Inflaming the diseased brain: a role for tainted melanins.

Inflammation plays a crucial role in neurodegenerative diseases, but the irritants responsible for this response remain largely unknown. This report addressed the hypothesis that hypochlorous acid reacts with dopamine to produce melanic precipitates that promote cerebral inflammation. Spectrophotometric studies demonstrated that nM amounts of HOCl and dopamine react within seconds. A second-order rate constant for the reaction of HOCl and dopamine of 2.5 × 10(4)M(-1)s(-1) was obtained by measuring loss of dopaminergic fluorescence due to HOCl. Gravimetric measurements, electron microscopy, elemental analysis, and a novel use of flow cytometry confirmed that the major product of this reaction is a precipitate with an average diameter of 1.5 µm. Flow cytometry was also used to demonstrate the preferential reaction of HOCl with dopamine rather than albumin. Engulfment of the chlorodopamine particulates by phagocytes in vitro caused these cells to release TNFα and die. Intrastriatal administration of 10(6) particles also increased the content of TNFα in the brain and led to a 50% loss of the dopaminergic neurons in the nigra. These studies indicate that HOCl and dopamine react quickly and preferentially with each other to produce particles that promote inflammation and neuronal death in the brain.

Oxysterol derivatives of cholesterol in neurodegenerative disorders.

Cholesterol is essential to the functions of the brain, which contains approximately 20% of the body's stores of this sterol. Most brain cholesterol is found in compacted myelin. The operation of the blood brain barrier (BBB) precludes the uptake of cholesterol from the periphery and consequently this sterol is produced de novo in the brain. In contrast, oxysterols - a class of hydroxylated cholesterol catabolites - traverse the BBB readily and facilitate the elimination of cholesterol from the brain. Oxysterols not only act as a transport form of cholesterol, but serve as endogenous regulators of gene expression in lipid metabolism and behave as ligands to nuclear receptors. Two of the more important brain-derived oxysterols are 24S-hydroxycholesterol and 27-hydroxycholesterol. Aberrant cholesterol metabolism has been implicated in a number of neurological disorders. Since oxysterols are thought to reflect the cerebral cholesterol turnover there has been great interest in the diagnostic and prognostic value of these metabolites in neurodegenerative diseases of the brain. The following article provides an overview of the involvement of oxysterols in Alzheimer's disease, multiple sclerosis and spastic paraplegias.

N(epsilon)-(gamma-L-glutamyl)-L-lysine (GGEL) is increased in cerebrospinal fluid of patients with Huntington's disease.

Pathological-length polyglutamine (Q(n)) expansions, such as those that occur in the huntingtin protein (htt) in Huntington's disease (HD), are excellent substrates for tissue transglutaminase in vitro, and transglutaminase activity is increased in post-mortem HD brain. However, direct evidence for the participation of tissue transglutaminase (or other transglutaminases) in HD patients in vivo is scarce. We now report that levels of N(epsilon)-(gamma-L-glutamyl)-L-lysine (GGEL)--a 'marker' isodipeptide produced by the transglutaminase reaction--are elevated in the CSF of HD patients (708 +/- 41 pmol/mL, SEM, n = 36) vs. control CSF (228 +/- 36, n = 27); p < 0.0001. These data support the hypothesis that transglutaminase activity is increased in HD brain in vivo.

Mechanisms for the cytotoxicity of cysteamine.

The major aim of this study was to quantitatively assess the contribution of H2O2 generation to the cytotoxicity induced by cysteamine. Cysteamine produces H2O2 at levels that correlate with its toxicity between 23 and 160 microM. A maximum of 6.9 microM H2O2 is generated by 625 microM cysteamine. When compared to the toxicity of exogenous H2O2, cysteamine-derived peroxide accounted for 57% of its toxicity. This corresponded to the percent toxicity due to 23 to 91 microM cysteamine. The remaining 43% toxicity appears to involve the inhibition of glutathione peroxidase, because activity of both the cellular and purified enzyme were inhibited by 200 microM cysteamine concentrations. CCRF-CEM cells have no catalase activity, so the inhibition of glutathione peroxidase may sensitize these cells to the less than toxic levels of peroxide generated by this aminothiol. Cysteamine also stimulated the production of cellular glutathione in a manner that was not related to its H2O2 generation. The production of glutathione did not influence toxicity but may reflect the accumulation of cysteamine to levels that inhibit glutathione peroxidase.

A sensitive fluorometric assay for tissue transglutaminase.

