The Fat-like Cadherin CDH-4 Acts Cell-Non-Autonomously in Anterior-Posterior Neuroblast Migration

Directed migration of neurons is critical in the normal and pathological development of the brain and central nervous system. In C. elegans, the bilateral Q neuroblasts, QR on the right and QL on the left, migrate anteriorly and posteriorly, respectively. Initial protrusion and migration of the Q neuroblasts is autonomously controlled by the transmembrane proteins UNC-40/DCC, PTP-3/LAR, and MIG-21. As QL migrates posteriorly, it encounters and EGL-20/Wnt signal that induces MAB-5/Hox expression that drives QL descendant posterior migration. QR migrates anteriorly away from EGL-20/Wnt and does not activate MAB-5/Hox, resulting in anterior QR descendant migration. A forward genetic screen for new mutations affecting initial Q migrations identified alleles of cdh-4, which caused defects in both QL and QR directional migration similar to unc-40, ptp-3, and mig-21. Previous studies showed that in QL, PTP-3/LAR and MIG-21 act in a pathway in parallel to UNC-40/DCC to drive posterior QL migration. Here we show genetic evidence that CDH-4 acts in the PTP-3/MIG-21 pathway in parallel to UNC-40/DCC to direct posterior QL migration. In QR, the PTP-3/MIG-21 and UNC-40/DCC pathways mutually inhibit each other, allowing anterior QR migration. We report here that CDH-4 acts in both the PTP-3/MIG-21 and UNC-40/DCC pathways in mutual inhibition in QR, and that CDH-4 acts cell-non-autonomously. Interaction of CDH-4 with UNC-40/DCC in QR but not QL represents an inherent left-right asymmetry in the Q cells, the nature of which is not understood. We conclude that CDH-4 might act as a permissive signal for each Q neuroblast to respond differently to anterior-posterior guidance information based upon inherent left-right asymmetries in the Q neuroblasts

Fluorogenic tagging of protein 3-nitrotyrosine with 4-(aminomethyl)benzenesulfonate (ABS) in tissues: a useful alternative to immunohistochemistry for fluorescence microscopy imaging of protein nitration

Protein tyrosine nitration is a common biomarker of biological aging and diverse pathologies associated with the excessive formation of reactive oxygen and nitrogen species. Recently, we suggested a novel fluorogenic derivatization procedure for the detection of 3-nitrotyrosine (3-NT) using benzylamine derivatives to convert specifically protein or peptide bound 3-NT to a highly fluorescent benzoxazole product. In the current study, we applied this procedure to fluorogenic derivatization of protein 3-NT in sections from adult rat cerebellum in order to: (i) test this method in imaging nitrated proteins in fixed brain tissue sections, and (ii) compare the chemical approach to immunohistochemical labeling with anti-3-NT antibodies. Immunofluorescence analysis of cerebellar sections using anti-3-NT antibodies showed differential levels of immunostaining in the molecular, Purkinje, and granule cell layers of the cerebellar cortex; in agreement with previous reports, the Purkinje cells were most highly labeled. Importantly, fluorogenic derivatization reactions of cerebellar proteins with 4-(aminomethyl)benzenesulfonic acid (ABS) and K3Fe(CN)6 at pH 9, following sodium dithionite (SDT) reduction of 3-NT to 3-aminotyrosine (3-AT), showed a very similar pattern of relative intensity of cell labeling and improved resolution when compared with antibody labeling. Our data demonstrate that ABS-derivatization may be either a useful alternative or a complimentary approach to immunolabeling in imaging protein nitration in cells and tissues, including under conditions of dual labeling with antibodies to cell proteins, thus allowing for cellular co-localization of nitrated proteins and any protein of interest

The Fat-like Cadherin CDH-4 Acts Cell-Non-Autonomously in Anterior-Posterior Neuroblast Migration

Directed migration of neurons is critical in the normal and pathological development of the brain and central nervous system. In C. elegans, the bilateral Q neuroblasts, QR on the right and QL on the left, migrate anteriorly and posteriorly, respectively. Initial protrusion and migration of the Q neuroblasts is autonomously controlled by the transmembrane proteins UNC-40/DCC, PTP-3/LAR, and MIG-21. As QL migrates posteriorly, it encounters and EGL-20/Wnt signal that induces MAB-5/Hox expression that drives QL descendant posterior migration. QR migrates anteriorly away from EGL-20/Wnt and does not activate MAB-5/Hox, resulting in anterior QR descendant migration. A forward genetic screen for new mutations affecting initial Q migrations identified alleles of cdh-4, which caused defects in both QL and QR directional migration similar to unc-40, ptp-3, and mig-21. Previous studies showed that in QL, PTP-3/LAR and MIG-21 act in a pathway in parallel to UNC-40/DCC to drive posterior QL migration. Here we show genetic evidence that CDH-4 acts in the PTP-3/MIG-21 pathway in parallel to UNC-40/DCC to direct posterior QL migration. In QR, the PTP-3/MIG-21 and UNC-40/DCC pathways mutually inhibit each other, allowing anterior QR migration. We report here that CDH-4 acts in both the PTP-3/MIG-21 and UNC-40/DCC pathways in mutual inhibition in QR, and that CDH-4 acts cell-non-autonomously. Interaction of CDH-4 with UNC-40/DCC in QR but not QL represents an inherent left-right asymmetry in the Q cells, the nature of which is not understood. We conclude that CDH-4 might act as a permissive signal for each Q neuroblast to respond differently to anterior-posterior guidance information based upon inherent left-right asymmetries in the Q neuroblasts

Inactivation of rabbit muscle glycogen phosphorylase b by peroxynitrite revisited: does the nitration of Tyr613 in the allosteric inhibition site control enzymatic function?

