Previously, we reported that the time course for the rapid phosphorylation rate of mu-opioid receptor expressed in human embryonic kidney (HEK)293 cells did not correlate with the slow receptor desensitization rate induced by [D-Ala(2),N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO). However, others have suggested that receptor phosphorylation is the trigger for mu-opioid receptor desensitization. In this study, we demonstrated the relatively slow rate of receptor desensitization could be attributed partially to the recycling of internalized receptor as determined by fluorescence-activated cell-sorting analysis. However, the blockade of the endocytic and Golgi transport events in HEK293 cells with monensin and brefeldin A did not increase the initial rate of receptor desensitization. But the desensitization rate was increased by reduction of the mu-opioid receptor level with beta-furnaltrexamine (betaFNA). The reduction of the receptor level with 1 microM betaFNA significantly increased the rate of etorphine-induced receptor desensitization. By blocking the ability of receptor to internalize with 0.4 M sucrose, a significant degree of receptor being rapidly desensitized was observed in HEK293 cells pretreated with betaFNA. Hence, mu-opioid receptor is being resensitized during chronic agonist treatment. The significance of resensitization of the internalized receptor in affecting receptor desensitization was demonstrated further with human neuroblastoma SHSY5Y cells that expressed a low level of mu-opioid receptor. Although DAMGO could not induce a rapid desensitization in these cells, in the presence of monensin and brefeldin A, DAMGO desensitized the mu-opioid receptor's ability to regulate adenylyl cyclase with a t(1/2) = 9.9 +/- 2.1 min and a maximal desensitized level at 70 +/- 4.7%. Furthermore, blockade of receptor internalization with 0.4 M sucrose enhanced the DAMGO-induced receptor desensitization, and the inclusion of monensin prevented the resensitization of the mu-opioid receptor after chronic agonist treatment in SHSY5Y cells. Thus, the ability of the mu-opioid receptor to resensitize and to recycle, and the relative efficiency of the receptor to regulate adenylyl cyclase activity, contributed to the observed slow rate of mu-opioid receptor desensitization in HEK293 cells.

The effects of BRL 34915, (+/-) 6-cyano-3,4-dihydro-2,2-dimethyl-trans-4-(2-oxo-1-pyrrolidyl)-2H-b enzo [b]-pyran-3-ol, on blood pressure and other haemodynamic parameters in animals have been investigated in comparison with those of nifedipine. In conscious spontaneously hypertensive rats and renal hypertensive cats and dogs the oral doses of BRL 34915 lowering blood pressure are 10 to 30 times lower than those of nifedipine. Tachycardia evoked by BRL 34915 tends to be less than that produced by nifedipine in the cat and of similar magnitude in the dog. The antihypertensive response to BRL 34915 in these models is reproducible on repeat once daily dosing without rebound hypertension on cessation of dosing. In studies using electromagnetic flow probes to measure regional blood flow in anaesthetised cats the intravenous administration of BRL 34915, unlike that of nifedipine, markedly increases renal blood flow yet BRL 34915 lacks the marked effect of nifedipine on femoral blood flow. BRL 34915, a compound structurally unrelated to existing cardiovascular drugs, is a potent new antihypertensive agent having an interesting profile of activity that renders this compound of clinical interest.

We cloned a novel protocadherin cDNA, which we named OL-protocadherin (OL-pc), from mouse brain cDNA libraries. Its cytoplasmic region showed no similarities to other protocadherins, indicating that it belongs to a novel subfamily of protocadherins. Experiments using transfectants showed that OL-pc is a homophilic cell-cell adhesion molecule. The molecular mass of OL-pc was 140 kDa in the brain. Expression of OL-pc mRNA was specific to the nervous system, changing over time from the embryonic stage to the adult stage. The OL-pc expression seemed to be restricted to a subset of functionally related brain nuclei and regions such as the nuclei in the main olfactory system, the limbic system, and the olivocortical projection. There were at least two distinct patterns of distribution for the OL-pc protein. First, it was localized in particular brain nuclei or compartments, such as the stripes of the developing cerebellum. Second, it was found at the synapse in regions such as the glomeruli of the olfactory bulb. In addition, the OL-pc protein seemed not to be detected or was detected only weakly in some regions, such as hippocampus in which the mRNA was expressed at high levels. These results indicate that the expression of OL-pc is developmentally regulated in a subset of the functional systems and that it may be involved in the formation of the neural network by segregation of the brain nuclei and mediation of the axonal connections.