We have devised a highly sensitive fluorometric well plate assay for tissue transglutaminase that is suitable for multiple kinetic analyses/high-throughput screening of chemical inventories for inhibitors of this enzyme. The procedure measures the rate of fluorescence enhancement (lambda(exc) 260 nm, lambda(em) 538 nm) when 1-N-(carbobenzoxy-l-glutaminylglycyl)-5-N-(5'N'N'-dimethylaminonaphthalenesulfonyl)diamidopentane (glutaminyl substrate) is cross-linked to dansyl cadaverine (amine substrate). The assay procedure can be used to measure the activity of as little as 60 microU of purified guinea pig liver tissue transglutaminase (4.2 ng or 54 fmol of enzyme).

Oleic acid increases cell surface expression and activity of CD11b on human neutrophils.

Traumatic bone injury frequently results in the release of marrow-derived fatty material into the circulation. This may lead to the syndrome of fat embolism, associated with the generation of free fatty acids, the sequestration of neutrophils in the lungs, and the subsequent development of acute respiratory distress. Neutrophil accumulation in tissues requires their adherence to vascular endothelial cells and involves the beta2 integrin, CD11b/CD18 (Mac-1). We now report that the exposure of isolated human neutrophils to oleic acid causes a rapid increase in the cell surface expression and affinity state of CD11b, particularly under acidic conditions that are typical of inflammatory sites. Oleic acid also triggers neutrophil aggregation and neutrophil adherence to both fibrinogen-coated surfaces and confluent cultures of HUVEC. These processes are blocked by CD11b-specific inhibitors, including neutrophil-inhibitory factor and mAbs to CD11b. These observations may help explain the etiology of so-called fat embolism wherein trauma-induced release of fatty material causes pulmonary neutrophil accumulation and the development of acute respiratory distress.

Inhibition of drug-naive and -resistant leukemia cell proliferation by low molecular weight thiols.

The aim of these studies was to investigate the ability of cysteamine and its congeners to arrest the proliferation of leukemic cells and to determine the physico-chemical properties responsible for this ability. Fifteen low molecular weight thiol-bearing compounds were shown to arrest the proliferation of CCRF-CEM cells and a methotrexate-resistant subline, with IC50 values between 10(-5) and 10(-4) M. Cysteamine arrested proliferation by slowing the passage of cells through S phase. These cells subsequently resumed cycling, although a proportion went on to die by apoptosis. The antiproliferative action of cysteamine was shown to depend, in part, on H2O2 production. This ability to generate peroxide is shared by many thiol compounds, and molecular modeling indicated that thiol groups were required for the antiproliferative actions of the congeners of cysteamine. Molecular modeling also revealed that the most efficacious antiproliferative agents were those that had their amino acid and thiol moieties separated by an intramolecular distance of 3.17 to 5.9 A, as exemplified by WR 1065 and the aminothiophenols. These findings indicate that thiol-bearing compounds may have some efficacy in the treatment of drug-naive and -resistant leukemia cells.

Induction of magnetic resonance-visible lipid in a transformed human breast cell line by tetraphenylphosphonium chloride.

Proton magnetic resonance spectroscopy (1H MRS) and DNA flow cytometry were used to monitor the effects of the cationic lipophilic phosphonium salt and potential antineoplastic agent tetraphenylphosphonium chloride (TPP) on the transformed human breast cell line HBL-100. TPP treatment for 48 hr was cytostatic at low concentrations and cytotoxic at higher concentrations with an IC50 of 55 microM as measured by Trypan blue exclusion. At micromolar concentrations, TPP caused a significant increase in the methylene MR signal arising from mobile lipid as measured by the ratio of the lipid CH2 peak height to either the CH3 peak height (internal referencing) or the peak height for p-aminobenzoic acid (PABA) as an external reference in a co-axial capillary within the sample. Over the same concentration range, TPP caused a slowing of passage through S phase as demonstrated by a significant depletion of cells in G2/M phase with a concurrent but non-significant increase in cells in S. Time-dependent increases in MR-visible lipid were observed with 2 microM TPP treatment, and the removal of TPP from the culture medium caused no significant reduction in mobile lipid. Two-dimensional 1H-1H COSY spectra of TPP-treated HBL-100 cells revealed concentration-dependent increases in cross-peak volume ratios arising from lipid acyl chains relative to both internal (lysine, polyamines) and external (PABA) standards. Increases in choline and glycerophosphocholine cross-peak volume ratios were observed, indicating that the catabolism or rearrangement of phospholipids may be responsible for the observed MR-visible lipid increases.

