Binding of interferon gamma by glycosaminoglycans: a strategy for localization and/or inhibition of its activity.

Glycosaminoglycans (GAGs) are a group of negatively charged molecules present in many tissues as components of the extracellular matrix, basement and cellular membranes. This work analysed the ability of this group of substances to interact with human interferon gamma and the effect of those interactions on its biologic activity. A variety of GAGs (heparin, heparan sulfate, chondroitin sulfate and hyaluronic acid), and a related sulfated polysaccharide (dextran sulfate), were found to interact with IFN-gamma as determined by inhibition of the binding of [125I]IFN-gamma to COLO-205 cells and binding to wells coated with GAGs. These interactions were inhibited by synthetic peptides mimicking the sequences of the basic amino acid cluster located at the C-terminal end of mouse and human IFN-gamma, or by poly-L-lysine, suggesting that ionic interactions between the positively-charged C-terminus and negatively charged groups in GAGs were involved. IFN-gamma molecules bound to plate-immobilized or endothelial cell surface GAGs retained biological activity, since they could induce major histocompatibility complex (MHC) class II expression on COLO-205 cells, suggesting that cell surface GAGs might be able to present IFN-gamma to its receptors. These results suggest important regulatory roles for GAGs on the activity of IFN-gamma in vivo.     

Reversible inhibition of mitochondrial complex I activity following chronic dopaminergic glutathione depletion in vitro: implications for Parkinson's disease

The pathogenesis underlying the selective degeneration of nigral dopaminergic neurons in Parkinson's disease is not fully understood but several lines of evidence implicate the role of oxidative stress and mitochondrial dysfunction. Depletion in levels of the thiol reducing agent glutathione (GSH + GSSG) is the earliest reported biochemical event to occur in the Parkinsonian substantia nigra prior to selective loss of complex I (CI) activity associated with the disease believed to contribute to subsequent dopaminergic cell death. Recent studies from our laboratory have demonstrated that acute reduction in both cellular and mitochondrial glutathione levels results in increased oxidative stress and a decrease in mitochondrial function linked to a selective decrease in CI activity through an NO-mediated mechanism (Jha, N.; Jurma, O.; Lalli, G.; Liu, Y.; Pettus, E. H.; Greenamyre, J. T.; Liu, R. M.; Forman, H. J.; Andersen, J. K. Glutathione depletion in PC12 results in selective inhibition of mitochondrial complex I activity. Implications for Parkinson's disease J. Biol. Chem. 275: 26096-26101; 2000. Hsu, M.; Srinivas, B.; Kumar, J.; Subramanian, R.; Andersen, J. Glutathione depletion resulting in selective mitochondrial complex I inhibition in dopaminergic cells is via an NO-mediated pathway not involving peroxynitrite: implications for Parkinson's disease J. Neurochem. 92: 1091-1103.2005.). However, the effect of prolonged glutathione depletion on dopaminergic cells is not known. In this present study, using low concentrations of buthionine-S-sulfoximine, a chemical inhibitor of the de novo glutathione synthesizing enzyme glutamate cysteine ligase, we developed a chronic model in which glutathione depletion in dopaminergic N27 cells for a 7-day period was found to lead to inhibition of CI activity via a peroxynitrite-mediated event which is reversible by the thiol reducing agent, dithiothreitol, and coincides with increased S-nitrosation of mitochondrial proteins.     

Trypsin inhibition activity of heat-denatured ovomucoid: a kinetic study

A kinetic study was conducted on the effect of heating in the temperature range of 75-110 degrees C on the trypsin inhibition activity of ovomucoid. Heat treatment of isolated ovomucoid resulted in a time-dependent decrease in trypsin inhibition activity that could accurately be described by a first-order kinetic model. The magnitude and the temperature dependence of the rate constants was affected by the pH during heat treatment. The heat stability of ovomucoid was the lowest at pH 7.6. Heat treatments intended to decrease the trypsin inhibition activity should therefore be carried out as soon as possible after laying, because the ovomucoid was inactivated faster at the pH of fresh egg white (pH 7.6). The presence of the other egg white constituents decreased the heat stability of ovomucoid compared to that of the model system of ovomucoid in buffer, presumably by the formation of ovomucoid-lysozyme complexes in the former.     