The Cro protein specified by bacteriophage lambda is a repressor of the genes expressed early in phage development and is required for a normal late stage of lytic growth. We have purified Cro protein to virtual homogeneity and analyzed its structure and properties as a DNA-binding protein and repressor of RNA synthesis. To confirm that the protein is the product of the cro gene, we have also shown that a missense mutation in the cro gene leads to a product that is more temperature- and salt-sensitive in its DNA-binding property. As purified, Cro protein is a dimer of identical subunits of molecular weight 8600. The purified protein binds to lambda-DNA carrying the specific binding sites (operators oL and oR) with an estimated dissociation constant of 10(-10) M to 10(-11) M; there is also weaker binding to other sites on DNA, as found for other DNA-binding regulatory proteins. In a purified transcription system, the Cro protein is an effective and specific repressor of RNA synthesis from the N and cro genes; thus Cro is an autorepressor which regulates its own synthesis. A comparison of the properties of the two lambda repressor proteins, cI and Cro, indicates that cI is a "strong repressor" specialized for complete turnoff of lytic functions needed for the maintenance of lysogeny, whereas Cro is a "weak repressor" specialized for a gradual turnoff of early viral genes that potentiates the late stage of lytic development.

Matrix metalloproteinases (MMPs), the key effectors of extracellular matrix remodeling, have been demonstrated to regulate the extension of neurites from neuronal cell bodies. In this report we have addressed the hypothesis that oligodendrocytes (OLs) may utilize a similar mechanism in extending their processes during the initial phase of myelination. Furthermore, given our previous findings linking protein kinase C (PKC) to the OL process outgrowth, we tested the postulate that this signal transduction pathway may regulate MMPs and thus the process outgrowth phenotype. We demonstrate that in response to pharmacologic activators of PKC, cultured human OLs augment their process extension with a concomitant increase in the activity of an MMP, MMP-9, as measured by gelatin zymography. Similarly, the phorbol ester-enhanced process extension and increased MMP-9 activity were both inhibited by calphostin C, a selective PKC inhibitor. Also, MMP inhibitors such as 1,10-phenanthroline and synthetic dipeptides that inactivate the MMP catalytic site negated the 4beta-phorbol-12,13-dibutyrate (PDB)-mediated process extension, further supporting the key role of MMPs in process extension in vitro. Finally, the elevation of MMP-9 protein expression in the mouse corpus callosum, a tissue rich in OL and myelin, coincided with the previously documented temporal increase in myelination that occurs postnatally. Taken together, these data suggest that MMP-9 constitutes an important mediator of OL process outgrowth, and that this protease in turn can be regulated by PKC. The results are relevant not only to the initial steps of myelination during development, but also to the attempted remyelination that has been shown to occur in pathologic conditions such as MS.

The acute effect of the somatostatin analog SMS 201-995 (SMS) was investigated in eight acromegalic patients. This substance is an octapeptide [DPhe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-(ol)] that inhibits GH release in experimental animals and man. After a control day, 50 micrograms SMS were injected sc, and plasma GH and insulin and blood glucose levels were measured at multiple intervals for 24 h. GH significantly (P less than 0.001) decreased in seven of eight acromegalic patients from 30 +/- 5 (+/- SE) to an average of 10.7 +/- 4 micrograms/l from 1-10 h after drug administration. No rebound effect occurred. Postprandial blood glucose levels were significantly (P less than 0.01) higher between 2 and 4 h after SMS treatment compared with control day values, and there was a substantial reduction in insulin secretion, as estimated by the area under the curve (P less than 0.01), during the first 3 h after SMS administration. Circulating GH was not altered by SMS or the dopamine agonist mesulergine in one patient, but the combination of both substances (50 micrograms SMS, sc, and 0.5 mg mesulergine, orally) reduced GH to below 50% of basal. In vitro studies showed that 1 PM, 0.1 nM, and 10 nM SMS or natural somatostatin exerted a similar inhibitory effect (12-39% reduction; P less than 0.01 for all three strengths) on GH release by cultured human pituitary tumor cells. In conclusion, the somatostatin derivative SMS exerts a potent and prolonged inhibitory action on GH secretion and a shorter lasting suppression of insulin in acromegalic patients. Therefore, it may represent a useful tool in the chronic management of this condition.