Tetraphenylphosphonium chloride induced MR-visible lipid accumulation in a malignant human breast cell line.

The effect of the cationic lipophilic phosphonium salt tetraphenylphosphonium chloride (TPP) on a human malignant breast cell line, DU4475, was monitored with proton nuclear magnetic resonance (1H MRS). TPP caused a dose- and time- dependent increase in resonances arising from MR-visible lipid as measured by the CH2/CH3 ratio in the 1-dimensional 1H MR spectrum. Two-dimensional MRS identified increases in the glycerophosphocholine/lysine cross-peak ratio and corresponding decreases in the phosphocholine/lysine ratio in a dose- dependent fashion in TPP-treated cells. Lipid metabolic changes are discussed in the light of other MR experiments, and the data indicate that accumulation of MR-visible lipids may arise from the rearrangement of phospholipids accompanying mitochondrial destruction or from the catabolism of phospholipids associated with early events in the cytotoxic process.

Cell cycle-dependent inhibition of the proliferation of human neural tumor cell lines by iron chelators.

The current studies were designed to examine the conditions under which the ferric iron chelator desferrioxamine (DFO) arrested cell cycle progression and hence the proliferation of neural cell lines in vitro. DFO arrested proliferation at different stages of the cell cycle depending on the concentration and duration of drug exposure. Twenty-four-hour treatment with 160 microM DFO arrested glioma cells in G1, whereas 72-hr treatment with 10 microM DFO acted to slow the passage of glioma cells through the cell cycle, eventually accumulating in G2/M. Another iron chelator, ADR 529, also inhibited the proliferation of glioma cells by lengthening the period of the cycle and causing the cells to arrest in G2/M. The effects of 10 and 160 microM DFO were irreversible after 24 and 48 hr, respectively, and 10 microM DFO became cytotoxic after 3 days. These observations demonstrate that DFO has different effects on the proliferation of neural tumor cell lines depending on the concentration and time of exposure, which result in different sites of cell cycle arrest. These different in vitro actions of DFO may have ramifications for the successful application of iron chelator therapy in vivo.

Inhibition of the proliferation of human neural neoplastic cell lines by cysteamine.

Cysteamine (CySH), a thiol compound that crosses the blood-brain barrier, inhibited the proliferation of neural neoplastic cells in vitro. The IC50 of cysteamine with respect to inhibition after 72 h of drug exposure, was approximately 70 microM in the glioma cell line, 2607, and approximately 80 microM in the neuroblastoma cell line, DAOY. Interestingly, the inhibition of proliferation of 2607 cells produced by 72 h treatment with CySH could also be induced with exposure periods as short as 8 h. Another thiol bearing compound, penicillamine methyl ester, also arrested the proliferation of 2607 cells with IC50 approximately 160 microM. Cell cycle analysis revealed that CySH acted to lengthen the cell cycle period of 2607 cells by slowing the passage of cells through S phase and caused the cells to finally arrest in G2/M. In the other cell lines tested, CySH arrested cells in all phases of the cell cycle. These observations suggest that CySH and its congeners may have some utility in the treatment of neoplasia in vivo.

Modulation of MR-visible mobile lipid levels by cell culture conditions and correlations with chemotactic response.

A transformed murine fibroblast cell line has been used to assess which criteria govern the appearance of a lipid pool that is mobile on the MR time scale. A high-resolution proton MR signal arising from neutral lipids, including triglyceride and cholesteryl esters, has previously been associated with membrane events in stimulated, transformed and malignant cells. We report that the attenuation of cellular proliferation by confluence or low pH caused significant increases in MR-visible lipid and that the lipid signal could be amplified at high density by the removal of serum. A significant decrease in chemotactic response accompanied the culture of cells at high density, but chemotactic response was not generally linked to alteration of the lipid signal. The appearance of the signal was also not correlated with the proportion of cells in any phase of the cell cycle. Significant changes in the MR-visible pools of the lipid metabolites choline, phosphocholine and glycerophosphocholine were measured under the culture conditions employed with 2D MRS and suggest that MR-visible lipid may arise from the catabolism of phospholipids.

Transcription factors as targets for oxidative signalling during lymphocyte activation.