Suppression of nigrostriatal and mesolimbic dopamine release in vivo following noradrenaline depletion by DSP-4: a microdialysis study.

Pretreatment of rats with the noradrenergic neurotoxin DSP-4 selectively reduced regional levels of noradrenaline in the brain by more than 75%, and decreased the concentration of endogenous DA in microdialysates of the caudate nucleus and nucleus accumbens by 52% and 28%, respectively. Results support the hypothesis that central noradrenergic mechanisms facilitate nigrostriatal and mesolimbic dopamine transmission in vivo.     

A chronic stress that impairs reactivity in rats also decreases dopaminergic transmission in the nucleus accumbens: a microdialysis study

Chronic stress induces in rats a decreased reactivity toward noxious stimuli (escape deficit), which can be reverted by antidepressant treatments. The present study reports that this condition of behavioral deficit is accompanied by a decreased level of extracellular dopamine in the nucleus accumbens shell. To assess whether this finding was the result of a decreased release or of an enhanced removal of dopamine, we acutely administered cocaine, and 2 h later d-amphetamine, to stressed and control rats. The increases in dopamine output observed in stressed animals after cocaine administration were significantly lower than those observed in control rats; whereas the total amount of dopamine released after d-amphetamine administration was similar in both groups of rats. These data suggest a reduced activity of dopaminergic neurons as the possible mechanism underlying dopamine basal level reduction in stressed animals. It is interesting that the stress group showed a locomotor response to cocaine not different from control rats, thus suggesting a condition of sensitization to dopamine receptor stimulation. Imipramine administered daily concomitantly with stress exposure completely reverted the escape deficit condition of chronically stressed rats. Moreover, stressed rats treated with imipramine showed basal and cocaine stimulated levels of extraneuronal dopamine similar to those observed in control animals.     

Mechanism of the inhibition of milk xanthine oxidase activity by metal ions: a transient kinetic study

The nature and mechanism of the inhibition of the oxidoreductase activity of milk xanthine oxidase (XO) by Cu(2+), Hg(2+) and Ag(+) ions has been studied by steady state and stopped flow transient kinetic measurements. The results show that the nature of the inhibition is noncompetitive. The inhibition constants for Cu(2+) and Hg(2+) are in the micromolar and that for Ag(+) is in the nanomolar range. This suggests that the metal ions have strong affinity towards XO. pH dependence studies of the inhibition indicate that at least two ionisable groups of XO are involved in the binding of these metal ions. The effect of the interaction of the metal ions on the reductive and oxidative half reactions of XO has been investigated, and it is observed that the kinetic parameters of the reductive half reaction are not affected by these metal ions. However, the interaction of these metal ions with XO significantly affects the kinetic parameters of the oxidative half reaction. It is suggested that this may be the main cause for the inhibition of XO activity by the metal ions.     

Regulation of axotomy-induced dopaminergic neuron death and c-Jun phosphorylation by targeted inhibition of cdc42 or mixed lineage kinase

Mechanical transection of the nigrostriatal dopamine pathway at the medial forebrain bundle (MFB) results in the delayed degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). We have previously demonstrated that c-Jun activation is an obligate component of neuronal death in this model. Here we identified the small GTPase, cdc42, and mixed lineage kinases (MLKs) as upstream factors regulating neuronal loss and activation of c-Jun following MFB axotomy. Adenovirus-mediated expression of a dominant-negative form of cdc42 in nigral neurons blocked MFB axotomy-induced activation (phosphorylation) of MAP kinase kinase 4 (MKK4) and c-Jun, resulting in attenuation of SNpc neuronal death. Pharmacological inhibition of MLKs, MKK4-activating kinases, significantly reduced the phosphorylation of c-Jun and abrogated dopaminergic neuronal degeneration following MFB axotomy. Taken together, these findings suggest that death of nigral dopaminergic neurons following axotomy can be attenuated by targeting cell signaling events upstream of c-Jun N-terminal mitogen-activated protein kinase/c-Jun.     

