The oxidative mechanism of action of ortho-quinone inhibitors of protein-tyrosine phosphatase alpha is mediated by hydrogen peroxide

Here, we report the identification and characterization of five ortho-quinone inhibitors of PTPalpha. We observed that the potency of these compounds in biochemical assays was markedly enhanced by the presence of DTT. A kinetic analysis suggested that they were functioning as irreversible inhibitors and that the inhibition was targeted to the catalytic site of PTPalpha. The inhibition observed by these compounds was sensitive to superoxide dismutase and catalase, suggesting that reactive oxygen species may be mediators of their inhibition. We observed that in the presence of DTT, these compounds would produce up to 2.5mM hydrogen peroxide (H(2)O(2)). The levels of H(2)O(2) produced were sufficient to completely inactivate PTPalpha. In contrast, without a reducing agent the compounds did not generate H(2)O(2) and showed little activity towards PTPalpha. In addition, these compounds inhibited PTPalpha-dependent cell spreading in NIH 3T3 cells at concentrations that were similar to their activity in biochemical assays. The biological implications of these results are discussed as they support growing evidence that H(2)O(2) is a key regulator of PTPs.     

A regulatory domain in the C-terminal extension of the yeast glycerol channel Fps1p

The Saccharomyces cerevisiae gene FPS1 encodes an aquaglyceroporin of the major intrinsic protein (MIP) family. The main function of Fps1p seems to be the efflux of glycerol in the adaptation of the yeast cell to lower external osmolarity. Fps1p is an atypical member of the family, because the protein is much larger (669 amino acids) than most MIPs due to long hydrophilic extensions in both termini. We have shown previously that a short domain in the N-terminal extension of the protein is required for restricting glycerol transport through the channel (Tamás, M. J., Karlgren, S., Bill, R. M., Hedfalk, K., Allegri, L., Ferreira, M., Thevelein, J. M., Rydstr?m, J., Mullins, J. G. L., and Hohmann, S. (2003) J. Biol. Chem. 278, 6337-6345). Deletion of the N-terminal domain results in an unregulated channel, loss of glycerol, and osmosensitivity. In this work we have investigated the role of the Fps1p C terminus (139 amino acids). A set of eight truncations has been constructed and tested in vivo in a yeast fps1Delta strain. We have performed growth tests, membrane localization following cell fractionation, and glycerol accumulation measurements as well as an investigation of the osmotic stress response. Our results show that the C-terminal extension is also involved in restricting transport through Fps1p. We have identified a sequence of 12 amino acids, residues 535-546, close to the sixth transmembrane domain. This element seems to be important for controlling Fps1p function. Similar to the N-terminal domain, the C-terminal domain is amphiphilic and has a potential to dip into the membrane.     

Immunomagnetic enrichment of interstitial cells of Cajal

Disruptions of networks of interstitial cells of Cajal (ICC), gastrointestinal pacemakers and mediators of neurotransmission, can lead to disordered phasic contractions and peristalsis by reducing and uncoupling electrical slow waves. However, detailed analysis of the ICC network behavior has been hampered by their scarcity, limited accessibility in intact tissues, and contamination with other cell types in culture. Our goal was to develop a simple technique to purify ICC from murine gastrointestinal muscles for functional studies. We identified ICC in live small intestinal muscles or primary cell cultures by Kit immunoreactivity using fluorescent antibodies. Because this technique also labels resident macrophages nonspecifically, parallel studies were performed in which nonfluorescent Kit antibodies and macrophages labeled with fluorescent dextran were used for subtractive analysis of ICC. In both groups, Kit-positive cells were tagged with superparamagnetic antibodies and sorted on magnetic columns. Efficacy was assessed by flow cytometry. ICC enrichment from primary cultures and freshly dissociated tissues was approximately 63-fold and approximately 8-fold, respectively. Unlike the cells derived directly from tissues, cells sorted from cultures frequently yielded extensive, nearly homogenous ICC networks on reseeding. Monitoring oscillations in mitochondrial Ca(2+) or membrane potential by imaging revealed spontaneous rhythmicity in these networks. Cells that did not bind to the columns yielded cultures that were depleted of ICC and dominated by smooth muscle cells. In conclusion, immunomagnetic sorting of primary cultures of ICC results in relatively homogenous, functional ICC networks. This technique is less suitable for obtaining ICC from freshly dispersed cells.     