There is increasing evidence that sequence-specific formation of 3-nitrotyrosine (3-NT) may cause functional changes in target proteins. Recently, the nitration of Tyr residues in glycogen phosphorylase b (Ph-b) was implicated in the age-associated decline of protein function (Sharov et al., Exp. Gerontol. 41, 407–416; 2006); in another report, the nitration of one specific residue, Tyr613, located in the allosteric inhibition site was hypothesized as a rationale for peroxynitrite inactivation (Dairou et al., J. Mol. Biol. 372, 1009–1021; 2007). In the present study, we have optimized the analysis of in-gel Ph-b digests by high performance liquid chromatography-electro spray ionization-tandem mass spectrometry, in order to achieve a quantitative analysis of nitration of individual Tyr residues at a high coverage of Tyr-containing sequences (92%). Our data do not confirm the role of Tyr613 nitration in the control of enzymatic function. Furthermore, we show here that the enzymatic activity of Ph-b does not directly correlate with the protein nitration levels, and that the modification of Cys and, potentially, other amino acid residues can better rationalize Ph-b inactivation by peroxynitrite

A Methodology for Simultaneous Fluorogenic Derivatization and Boronate Affinity Enrichment of 3-Nitrotyrosine Containing Peptides

We synthesized and characterized a new tagging reagent, (3R,4S)-1-(4-(aminomethyl)phenylsulfonyl)pyrrolidine-3,4-diol (APPD), for the selective fluorogenic derivatization of 3-nitrotyrosine (3-NT) residues in peptides (after reduction to 3-aminotyrosine) and affinity enrichment. The synthetic 3-NT-containing peptide, FSAY(3-NO2)LER, was employed as a model for method validation. Further, this derivatization protocol was successfully tested for analysis of 3-NT-containing proteins exposed to peroxynitrite in the total protein lysate of cultured C2C12 cells. The quantitation of 3-NT content in samples was achieved through either fluorescence spectrometry or boronate affinity chromatography with detection by specific fluorescence (excitation and emission wavelengths of 360 and 510 nm, respectively); the respective limits of detection were 95 and 68 nM (19 and 13 pmol total amount) of 3-NT. Importantly, the derivatized peptides show a strong retention on a synthetic boronate affinity column, containing sulfonamide-phenylboronic acid, under mild chromatographic conditions, affording a route to separate the derivatized peptides from large amounts (milligrams) of non-derivatized peptides, and to enrich them for fluorescent detection and MS identification. Tandem MS analysis identified chemical structures of peptide 3-NT fluorescent derivatives and revealed that the fluorescent derivatives undergo efficient backbone fragmentations, permitting sequence-specific identification of protein nitration at low concentrations of 3-NT in complex protein mixtures

Designing Formulation Strategies for Enhanced Stability of Therapeutic Peptides in Aqueous Solutions: A Review

Over the past few decades, there has been a tremendous increase in the utilization of therapeutic peptides. Therapeutic peptides are usually administered via the parenteral route, requiring an aqueous formulation. Unfortunately, peptides are often unstable in aqueous solutions, affecting stability and bioactivity. Although a stable and dry formulation for reconstitution might be designed, from a pharmaco-economic and practical convenience point of view, a peptide formulation in an aqueous liquid form is preferred. Designing formulation strategies that optimize peptide stability may improve bioavailability and increase therapeutic efficacy. This literature review provides an overview of various degradation pathways and formulation strategies to stabilize therapeutic peptides in aqueous solutions. First, we introduce the major peptide stability issues in liquid formulations and the degradation mechanisms. Then, we present a variety of known strategies to inhibit or slow down peptide degradation. Overall, the most practical approaches to peptide stabilization are pH optimization and selecting the appropriate type of buffer. Other practical strategies to reduce peptide degradation rates in solution are the application of co-solvency, air exclusion, viscosity enhancement, PEGylation, and using polyol excipients

Sarcoendoplasmic Reticulum Ca2+ ATPase. A Critical Target in Chlorine Inhalation–Induced Cardiotoxicity

