Mass tagging approach for mitochondrial thiol proteins

A mass tagging approach is described for mitochondrial thiol proteins under nondenaturing conditions. This approach utilizes stable isotope-coded, thiol-reactive (4-iodobutyl)triphenylphosphonium (IBTP) reagents, i.e., the isotopomers IBTP-d(0) and IBTP-d(15). The mass spectrometric properties of IBTP-labeled peptides were evaluated using an ESI-q-TOF and a MALDI-TOF/TOF instrument. High energy collision induced dissociation (CID) in the TOF/TOF instrument caused side-chain fragmentation in the butyltriphenylphosphonium moiety-containing Cys-residue. By contrast, low energy CID in the qTOF instrument yielded sequence tags of IBTP-labeled peptides that were suitable for automated database searching. The IBTP labeling strategy was then applied to the analysis of a protein extract obtained from cardiac mitochondria. The relative abundance measurements for identified IBTP-labeled peptides showed an average variability for peptide quantitation of approximately 10% based on peak area ratios of ion signals for the d(0)/d(15)-tagged peptide pairs. The reactivity of the IBTP reagents was further studied by molecular modeling and visualization. The present study suggests that the IBTP reagent seems to show a bias toward highly surface-exposed protein thiols. Hence, the described mass tagging approach might become potentially useful in redox proteomics studies designed to identify protein thiols that are particularly prone to oxidative modifications.     

Effects of Moderate Aerobic Exercise Training on Hemorheological and Laboratory Parameters in Ischemic Heart Disease Patients

Background and Design

In this study we set out to determine the effects of long-term physical training on hemorheological, laboratory parameters, exercise tolerability, psychological factors in cardiac patients participating in an ambulatory rehabilitation program.

Methods

Before physical training, patients were examined by echocardiography, tested on treadmill by the Bruce protocol, and blood was drawn for laboratory tests. The enrolled 79 ischemic heart disease patients joined a 24-week cardiac rehabilitation training program. Blood was drawn to measure hematocrit (Hct), plasma and whole blood viscosity (PV, WBV), red blood cell (RBC) aggregation and deformability. Hemorheological, clinical chemistry and psychological measurements were repeated 12 and 24 weeks later, and a treadmill test was performed at the end of the program.

Results

After 12 weeks Hct, PV, WBV and RBC aggregation were significantly decreased, RBC deformability exhibited a significant increase (p<0.05). Laboratory parameters (triglyceride, uric acid, hsCRP and fibrinogen) were significantly decreased (p<0.05). After 24 weeks the significant results were still observed. By the end of the study, IL-6 and TNF-α levels displayed decreasing trends (p<0.06). There was a significant improvement in MET (p<0.001), and the BMI decrease was also significant (p<0.05). The vital exhaustion parameters measured on the fatigue impact scale indicated a significant improvement in two areas of the daily activities (p<0.05).

Conclusions

Regular physical training improved the exercise tolerability of patients with ischemic heart disease. Previous publications have demonstrated that decreases in Hct and PV may reduce cardiovascular risk, while a decrease in RBC aggregation and an increase in deformability improve the capillary flow. Positive changes in laboratory parameters and body weight may indicate better oxidative and inflammatory circumstances and an improved metabolic state. The psychological findings point to an improvement in the quality of life.     

Effect of Potassium on the Release of [3H]Noradrenaline from Rabbit and Human Pulmonary Artery

The release of [3H]noradrenaline ( [3H]NA) from rabbit and human isolated pulmonary artery has been measured. Removal of external potassium ions enhanced both the resting and stimulated release of [3H]NA from the strips. On adding K+ to tissues which had been suspended in K+-free Krebs solution, the release of [3H]NA was reduced in both stimulated and unstimulated tissues. Selective inhibition of presynaptic alpha 2-adrenoceptors by yohimbine significantly potentiated the release of [3H]NA evoked by stimulation in K+-free solution. The presynaptic inhibitory effect of NA was much less pronounced when the release was enhanced by the removal of external K+. Since the activity of NA, K-ATPase may be affected by removing K+ or by adding it to tissue previously kept in K+-free solution, the results may indicate involvement of the sodium pump in NA release.     

Framing Political Messages to Fit the Audience’s Regulatory Orientation: How to Improve the Efficacy of the Same Message Content

This research investigates how the impact of persuasive messages in the political domain can be improved when fit is created by subliminally priming recipients’ regulatory focus (either promotion or prevention) and by linguistic framing of the message (either strategic approach framing or strategic avoidance framing). Results of two studies show that regulatory fit: a) increases the impact of a political message favoring nuclear energy on implicit attitudes of the target audience (Study 1); and b) induces a more positive evaluation of, and intentions to vote for, the political candidate who is delivering a message concerning immigration policies (Study 2).     