We previously have demonstrated a requirement for oxidative events during cell cycle entry in T lymphocytes and have hypothesised that reactive oxygen species may act as intracellular signalling agents during lymphocyte activation. In the current study, cysteamine, an aminothiol compound with antioxidant activity, has been used to further investigate the role of oxidative signalling during lymphocyte activation. Treatment of normal human peripheral blood lymphocytes with cysteamine in vitro was found to inhibit proliferation in a dose-dependent manner, with essentially complete inhibition occurring at a dose of 400 microM. This inhibitory effect was limited to the first 2 h after mitogenic activation, localizing the time-frame of action of cysteamine to within the commitment period. It therefore was of interest to establish which, if any, commitment events were affected by oxidative signalling during cell cycle entry. Taking the IL-2 gene as a candidate, we examined the effect of cysteamine treatment on early gene expression during lymphocyte activation, and on the activity of transcription factors AP-1, NF-kappa B, NF-AT and Oct1, whose functions are required for expression of the IL-2 mRNA. Cysteamine treatment inhibited both expression of the IL-2 mRNA and secretion of IL-2 into the culture medium. The inhibitory effect of cysteamine may be mediated at least in part by an effect on transcription factor function, as the DNA binding activities of AP-1 and NF-kappa B extracted from mitogen-stimulated cells were significantly inhibited by cysteamine treatment. Interestingly, Oct1 and NF-AT DNA binding activity were not affected by cysteamine treatment, suggesting that oxidative signalling processes operate in a selective manner. The identification of regulatory proteins, such as transcription factors, as molecular targets for oxidative signalling provides further evidence to implicate oxidative signalling as being intimately involved in the G0 to G1 phase transition in T lymphocytes.

Thiol-bearing compounds selectively inhibit protein kinase C-dependent oxidative events and proliferation in human T cells.

The aminothiol cysteamine at 10(-5) to 10(-4) M concentrations inhibited both the proliferation of mitogenically stimulated human peripheral mononuclear cells and the phorbol myristate acetate-mediated oxidation of 2',7'-dichlorofluorescein within these cells. Both 2',7'-dichlorofluorescein oxidation and the proliferative response were maximally sensitive to cysteamine-induced inhibition during the first 2 h of mitogenic stimulation. This period of sensitivity indicates that cysteamine preferentially arrests cells transiting from G0 to G1 and is the first such demonstration, of an early cell cycle site of arrest for this compound. 2,3-Dimercapto-1-propane-sulfonic acid and WR 1065 were found to be more effective than cysteamine in attenuating T cell replication but not N-acetylcysteine. Aminothiols preferentially inhibited the intracellular oxidation of 2',7'-dichlorofluorescein, rather than the activity of protein kinase C, which initiates the oxidation, indicating that oxidative events are one of a number of crucial and independent events required for the successful transition through G0-G1. Since aminothiols affect both lectin and PMA/ionomycin-directed proliferation, these aminothiol-sensitive events may serve to integrate and regulate common pathways in T cell activation.

Identification of apoptotic and necrotic human leukemic cells by flow cytometry.

Glucocorticoids kill human leukemic cells, CCRF-CEM.f2, by apoptosis. Cell death is preceded by DNA fragmentation into nucleosomal subunits which can be evaluated by DNA gel electrophoresis or by determining the proportion of cleaved DNA in whole cell lysates. Hyperthermic treatment of CCRF-CEM.f2 cells induces necrotic cell death which is characterized by a different DNA gel electrophoretic pattern. However, these techniques cannot distinguish the individual contributions of both forms of death in a heterogeneous population. Therefore, we have developed a flow cytometric method that readily distinguishes apoptotic and necrotic cells on the basis of propidium iodide staining and cellular light scatter characteristics. This method can be used to analyze factors influencing the mechanisms of cell death after cytotoxic drug treatment.

Protein phosphorylation in guinea-pig myenteric ganglia and brain: presence of calmodulin kinase II. protein kinase C and cyclic AMP kinase and characterization of major phosphoproteins.