The central nucleus of the amygdala (CeA) plays an important role both in stimulus-reward learning for the reinforcing effects of drugs of abuse and in environmental condition-induced analgesia. Both of these two CeA functions involve the opioid system within the CeA. However, the pharmacological profiles of its opioid receptor system have not been fully studied and the synaptic actions of opioid receptors in the CeA are largely unknown. In this study with whole-cell voltage-clamp recordings in brain slices in vitro, we examined actions of opioid agonists on glutamate-mediated excitatory postsynaptic currents (EPSCs) in CeA neurons. Opioid peptide methionine-enkephalin (ME; 10 microM) produced a significant inhibition (38%) in the amplitude of evoked EPSCs, an action mimicked by the mu-opioid receptor agonist [D-Ala(2),N-MePhe(4),Gly-ol(5)]-enkephalin (DAMGO; 1 microM, 44%). Both effects of ME and DAMGO were abolished by the mu receptor antagonist CTAP (1 microM), suggesting a mu receptor-mediated effect. Neither delta-opioid receptor agonist [D-Pen(2),D-Pen(5)]-enkephalin (1 microM) nor kappa-opioid receptor agonist U69593 (300 nM) had any effect on the glutamate EPSC. ME significantly increased the paired-pulse ratio of the evoked EPSCs and decreased the frequency of miniature EPSCs without altering the amplitude of miniature EPSCs. Furthermore, the mu-opioid inhibition of the EPSC was blocked by 4-aminopyridine (4AP; 100 microM), a voltage-dependent potassium channel blocker, and by phospholipase A(2) inhibitors AACOCF(3) (10 microM) and quinacrine (10 microM). These results indicate that only the mu-opioid receptor is functionally present on presynaptic glutamatergic terminals in normal CeA neurons, and its activation reduces the probability of glutamate release through a signaling pathway involving phospholipase A(2) and the presynaptic, 4AP-sensitive potassium channel. This study provides evidence for the presynaptic regulation of glutamate synaptic transmission by mu-opioid receptors in CeA neurons.

We have investigated the structure of the glycosylphosphatidylinositol (GPI) anchor and the O-linked glycan chains of the 40/45-kDa glycoprotein from the cell surface of the protozoan parasite Trypanosoma cruzi. This glycoconjugate is the major acceptor for sialic acid transferred by trans-sialidase of T. cruzi Y-strain, epimastigote form. The GPI anchor was liberated by treatment with hot alkali, and the phosphoinositol-oligosaccharide moiety was characterized and shown to have the following structure. [formula: see text] Unusually the glucosamine was 6-O-substituted with 2-aminoethylphosphonate, and 2-aminoethylphosphonate was also present on the third mannose residue distal to glucosamine, partially replacing the ethanolamine phosphate. The beta-eliminated reduced oligosaccharide chains showed that two novel classes of O-linked N-acetylglucosamine oligosaccharide were present. The first series had the structures Galp beta 1-3GlcNAc-ol; Galp beta 1-6(Galp beta 1-3)GlcNAc-ol; and Galp beta 1-2Galp beta 1-6(Galp beta 1-3)GlcNAc-ol, whereas the other series had a 1-4 linkage to N-acetylglucosaminitol and had structures Galp beta 1-4GlcNAc-ol, Galp beta 1-6(Galp beta 1-4)GlcNAc-ol, and Galp beta 1-2Galp beta 1-6(Galp beta 1-4)GlcNAc-ol. We have also investigated the kinetics of in vitro sialylation of these O-linked oligosaccharides by the T. cruzi transsialidase and have shown that incorporation of one molecule of sialic acid hinders entry of a second molecule when two potential acceptor sites are present.

OBJECTIVE

The objective of this study was to investigate the long-term safety and efficacy of risperidone in disruptive behavior disorders in children with subaverage IQs. Disruptive behavior disorders were defined as oppositional defiant disorder, disruptive behavior disorder, and conduct disorder as per the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) criteria.