Autopsy specimens from human victims or experimental animals that die due to acute chlorine gas exposure present features of cardiovascular pathology. We demonstrate acute chlorine inhalation–induced reduction in heart rate and oxygen saturation in rats. Chlorine inhalation elevated chlorine reactants, such as chlorotyrosine and chloramine, in blood plasma. Using heart tissue and primary cardiomyocytes, we demonstrated that acute high-concentration chlorine exposure in vivo (500 ppm for 30 min) caused decreased total ATP content and loss of sarcoendoplasmic reticulum calcium ATPase (SERCA) activity. Loss of SERCA activity was attributed to chlorination of tyrosine residues and oxidation of an important cysteine residue, cysteine-674, in SERCA, as demonstrated by immunoblots and mass spectrometry. Using cardiomyocytes, we found that chlorine-induced cell death and damage to SERCA could be decreased by thiocyanate, an important biological antioxidant, and by genetic SERCA2 overexpression. We also investigated a U.S. Food and Drug Administration–approved drug, ranolazine, used in treatment of cardiac diseases, and previously shown to stabilize SERCA in animal models of ischemia–reperfusion. Pretreatment with ranolazine or istaroxime, another SERCA activator, prevented chlorine-induced cardiomyocyte death. Further investigation of responsible mechanisms showed that ranolazine- and istaroxime-treated cells preserved mitochondrial membrane potential and ATP after chlorine exposure. Thus, these studies demonstrate a novel critical target for chlorine in the heart and identify potentially useful therapies to mitigate toxicity of acute chlorine exposure.This work was supported by the CounterACT Program, National Institutes of Health, Office of the Director, and the National Institute of Environmental Health Sciences grant U54 ES015678 (C.W.W.), and by Children’s Hospital of Colorado/Colorado School of Mines Pilot Award G0100394 and a Children’s Hospital of Colorado Research Institute’s Pilot Award (S.A.)

Fe-Chlorophyllin Promotes the Growth of Wheat Roots Associated with Nitric Oxide Generation

Effects of Fe-chlorophyllin on the growth of wheat root were investigated in this study. We found that Fe-chlorophyllin can promote root growth. The production of nitric oxide in wheat root was detected using DAF-2DA fluorescent emission. The intensity of fluorescent in the presence of 0.1 mg/L Fe-chlorophyllin was near to that observed with the positive control of sodium nitroprusside (SNP), the nitric oxide donor. IAA oxidase activity decreased with all treatments of Fe-chlorophyllin from 0.01 to 10 mg/L. At the relatively lower Fe-chlorophyllin concentration of 0.1 mg/L, the activity of IAA oxidase displayed a remarkable decrease, being 40.1% lower than the control. Meanwhile, Fe-chlorophyllin treatment could increase the activities of reactive oxygen scavenging enzymes, such as superoxide dismutase (SOD) and peroxidase (POD), as determined using non-denaturing polyacrylamide gel electrophoresis. These results indicate that Fe-chlorophyllin contributes to the growth of wheat root associated with nitric oxide generation

A mother's sacrifice: what is she keeping for herself?

Individual cells of the budding yeast, Saccharomyces cerevisiae, have a limited life span and undergo a form of senescence termed replicative aging. Replicative life span is defined as the number of daughter cells produced by a yeast mother cell before she ceases dividing. Replicative aging is asymmetric: a mother cell ages but the age of her daughter cells is 'reset' to zero. Thus, one or more senescence factors have been proposed to accumulate asymmetrically between mother and daughter yeast cells and lead to mother-specific replicative senescence once a crucial threshold has been reached. Here we evaluate potential candidates for senescence factors and age-associated phenotypes and discuss potential mechanisms underlying the asymmetry of replicative aging in budding yeast

Free radical generation by skeletal muscle of adult and old mice: effect of contractile activity

Oxidative modification of cellular components may contribute to tissue dysfunction during aging. In skeletal muscle, contractile activity increases the generation of reactive oxygen and nitrogen species (ROS). The question of whether contraction-induced ROS generation is further increased in skeletal muscle of the elderly is important since this influences recommendations on their exercise participation. Three different approaches were used to examine whether aging influences contraction-induced ROS generation. Hind limb muscles of adult and old mice underwent a 15-min period of isometric contractions and we examined ROS generation by isolated skeletal muscle mitochondria, ROS release into the muscle extracellular fluid using microdialysis techniques, and the muscle glutathione and protein thiol contents. Resting skeletal muscle of old mice compared with adult mice showed increased ROS release from isolated mitochondria, but no changes in the extracellular levels of superoxide, nitric oxide, hydrogen peroxide, hydroxyl radical activity or muscle glutathione and protein thiol contents. Skeletal muscle mitochondria isolated from both adult and old mice after contractile activity showed significant increases in hydrogen peroxide release compared with pre-contraction values. Contractions increased extracellular hydroxyl radical activity in adult and old mice, but had no significant effect on extracellular hydrogen peroxide or nitric oxide in either group. In adult mice only, contractile activity increased the skeletal muscle release of superoxide. A similar decrease in muscle glutathione and protein thiol contents was seen in adult and old mice following contractions. Thus, contractile activity increased skeletal muscle ROS generation in both adult and old mice with no evidence for an age-related exacerbation of ROS generation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72706/1/j.1474-9726.2006.00198.x.pd