Effect of nicotine on extracellular levels of neurotransmitters assessed by microdialysis in various brain regions: Role of glutamic acid

We studied the effect of local administration of nicotine on the release of monoamines in striatum, substantia nigra, cerebellum, hippocampus, cortex (frontal, cingulate), and pontine nucleus and on the release of glutamic acid in striatum of rats in vivo, using microdialysis for nicotine administration and for measuring extracellular amine and glutamic acid levels. Following nicotine administration the extracellular concentration of dopamine, increased in all regions except cerebellum; serotonin increased in cingulate and frontal cortex; and norepinephrine increased in substantia nigra, cingulate cortex, and pontine nucleus. Cotinine, the major nicotine metabolite, had no effect at similar concentrations. The cholinergic antagonists mecamylamine and atropine, the dopaminergic antagonists haloperidol and sulpiride, and the excitatory amino acid antagonist kynurenic acid all inhibited the nicotine-induced increase of extracellular dopamine in the striatum. The fact that kynurenic acid almost completely prevented the effects of nicotine, and nicotine at this concentration produced a 6-fold increase of glutamic acid release, suggests that the effect of nicotine is mainly mediated via glutamic acid release.     

Inhibition of noxious stimulus-induced spinal prostaglandin E2 release by flurbiprofen enantiomers: a microdialysis study

Peripheral noxious stimuli have been shown to induce prostaglandin (PG) E2 release at the site of inflammation and in the spinal cord. The antiinflammatory and antinociceptive effects of cyclooxygenase-inhibiting drugs are thought to depend on the inhibition of PG synthesis. R-Flurbiprofen, however, does not inhibit cyclooxygenase activity in vitro but still produces antinociceptive effects. To find out whether R-flurbiprofen acts via inhibition of spinal PG release, concentrations of PGE2 and flurbiprofen in spinal cord tissue were assessed by microdialysis. The catheter was transversally implanted through the dorsal horns of the spinal cord at level L4. R- and S-flurbiprofen (9 and 27 mg kg(-1), respectively) were administered intravenously 10-15 min before subcutaneous injection of formalin into the dorsal surface of one hindpaw. Flurbiprofen was rapidly distributed into the spinal cord with maximal concentrations after 30-45 min. Baseline PGE2 dialysate concentrations were 100.6 +/- 6.4 pg ml(-1) (mean +/- SEM). After formalin injection they rose about threefold with a maximum of 299.4 +/- 68.4 pg ml(-1) at 7.5 min. After approximately 1 h PGE2 levels returned to baseline. Both flurbiprofen enantiomers completely prevented the formalin-induced increase of spinal PGE2 release and reduced PGE2 concentrations below basal levels. S- and R-flurbiprofen at 9 mg kg(-1) produced a minimum of 15.8 +/- 5.2 and 27.7 +/- 14.9 pg ml(-1), respectively, and 27 mg kg(-1) S- and R-flurbiprofen resulted in 11.7 +/- 1.7 and 9.3 +/- 4.7 pg ml(-1), respectively. PGE2 levels remained at the minimum up to the end of the observation period at 5 h. When 27 mg kg(-1) R-flurbiprofen was injected intravenously without subsequent formalin challenge, baseline immunoreactive PGE2 concentrations were not affected. S-Flurbiprofen (27 mg kg(-1)), however, led to a moderate reduction (approximately 40%). The data suggest that antinociception produced by R-flurbiprofen is mediated at least in part by inhibition of stimulated spinal PGE2 release and support the current view that increased spinal PGE2 release significantly contributes to nociceptive processing.     