METHODS

This was a 48-week open-label (OL) extension study of risperidone in 77 children diagnosed with a disruptive behavior disorder, and either borderline intellectual function or mild or moderate mental retardation who had participated in a previous 6-week, double-blind (DB) study and completed at least 2 weeks of DB therapy. Children, aged 5 to 12 years inclusive, who had: 1) a DSM-IV Axis I diagnosis of conduct disorder, oppositional defiant disorder, or disruptive behavior disorder- not otherwise specified; 2) a parent-assessed rating of>> or =24 in the Conduct Problem Subscale of the Nisonger-Child Behavior Rating Form(28); 3) a DSM-IV Axis II diagnosis of mild or moderate mental retardation or borderline intellectual functioning with an IQ>> or =36 and < or =84; and 4) a score of < or =84 on the Vineland Adaptive Behavior Scale. Participants received oral solution risperidone given at a once daily dose of between 0.02 and 0.06 mg/kg for a maximum of 48 weeks. Participants in the DB study who had been randomized would have had a maximum of 54 weeks of risperidone therapy. Study visits were scheduled at entry, weekly for the first month, and monthly for the remaining 11 months.

RESULTS

Baseline scores on the conduct problem subscale at the start of the previous DB study were similar for both treatment groups: mean values of 33.5 and 33.3 were recorded for placebo- and risperidone-treated participants, respectively. At the time of the OL baseline visit, mean Conduct Problem Subscale scores were lower in those who had been treated with risperidone than in those who remained risperidone-naïve (17.5 and 26.1, respectively). Within 1 week of receiving daily risperidone therapy (mean daily dose: 1.38 mg), those participants who had been risperidone-naïve at OL entry showed a rapid improvement in the Conduct Problem Subscale score. At the week 1 assessment, the mean change from baseline for those who had been risperidone-naïve at OL entry was similar in magnitude to the change from DB baseline recorded for participants who had received risperidone in the DB study. This mean improvement was sustained in both groups throughout the remainder of the OL study. At study endpoint, those participants who had been risperidone-naïve at OL entry experienced a highly significant mean decrease from OL baseline in the mean Conduct Problem Subscale score of 10.6 +/- 2.18. The response to risperidone in the OL trial remained stable in those participants who had been treated with risperidone in the previous DB trial; in this group, the mean change at study endpoint from OL baseline was a nonsignificant decrease of 1.26 +/- 1.45. At DB baseline, 68% of participants had a Clinical Global Impression assessment rated as marked, severe, or extremely severe. By DB study endpoint, only 17% of participants (15% of whom had received placebo and 19% of whom had been treated with risperidone in the previous study) had this severe an assessment; 63% of participants had symptoms rated as either none, very mild, or mild. Similarly, highly significant decreases from baseline in the Vineland Adaptive Behavior Scale rating of the most troublesome symptom (often identified as either aggression (hitting, fighting, or temper tantrums) were observed by study endpoint after 48 weeks of risperidone therapy. For those participants who had received placebo in the previous study, a mean decrease of 47.1 +/- 4.87 mm from a DB baseline of 79.4 +/- 2.69 mm was observed. In those who had received risperidone, a mean decrease of 43.5 +/- 4.57 mm from a DB baseline of 79.3 +/- 3.66 mm was observed. Five subgroup analyses of the primary efficacy outcome were performed. These included analysis by diagnosis (conduct disorder, oppositional defiant disorder, and disruptive behavior disorder-not otherwise specified), degree of mental retardation (borderline, mild, moderate), and presence or absence of somnolence, attention-deficit/hyperactivity disorder, and psychostimulants. The results showed that the efficacy of risperidone was not affected by type of disorder, level of retardation, presence/absence of somnolence or attention-deficit/hyperactivity disorder, or use of psychostimulants. Adverse events were reported for 76 participants; none were serious and most were mild/moderate in severity. Somnolence (52%), headache (38%), and weight gain (36%) were the most common adverse events. The degree of sedation was mild and not associated with cognitive deterioration. In fact, for most parameters assessed on the modified California Verbal Learning Test (a test for verbal learning and memory), there were statistically significant improvements relative to both OL and DB baselines in the mean scores. In addition, statistically significant improvements over baseline were also seen for some Continuous Performance Task (which is a test for attention and impulsivity) parameters. Overall, no deterioration of cognitive function was observed while participants were treated with risperidone. Almost half of the 8.5 kg gained was attributable to normal growth. Asymptomatic peak prolactin levels were observed within 4 weeks of beginning risperidone treatment and declined over time to within normal range. At study endpoint, mean prolactin levels were statistically significantly greater than baseline only in male participants but still <20 ng/mL, which is within the normal range. Twenty participants experienced mild or moderate extrapyramidal symptoms, although none withdrew for this reason.