Distribution of PACAP-38 in the central nervous system of various species determined by a novel radioimmunoassay

Pituitary adenylate cyclase activating polypeptide (PACAP) occurs in two molecular forms: PACAP-38 and PACAP-27. Soon after the isolation and chemical characterization of PACAP, the first radioimmunoassay (RIA) methods have been developed, but it is a still rarely used laboratory technique in the field of PACAP research. The aim of the present study was to develop a novel, highly specific PACAP-38 assay to investigate the quantitative distribution of PACAP-38 in the central nervous system of various vertebrate species under the same technical and experimental conditions. Different areas of the brain and the spinal cord were removed from rats, chickens and fishes and the tissue samples were processed for PACAP-38 RIA. Our results indicate that the antiserum used in the RIA is C-terminal specific, without affinity for other members of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon peptide family. The average ID50 value was 48.6+/-3.4 fmol/ml determined in 10 consecutive assays. Detection limit for PACAP-38 proved to be 2 fmol/ml. PACAP-38 immunoreactivity was present in the examined brain areas of each species studied, with highest concentration in the rat diencephalons. High levels of PACAP-38 were also detected in the rat telencephalon, followed by spinal cord and brainstem. The central nervous system of the fish also contained considerable concentrations of PACAP-38, whereas lowest concentrations were measured in the central nervous system of the chicken.     

Peroxidase mediated hydrogen peroxide production in barley roots grown under stress conditions

All applied metals (Co, Al, Cu, Cd) and NaCl inhibited barley root growth. No root growth inhibition was caused by drought exposure, in contrast to cold treatment. 0.01 mM H₂O₂ stimulated root growth and GA application did not affect root growth at all. Other activators and inhibitors of H₂O₂ production (SHAM, DTT, 10 mM H₂O₂, 2,4-D) inhibited root growth. Loss of cell viability was most significant after Al treatment, followed by Cd and Cu, but no cell death was induced by Co. Drought led to slight increase in Evans blue uptake, whereas neither NaCl nor cold influenced this parameter. DTT treatment caused slight increase in Evans blue uptake and significant increases were detected after 2,4-D and 10 mM H₂O₂ treatment, but were not induced by others stressors. Metal exposure increased guaiacol-POD activity, which was correlated with oxidation of NADH and production of H₂O₂. Exposure to drought caused a minor change in NADH oxidation, but neither H₂O₂ production nor guaiacol-POD activity was increased. Cold and NaCl application decreased all monitored activities. Increase in NADH oxidation and guaiacol-POD activity was caused by 10 mM H₂O₂ and 0.01 mM 2,4-D treatment, which also caused enhancement of H₂O₂ production. Slight inhibition of all activities was caused by 0.01 mM H₂O₂, GA, DTT; more pronounced inhibition was detected after SHAM treatment. The role of H₂O₂ production mediated by POD activity in relation to root growth and cell viability under exposure to some abiotic stress factors is discussed.     

Nitric oxide synthesis is blocked in the enteral nervous system during dormant periods of the snail<Emphasis Type="Italic"> Helix lucorum</Emphasis>