Developmental capacity of bovine oocytes following inhibition of meiotic resumption by cycloheximide or 6-dimethylaminopurine

This study was conducted to assess the fertilizability and developmental capacity of bovine oocytes which had been maintained in meiotic arrest by either a protein synthesis inhibitor, cycloheximide (CHX), or an inhibitor of serine/threonine protein kinases, 6-dimethylaminopurine (6-DMAP). Both CHX and 6-DMAP reversibly prevented nuclear maturation of nearly all oocytes for 24 h. After the reversal of arrest, CHX-treated oocytes could be successfully matured and fertilized. They developed to the blastocyst stage at slightly lower rates than oocytes cultured without inhibition for 22 h prior to sperm addition but at higher rates than those incubated in a medium containing no inhibitors for 46 h prior to fertilization. Oocytes inhibited by CHX for 48 h matured and fertilized normally but failed to develop into blastocysts. Even though 6-DMAP-treated oocytes completed meiosis I after removal from the drug, the rates of fertilization and blastocyst formation were lower than for untreated oocytes or CHX-treated oocytes. Effects of adding FSH and/or estradiol-17 beta (E(2)) during CHX-inhibition for 24 h were also examined. Embryos from oocytes treated with CHX and E(2) or with CHX and FSH + E(2) developed into blastocysts at similar rates as the controls. Further development of inhibited oocytes was examined by transferring blastocysts derived from oocytes inhibited by CHX with FSH and E(2) for 24 h to recipient heifers. Two calves were obtained following transfer. These results indicate that CHX-inhibited oocytes retain developmental competence, while 6-DMAP-inhibited oocytes after the reversal of arrest have reduced capacities for fertilization and further development. The addition of FSH and E(2) during CHX-inhibition improves development to the blastocyst stage of the oocytes that are capable of initiating and maintaining pregnancy after embryo transfer to recipient animals.     

Role of calcium in gamma interferon induction: inhibition by calcium entry blockers

Mitogen-induced gamma interferon production by human lymphoid cell cultures was studied in the presence of calcium entry blockers. A dose-dependent inhibition was found in the presence of drug concentrations down to 10(-5) M. This finding shows that calcium flow through lymphocyte membranes after oxidation of membrane-bound galactose residues is also critical for triggering interferon production.     

Benzodiazepine-sensitive GABA(A) receptors limit the activity of the NMDA/NO/cyclic GMP pathway: a microdialysis study in the cerebellum of freely moving rats

In the cerebellum, infusion of NMDA (200 microM) for 20 min evoked a marked (200%) increase of extracellular cyclic GMP (cGMP) levels. The selective GABA(A) receptor agonist muscimol (0.01-100 microM) was able to counteract the NMDA effect with an EC(50) of 0.65 microM; the inhibitory effect of muscimol (10 microM) was prevented by bicuculline (50 microM). Diazepam (10 microM) significantly potentiated the muscimol (1 microM) inhibition; furthermore, when coinfused with 0.1 microM muscimol (a concentration not affecting, on its own, the cGMP response to NMDA), diazepam (10 microM) reduced the NMDA effect. Similar results were obtained with zolpidem (0.1-1 microM). Finally, local infusion of the benzodiazepine site antagonist flumazenil (10 microM), together with muscimol and diazepam, almost completely restored the effect of NMDA on extracellular cGMP levels. It is concluded that GABA(A) receptors potently control the NMDA/nitric oxide/cGMP pathway in the cerebellum in vivo. In terms of the alpha subunit composition, we can deduce that the cerebellar GABA(A) receptor does not contain alpha(6) or beta(4) subunits because it is diazepam-sensitive. Moreover, the observation that zolpidem is active at a rather low concentration, in combination with localization studies present in the literature, tend to exclude the presence of alpha(5) subunits in the receptor composition and suggest the involvement of an alpha(1) subunit.     