CONCLUSIONS

Risperidone, administered as an oral solution at a mean dose of 1.38 mg/d (range: 0.02-0.06 mg/kg/d) for 1 year, was well tolerated, safe, and showed maintenance of effect in the treatment of disruptive behavior disorders in children aged 5 to 12 years with subaverage IQs.

Activation of opioid receptors in the periphery and centrally in the brain results in inhibition of gastric and other vagally mediated functions. The aim of this study was to examine the role of the endogenous opioid agonist endomorphin 1 (EM-1) in regulating synaptic transmission within the nucleus tractus solitarius (NTS), an integration site for autonomic functions. We performed whole cell patch-clamp recordings from coronal brain slices of the rat medulla. A subset of the neurons studied was prelabeled with a stomach injection of the transsynaptic retrograde virus expressing EGFP, PRV-152. Solitary tract stimulation resulted in constant latency excitatory postsynaptic currents (EPSCs) that were decreased in amplitude by EM-1 (0.01-10 microM). The paired-pulse ratio was increased with little change in input resistance, suggesting a presynaptic mechanism. Spontaneous EPSCs were decreased in both frequency and amplitude by EM-1, and miniature EPSCs were reduced in frequency but not amplitude, suggesting a presynaptic mechanism for the effect. Spontaneous inhibitory postsynaptic currents (IPSCs) were also reduced in frequency by EM-1, but the effect was blocked by TTX, suggesting activity at receptors on the somata of local inhibitory neurons. Synaptic input arising from local NTS neurons, which were activated by focal photolysis of caged glutamate, was inhibited by EM-1. The actions of EM-1 were similar to those of D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) and were blocked by naltrexone, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), or D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP). These results suggest that EM-1 acts at mu-opioid receptors to modulate viscerosensory input and specific components of local synaptic circuitry in the NTS.

Opioid peptides produce gastrointestinal inhibition and increase feeding when applied to the brainstem. The present studies were designed to determine the actions of opioid peptides on synaptic transmission within the dorsal motor nucleus of the vagus (DMV) and the localization of mu-opioid receptors. Whole-cell recordings were made from identified gastrointestinal-projecting DMV neurons in thin brainstem slices of the rat. Electrical stimulation of the nucleus of the tractus solitarius evoked EPSCs and IPSCs. In all neurons tested, methionine (Met)-enkephalin (0.003-30 microm) inhibited the peak amplitude of the EPSCs. The effect was prevented by naloxone (1 microm) as well as by naloxonazine (0.2 microm). An increase in the ratio of the evoked paired pulses indicated that the inhibition was attributable to actions at presynaptic receptors. This presynaptic inhibitory action was mimicked by [d-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (0.1 microm) and the analgesic dipeptide kyotorphin (10 microm) but not by cyclic[d-Pen(2), d-Pen(5)]-enkephalin (1 microm) and trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methanesulfonate (1 microm). In contrast, the amplitude of evoked IPSCs was not altered either by Met-enkephalin or by any of the opioid receptor-selective agonists. Immunohistochemical studies revealed that nerve terminals apposing DMV neurons showed immunoreactivity to mu-opioid receptors colocalized with glutamate immunoreactivity but not glutamic acid decarboxylase immunoreactivity. These results suggest that within the DMV, mu-opioid receptors are present on the nerve terminals of excitatory but not inhibitory inputs to GI motoneurons. Such specificity may imply that the central inhibitory action of opioid peptides on gastrointestinal function targets selected pathways.