During dormancy of terrestrial snails, the whole neuromodulation of the nervous system is deeply modified. In this work we studied the adaptation of a previously described, putatively nitric oxide (NO) forming enteral network to the long-term resting periods of the snail Helix lucorum. The standard NADPH diaphorase (NADPHd) technique, which is an accepted method for histochemical NO synthase (NOS) detection, labeled the same enteric neurons of the midintestine in active or hibernated snails. Quantification of the NO-derived nitrite by the Griess reaction established that the nitrite formation is confined to the NADPHd-reactive network containing the midintestinal segment. In active snails, the nitrite formation could be enhanced by the NOS substrate l-arginine (10 M–1 mM), but decreased by the known NOS inhibitors 1 mM N-nitro-l-arginine (NOARG) and 10 mM aminoguanidine (AG). Application of 1 mM l-arginine and 1 mM NOARG decreased the amplitude of the midintestinal muscle contractile activity, but did not affect the rectal motility. In dormancy, the nitrite formation was reduced in the NADPHd-reactive midintestinal network. Application of l-arginine could not provoke nitrite production and did not influence the midintestinal motility. Our findings indicate that NO is involved in the neural transmission to intestinal muscles of gastropods, but enteric release of NO is blocked during dormancy. The decreased NO synthesis is possibly due to an as yet undefined mechanism, by which the l-arginine/NO conversion ability of NOS could temporarily be inhibited in the long-term resting period of H. lucorum.T. Röszer and Zs. Czimmerer contributed equally to this work as lead authors. This research was supported by OTKA grant no. T42762 (G.B.) and PRCH Student Science Foundation grants 2000, 2002 (T.R.) and 2003 (Zs. Cz). The study is dedicated to Borbála Vecsei-Czimmerer, Elemér Czimmerer, Ágnes M. Fodor-Röszer and József S. Röszer.     

The effect of LIGHT in inducing maturation of monocyte-derived dendritic cells from MDS patients

LIGHT is a recently cloned novel cytokine belonging to the TNF family that is selectively expressed on immature dendritic cells (iDCs) generated from monocytes isolated from human PBMCs. In these studies, we demonstrate that exogenous soluble LIGHT or soluble CD40 ligand (CD40L) can promote monocyte-derived dendritic cell maturation in vitro by the up-regulation of CD86, CD80, CD83, and HLA-DR antigen expression. Immature dendritic cells differentiated from monocytes of MDS patients displayed lower levels of costimulatory and HLA-DR molecules compared with iDCs differentiated from monocytes of normal subjects. However, upon induction of maturation by LIGHT or CD40L, the expression of costimulatory and HLA-DR molecules is comparable between DCs from MDS and normal subjects. Exogenous LIGHT- and CD40L-stimulated mature DCs (mDCs) also displayed increased antigen presentation to autologous T lymphocytes (tetanus toxin) or allogeneic T lymphocytes in mixed lymphocyte reactions. DCs matured by LIGHT showed increased secretion of IL-6, IL-12p75, and TNF-, but not IL-1. We conclude that both LIGHT and CD40L are immunoregulating factors that induce monocyte-derived iDCs from MDS patients to undergo maturation resulting in increased antigen presentation and T-cell activation. Monocyte-derived DCs can be stimulated to undergo phenotypic and functional changes with LIGHT that might be applied in the development of a DC-based vaccine for MDS treatment.     

c-Myc localization within the nucleus: evidence for association with the PML nuclear body

Definitive localization of c-Myc within the nucleus is important to fully understand the regulation and function of this oncoprotein. Studies of c-Myc distribution, however, have produced conflicting results. To overcome technical challenges inherent in c-Myc cytology, we use here three methods to visualize c-Myc and in addition examine the impact of proteasome inhibition. EYFP or HA-tagged Myc was reintroduced by stable transfection into myc null diploid rat fibroblasts, replacing endogenous Myc with tagged Myc expressed at or near normal levels. This tagged Myc is shown to functionally replace the endogenous Myc by restoration of normal cell morphology and growth rate. We were able to confirm key findings using antibodies to the endogenous c-Myc and/or its partner, Max. Contrary to some published reports, by all three methods the c-Myc protein in rat fibroblasts distributes predominantly throughout the nucleus in a dispersed granular pattern, avoiding the nucleolus. Importantly, however, several findings provide evidence for an unanticipated relationship between c-Myc and PML nuclear bodies, which is enhanced under conditions of proteasome inhibition. Evidence of Max concentration within PML bodies is shown both with and without proteasome inhibition, strengthening the relationship between PML bodies and Myc/Max. Some accumulation of Myc and Max in nucleoli upon proteasome inhibition is also observed, although co-localization of ubiquitin was only seen with PML bodies. This work provides a comprehensive study of c-Myc distribution and also presents the first evidence of a relationship between turnover of this oncoprotein and PML nuclear bodies, known to break down in certain cancers.