Crystal structure of the herpes simplex virus 1 DNA polymerase

Herpesviruses are the second leading cause of human viral diseases. Herpes Simplex Virus types 1 and 2 and Varicella-zoster virus produce neurotropic infections such as cutaneous and genital herpes, chickenpox, and shingles. Infections of a lymphotropic nature are caused by cytomegalovirus, HSV-6, HSV-7, and Epstein-Barr virus producing lymphoma, carcinoma, and congenital abnormalities. Yet another series of serious health problems are posed by infections in immunocompromised individuals. Common therapies for herpes viral infections employ nucleoside analogs, such as Acyclovir, and target the viral DNA polymerase, essential for viral DNA replication. Although clinically useful, this class of drugs exhibits a narrow antiviral spectrum, and resistance to these agents is an emerging problem for disease management. A better understanding of herpes virus replication will help the development of new safe and effective broad spectrum anti-herpetic drugs that fill an unmet need. Here, we present the first crystal structure of a herpesvirus polymerase, the Herpes Simplex Virus type 1 DNA polymerase, at 2.7 A resolution. The structural similarity of this polymerase to other alpha polymerases has allowed us to construct high confidence models of a replication complex of the polymerase and of Acyclovir as a DNA chain terminator. We propose a novel inhibition mechanism in which a representative of a series of non-nucleosidic viral polymerase inhibitors, the 4-oxo-dihydroquinolines, binds at the polymerase active site interacting non-covalently with both the polymerase and the DNA duplex.     

Novel Acyclic Derivatives of Aica-Riboside (1-(β-D-Ribopuranosyl)5-Amino-4-Imidazolecarboxamide), Potential Antiviral Agents

Abstract

A synthetic approach is described to obtain from AICA-riboside acyclic analogues of 2′-deoxyribosides in which the C(2′)-C(3′) bond is cleaved and the natural configuration (E) at the anomeric C (1′)-position is retained.     

Cell Wall Structure in Cells Adapted to Growth on the Cellulose-Synthesis Inhibitor 2,6-Dichlorobenzonitrile : A Comparison between Two Dicotyledonous Plants and a Graminaceous Monocot

Our previous work (E. Shedletzky, M. Shmuel, D.P. Delmer, D.T.A. Lamport [1990] Plant Physiol 94:980-987) showed that suspension-cultured tomato cells adapted to growth on the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB) have a markedly altered cell wall composition, most notably a markedly reduced level of the cellulose-xyloglucan network. This study compares the adaptation to DCB of two cell lines from dicots (tomato [Lycopersicon esculentum] and tobacco [Nicotiana tabacum]) and a Graminaceous monocot (barley [Hordeum bulbosum] endosperm). The difference in wall structures between the dicots and the monocot is reflected in the very different types of wall modifications induced by growth on DCB. The dicots, having reduced levels of cellulose and xyloglucan, possess walls the major integrity of which is provided by Ca²⁺-bridged pectates because protoplasts can be prepared from these cells simply by treatment with divalent cation chelator and a purified endopolygalacturonase. The tensile strength of these walls is considerably less than walls from nonadapted cells, but wall porosity is not altered. In contrast, walls from adapted barley cells contain very little pectic material and normal to elevated levels of noncellulosic polysaccharides compared with walls from nonadapted cells. Surprisingly, they have tensile strengths higher than their nonadapted counterpart, although cellulose levels are reduced by 70%. Evidence is presented that these walls obtain their additional strength by an altered pattern of cross-linking of polymers involving phenolic components. Such cross-linking may also explain the observation that the porosity of these walls is also considerably reduced. Cells of adapted lines of both the dicots and barley are resistant to plasmolysis, suggesting that they possess very strong connections between the wall and the plasma membrane.     

Regulation of MKP-1 expression and MAPK activation by PARP-1 in oxidative stress: a new mechanism for the cytoplasmic effect of PARP-1 activation

Previously, it was suggested that the release of nuclearly formed ADP-ribose polymers or ADP-ribosylated proteins could be responsible for the cytosolic and mitochondrial effects of poly(ADP-ribose) polymerase (PARP)-1 activation in oxidative stress. In this report, we provide a novel alternative mechanism. We found that reactive oxygen species-activated PARP-1 regulated the activation of JNK and p38 mitogen-activated protein kinases (MAPKs) because inhibition of PARP-1 by pharmacons, small interfering RNA silencing of PARP-1 expression, or the transdominant expression of enzymatically inactive PARP-1 resulted in the inactivation of these MAPKs. This regulation was achieved by increased expression and enlarged cytoplasmic localization of MAPK phosphatase-1 (MKP-1) upon PARP-1 inhibition in oxidative stress because changes in MKP-1 expression were reflected in the phosphorylation states of JNK and p38. Furthermore, we found that in MKP-1-silenced cells, PARP inhibition was unable to exert its protective effect, indicating the pivotal roles of JNK and p38 in mediating the oxidative-stress-induced cell death as well as that of increased MKP-1 expression in mediating the protective effect of PARP inhibition. We suggest that regulation of a protein that can directly influence cytoplasmic signaling cascades at the expression level represents a novel mechanism for the cytoplasmic action of PARP-1 inhibition.     

Von Hippel-Lindau disease: identification of deletion mutations by pulsed-field gel electrophoresis

Von Hippel-Lindau disease (VHL) is an inherited multisystem neoplastic disorder. We prepared a 2.5-megabase (Mb) restriction map of the region surrounding the VHL gene and identified and characterized overlapping deletions in three unrelated patients affected with VHL. The smallest nested deletion (100 kb) was located within a 510-kb NruI fragment detected by 19–63. The rearrangements detected will be useful in isolating and evaluating candidate cDNAs for the VHL gene. The detailed physical map will be useful in studying the organization and structure of genes in the VHL region.