Progesterone (P(4)) in the ventromedial hypothalamus (VMH) and ventral tegmental (VTA) is important for facilitation of lordosis; however, P(4)'s actions in these brain areas are different. Using lordosis in rodents as in vivo experimental models, we have examined the effects progestins exert in the midbrain and hypothalamus. Localization and blocker studies indicate that P(4)'s actions in the VMH require intracellular progestin receptors (PRs) but in the VTA they do not. Progestins that have rapid, membrane effects, and/or are devoid of affinity for PRs, facilitate lordosis when applied to the VTA. Manipulation of GABA and/or GABA(A)/benzodiazepine receptor complexes (GBRs) in the VTA alter lordosis, which suggests that progestins may interact with GBRs to facilitate receptivity by enhancing the function of GABAergic neurons. Interfering with P(4)'s metabolism to 5 alpha-pregnan-3 alpha-ol-20-one (3 alpha,5 alpha-THP), the most effective endogenous positive modulator of GBRs, or the biosynthesis of the neurosteroid 3 alpha,5 alpha-THP in the VTA attenuates female sexual behavior in rodents. Stimulation of mitochondrial benzodiazepine receptors (MBRs), which enhance neurosteroid production, by infusions of a MBR agonist to the VTA enhances lordosis. 3 alpha,5 alpha-THP is increased in the midbrain of mated>> proestrous>> diestrous rodents. These data suggest that 3 alpha,5 alpha-THP has a proximate modulatory role on lordosis. In summary, the actions of P(4) in the VTA are different from those in the VMH that involve PRs. In the VTA, P(4) may facilitate lordosis following metabolism to and/or biosynthesis of 3 alpha,5 alpha-THP, which may have subsequent actions at GBRs and/or MBRs to acutely modulate female sexual behavior in rodents.

Galactocerebroside and sulfatide, major galactosphingolipid components of oligodendrocyte plasma membranes and myelin, are first expressed at a critical point, when progenitors cease to proliferate and commence terminal differentiation. We showed previously that an antibody to galactocerebroside/sulfatide arrested terminal differentiation, suggesting a role for these galactolipids in oligodendrocyte differentiation. We have now investigated the differentiation of oligodendrocytes (1) in response to other anti-galactolipid antibodies, showing that anti-sulfatide O4 but not anti-galactocerebroside O1 blocks terminal differentiation, perhaps by mimicking an endogenous ligand, and (2) in a transgenic mouse unable to synthesize these lipids because of mutation of the gene for ceramide galactosyltransferase, a key enzyme for galactosphingolipid synthesis. We find that galactosyltransferase mRNA expression begins at the late progenitor [pro-oligodendroblast (Pro-OL)] stage of the lineage and that the late progenitor marker pro-oligodendroblast antigen is not synthesized in the absence of galactosyltransferase. The principal outcome of the elimination of these galactolipids is a two- to threefold enhancement in the number of terminally differentiated oligodendrocytes both in culture and in vivo. Because the general pattern of differentiation and the level of progenitor proliferation and survival appear to be unaltered in the mutant cultures, we conclude that the increased number of oligodendrocytes is caused by an increased rate and probability of differentiation. In agreement with these two experimental approaches, we present a model in which galactosphingolipids (in particular galactocerebroside and/or sulfatide) act as sensors and/or transmitters of environmental information, interacting with endogenous ligands to function as negative regulators of oligodendrocyte differentiation, monitoring the timely progress of Pro-OLs into terminally differentiating, myelin-producing oligodendrocytes.

Th1 cytokine-induced loss of oligodendrocytes (OLs) is associated with axonal loss in CNS demyelinating diseases such as multiple sclerosis (MS)that contributes to neurological disabilities in affected individuals. Recent studies indicated that, in addition to Th1-phenotype cytokines including tumor necrosis factor (TNF)-α, Th17 phenotype cytokine, interleukin (IL)-17 also involved in the development of MS. In this study, we investigated the direct effect of IL-17 on the survival of OLs in the presence of TNF-α and individually in vitro settings. Our findings suggest that IL-17 alone, however, was not able to affect the survival of OLs, but it exacerbates the TNF-α-induced OL apoptosis as compared with individual TNF-α treatment. This effect of cytokines was ascribed to an inhibition of cell-survival mechanisms, co-localization of Bid/Bax proteins in the mitochondrial membrane and caspase 8 activation mediated release of apoptosis inducing factor from mitochondria in treated OLs. In addition, cytokine treatment disturbed the mitochondrial membrane potential in OLs with corresponding increase in the generation of reactive oxygen species, which were attenuated by N-acetyl cysteine treatment. In addition, combining of these cytokines induced cell-cycle arrest at G1/S phases in OL-like cells and inhibited the maturation of OL progenitor cells that was attenuated by peroxisome proliferator-activated receptor-γ/-β agonists. Collectively, these data provide initial evidence that IL-17 exacerbates TNF-α-induced OL loss and inhibits the differentiation of OL progenitor cells suggesting that antioxidant- or peroxisome proliferator-activated receptor agonist-based therapies have potential to limit CNS demyelination in MS or other related demyelinating disorders.