A comparison of mediators released or generated by IFN-gamma-treated human mast cells following aggregation of Fc gamma RI or Fc epsilon RI

The high affinity receptor for IgG (Fc gamma RI, CD64) is expressed on human mast cells, where it is up-regulated by IFN-gamma and, thus, may allow mast cells to be recruited through IgG-dependent mechanisms in IFN-gamma-rich tissue inflammation. However, the mediators produced by human mast cells after aggregation of Fc gamma RI are incompletely described, and it is unknown whether these mediators are distinct from those produced after activation of human mast cells via Fc epsilon RI. Thus, we investigated the release of histamine and arachidonic acid metabolites and examined the chemokine and cytokine mRNA profiles of IFN-gamma-treated cultured human mast cells after Fc gamma RI or Fc epsilon RI aggregation. Aggregation of Fc gamma RI resulted in histamine release and PGD(2) and LTC(4) generation. These responses were qualitatively indistinguishable from responses stimulated via Fc epsilon RI. Aggregation of Fc epsilon RI or Fc gamma RI led to an induction or accumulation of 22 cytokine and chemokine mRNAs. Among them, seven cytokines (TNF-alpha, IL-1beta, IL-5, IL-6, IL-13, IL-1R antagonist, and GM-CSF) were significantly up-regulated via aggregation of Fc gamma RI compared with Fc epsilon RI. TNF-alpha mRNA data were confirmed by quantitative RT-PCR and ELISA. Furthermore, we confirmed histamine and TNF-alpha data using IFN-gamma-treated purified human lung mast cells. Thus, aggregation of Fc gamma RI on mast cells led to up-regulation and/or release of three important classes of mediators: biogenic amines, lipid mediators, and cytokines. Some cytokines, such as TNF-alpha, were released and generated to a greater degree after Fc gamma RI aggregation, suggesting that selected biologic responses of mast cells may be preferentially generated through Fc gamma RI in an IFN-gamma-rich environment.     

Protection from quinidine or physostigmine against in vitro inhibition by sarin of acetylcholinesterase activity

We have studied the relative effectiveness of quinidine and physostigmine in protecting against the inhibition of acetylcholinesterase (AChE) by sarin, an organophosphate (OP) compound. The protective effects of these compounds were studied in vitro in both synaptosomal and soluble samples obtained from various regions of sarin-administered or control isolated, perfused canine brain. Although AChE activities in the sarin-administered brain were substantially lower than in the control brain, we observed regional differences in the AChE activity in both. The AChE in the control brain and the AChE remaining in sarin-administered brain had different susceptibilities to inhibition from OP compounds in vitro and, therefore, have different properties. Quinidine partially protected AChE from the inhibitory effects of sarin in vitro possibly by altering the sarin binding sites. Addition of sarin to physostigmine-treated control brain samples allowed partial recovery of the AChE activity. The protective effects of quinidine or physostigmine were lost when samples from sarin-administered brain were treated in vitro with these compounds and then again exposed to sarin. Therefore, both quinidine and physostigmine provided partial protection against the inhibitory effects of sarin in vitro if they were added prior to sarin.     

Life shortening and disease incidence in BALB/c mice following a single d(50)-Be neutron or gamma exposure

Male BALB/c mice, 12 weeks old, were given a single exposure of either 137Cs gamma rays or d(50)-Be neutrons at a dose rate of 3 Gy/min. The animals were kept until death, and causes of death or possible causes of death were ascertained by autopsy and histology. The data were evaluated by competing risk methods. The survival time dose-effect curve for both types of exposure was linear and did not differ significantly (slopes: 55.8 +/- 4.0 days/Gy for neutrons and 46.2 +/- 4.3 days/Gy for gamma rays). The incidence of different diseases also was similar for both groups except that more carcinomas, sarcomas, and myeloid leukemias seemed to occur after neutron exposure and that nonstochastic lung and kidney diseases seemed to arise at lower doses.