (MHC) class I complexes and MHC class I receptors expressed on other cell types. Here, we analyzed a variant of the immunodominant human leukocyte antigen (HLA)-B2705–restricted HIV-1 Gag KK10 epitope (KRWIILGLNK) with an L to M amino acid substitution at position 6 (L6M), which arises as a CTL escape variant after primary infection but is sufficiently immunogenic to elicit a secondary, de novo HIV-1–specific CD8+ T cell response with an alternative TCR repertoire in chronic infection. In addition to altering recognition by HIV-1–specific CD8+ T cells, the HLA-B2705–KK10 L6M complex also exhibits substantially increased binding to the immunoglobulin-like transcript (ILT) receptor 4, an inhibitory MHC class I–specific receptor expressed on myelomonocytic cells. Binding of the B2705–KK10 L6M complex to ILT4 leads to a tolerogenic phenotype of myelomonocytic cells with lower surface expression of dendritic cell (DC) maturation markers and co-stimulatory molecules. These data suggest a link between CTL-driven mutational escape, altered recognition by innate MHC class I receptors on myelomonocytic cells, and functional impairment of DCs, and thus provide important new insight into biological consequences of viral sequence diversificatio

Liesegang patterns: Effect of dissociation of the invading electrolyte

The effect of dissociation of the invading electrolyte on the formation of Liesegang bands is investigated. We find, using organic compounds with known dissociation constants, that the spacing coefficient, 1+p, that characterizes the position of the n-th band as x_n ~ (1+p)^n, decreases with increasing dissociation constant, K_d. Theoretical arguments are developed to explain these experimental findings and to calculate explicitly the K_d dependence of 1+p.Comment: RevTex, 8 pages, 3 eps figure

Translational control of the SigR-directed oxidative stress response in streptomyces via IF3-mediated repression of a noncanonical GTC start codon

The major oxidative stress response in Streptomyces is controlled by the sigma factor SigR and its cognate antisigma factor RsrA, and SigR activity is tightly controlled through multiple mechanisms at both the transcriptional and posttranslational levels. Here we show that sigR has a highly unusual GTC start codon and that this leads to another level of SigR regulation, in which SigR translation is repressed by translation initiation factor 3 (IF3). Changing the GTC to a canonical start codon causes SigR to be overproduced relative to RsrA, resulting in unregulated and constitutive expression of the SigR regulon. Similarly, introducing IF3* mutations that impair its ability to repress SigR translation has the same effect. Thus, the noncanonical GTC sigR start codon and its repression by IF3 are critical for the correct and proper functioning of the oxidative stress regulatory system. sigR and rsrA are cotranscribed and translationally coupled, and it had therefore been assumed that SigR and RsrA are produced in stoichiometric amounts. Here we show that RsrA can be transcribed and translated independently of SigR, present evidence that RsrA is normally produced in excess of SigR, and describe the factors that determine SigR-RsrA stoichiometry.IMPORTANCE In all sigma factor-antisigma factor regulatory switches, the relative abundance of the two proteins is critical to the proper functioning of the system. Many sigma-antisigma operons are cotranscribed and translationally coupled, leading to a generic assumption that the sigma and antisigma factors are produced in a fixed 1:1 ratio. In the case of sigR-rsrA, we show instead that the antisigma factor is produced in excess over the sigma factor, providing a buffer to prevent spurious release of sigma activity. This excess arises in part because sigR has an extremely rare noncanonical GTC start codon, and as a result, SigR translation initiation is repressed by IF3. This finding highlights the potential significance of noncanonical start codons, very few of which have been characterized experimentally. It also emphasizes the limitations of predicting start codons using bioinformatic approaches, which rely heavily on the assumption that ATG, GTG, and TTG are the only permissible start codons