The aim of this study was to demonstrate the presence of calmodulin-stimulated protein kinase II, protein kinase C, and cyclic AMP-stimulated protein kinase in isolated myenteric ganglia and to characterize the major ganglia phosphoproteins using biochemical and immunochemical techniques. Ganglia from the small intestine of guinea-pigs were isolated, disrupted by sonication in Triton X-100, and phosphorylated. The phosphoprotein patterns obtained were compared with those of synaptosomes from guinea-pig and rat cerebral cortex. Myenteric ganglia were as rich in protein kinase C and cyclic AMP-stimulated protein kinase as brain tissue, but the level of calmodulin-stimulated protein kinase II was relatively lower. The alpha subunit of calmodulin-stimulated protein kinase II was detected by immunoblotting and the beta subunit by autophosphorylation. The ratio of beta to alpha subunit was considerably higher in ganglia than in brain and ganglia beta subunit had a lower apparent molecular weight than the brain enzyme. A number of neuronal phosphoproteins were found in ganglia including the 87,000 mol. wt phosphoprotein, synapsins 1a and 1b, and proteins IIIa and IIIb. A phosphoprotein of 48,000 mol. wt had many of the characteristics of the B-50 protein but was not the same. In addition, a number of other phosphoproteins not previously identified in neurons were found in ganglia including those with apparent molecular weights of 60,000 and 58,000 that were the major calmodulin kinase substrates. The guinea-pig enteric nervous system has been extensively studied but, unlike other parts of the mammalian nervous system, little is known about the intracellular mechanisms underlying its functions. A technique for isolating myenteric ganglia is now available and we have used this preparation to characterize the major protein kinase and phosphoproteins present in this tissue. The results obtained will allow the phosphorylation of the various proteins to be investigated after physiological or pharmacological manipulation of myenteric ganglia in situ and in vivo.

Possible oncostatic action of cysteamine on the pituitary glands of oestrogen-primed hyperprolactinaemic rats.

Cysteamine was investigated for its potential to reduce the size and secretion of oestrogen-primed hyperprolactinaemic rat pituitary glands. Subcutaneous administration of 80 and 120 mg cysteamine/kg significantly reduced plasma prolactin concentrations by 58 and 91% respectively, after 4h. Administration of cysteamine (60 mg s.c./kg body weight per day) for 10 days, to rats which had received an injection of 2 mg oestradiol benzoate on day 1, resulted in a significant reduction in pituitary mass (19%) and GH concentration (21%). Oral administration of 60 mg cysteamine/kg body weight to hyperprolactinaemic rats also produced a significant reduction in plasma prolactin of 94% after 2h. Oral administration of 60 mg cysteamine/kg body weight per day to rats for a 20-day period, during which they had received two injections of 2 mg oestradiol benzoate on day 1 and day 14 of treatment, resulted in a significant reduction in pituitary mass (29%) and the concentration of trunk blood prolactin concentration (35%). However, when oral cysteamine (60 mg cysteamine/kg body weight per day) was given for 20 days to rats which had been treated with 2 mg oestradiol benzoate once every 14 days over a 90-day period, it caused no change in pituitary weight, prolactin or GH concentration, or the concentration of prolactin in trunk blood.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of cysteamine on the lysosomal enzymes of the hyperprolactinaemic rat pituitary.

The effect of cysteamine on the activity of lysosomal enzymes and the prolactin content of isolated hyperprolactinaemic cells has been investigated. In broken cell preparations, cysteamine markedly stimulated acid prolactin protease activity. In intact cells, however, cysteamine inhibited acid prolactin protease activity and beta-galactosidase. Moreover, the activities of alpha-mannosidase, acid phosphatase, beta-glucuronidase, total arylsulphatase and hexosaminidase were not changed by the addition of cysteamine. Cysteamine significantly depleted the cells of prolactin, and this action was not compromized by the inclusion of either leupeptin, chloroquine or NH4Cl in the incubation media. Taken together, these results indicate that cysteamine does not promote degradation of prolactin and hence depletion of prolactin from the pituitary through a mechanism involving lysosomal enzyme degradation.

The depletion of plasma prolactin by pantethine in oestrogen-primed hyperprolactinaemic rats.

Pantethine was investigated for its potential to deplete prolactin in the plasma and pituitary cells of oestrogen-primed hyperprolactinaemic rats. This compound has been used in the past to deliver cysteamine systemically, through its congener pantetheine, a metabolic precursor for cysteamine. Cysteamine itself, specifically reduces plasma and pituitary prolactin. The addition of pantethine (2-10 mmol/l) to the media of isolated pituitary cells over 4 h did not appreciably alter the intracellular content of immunoreactive prolactin. Moreover, oral administration of pantethine at 0.5 and 1.0 g/kg body weight did not influence the concentration of immunoreactive plasma prolactin. However, the concentration of plasma prolactin fell by 48 and 67%, when pantethine was injected i.p. at 0.5 and 1.0 g/kg body weight, after 4 h. Intravenous administration of pantethine resulted in even greater losses of prolactin, in the order of 50 and 81% depletion for 0.5 and 1.0 g/kg body weight respectively and within 2 h of administration. However, cysteamine was found to be more efficacious than pantethine on a molar basis with regard to depleting the plasma concentration of prolactin in hyperprolactinaemic rats.