Myelin oligodendrocyte glycoprotein (MOG) is, quantitatively, a relatively minor component of the myelin membrane. Nevertheless, peritoneal administration of MOG evokes potent cellular and humoral immunoreactivity, resulting in an experimental allergic encephalitis with immunopathology similar to multiple sclerosis. Moreover, antibodies against MOG cause myelin destruction in situ. Therefore, it appears that MOG-related demyelination is dependent on anti-MOG antibody, but the mechanism(s) by which it occurs is unclear. Of potential significance are observations that some proteins are selectively partitioned into specialized plasma membrane microdomains rich in glycosphingolipids and cholesterol ("lipid rafts"). In particular, during ligand or antibody cross-linking, various plasma membrane receptors undergo enhanced partitioning into rafts as an obligatory first step toward participation in early signal transduction events. In contrast to mature myelin, in oligodendrocytes (<em>OLs</em>) in culture MOG is not raft associated [Triton X-100 (TX-100) soluble, 4 degrees C]. However, in this study we show that antibody cross-linking (anti-MOG plus secondary antibody) of MOG on the surface of <em>OLs</em> results in the repartitioning of approximately 95% of MOG into the TX-100-insoluble fraction. This repartitioning of MOG is rapid (<or=1 min), antibody dose dependent, requires an intact cytoskeleton, leads to phosphorylation or dephosphorylation of tyrosine, serine, and threonine residues in specific proteins (e.g., beta-tubulin, Gbeta1-2), and invokes a rapid retraction of <em>OL</em> processes. After removal of the cross-linking antibodies, these events are reversed. We hypothesize that antibody-mediated repartitioning of MOG into TX-100-insoluble glycosphingolipid-cholesterol-rich microdomains initiates specific cellular signaling that could be related to initial steps of MOG-mediated demyelination.

Cerebral white matter injury during prenatal maternal infection characterized as periventricular leukomalacia is the main substrate for cerebral palsy (CP) in premature infants. Previously, we reported that maternal LPS exposure causes oligodendrocyte (OL)-injury/hypomyelination in the developing brain which can be attenuated by an antioxidant agent, N-acetyl cysteine (NAC). Herein, we elucidated the role of peroxisomes in LPS-induced neuroinflammation and cerebral white matter injury. Peroxisomes are important for detoxification of reactive oxidative species (ROS) and metabolism of myelin-lipids in OLs. Maternal LPS exposure induced selective depletion of developing OLs in the fetal brain which was associated with ROS generation, glutathione depletion and peroxisomal dysfunction. Likewise, hypomyelination in the postnatal brain was associated with decrease in peroxisomes and OLs after maternal LPS exposure. Conversely, NAC abolished these LPS-induced effects in the developing brain. CP brains imitated these observed changes in peroxisomal/myelin proteins in the postnatal brain after maternal LPS exposure. In vitro studies revealed that pro-inflammatory cytokines cause OL-injury via peroxisomal dysfunction and ROS generation. NAC or WY14643 (peroxisome proliferators activated receptor (PPAR)-alpha agonist) reverses these effects of pro-inflammatory cytokines in the wild-type OLs, but not in PPAR-alpha(-/-) OLs. Similarly treated B12 oligodenroglial cells co-transfected with PPAR-alpha siRNAs/pTK-PPREx3-Luc, and LPS exposed PPAR-alpha(-/-) pregnant mice treated with NAC or WY14643 further suggested that PPAR-alpha activity mediates NAC-induced protective effects. Collectively, these data provide unprecedented evidence that LPS-induced peroxisomal dysfunction exacerbates cerebral white matter injury and its attenuation by NAC via a PPAR-alpha dependent mechanism expands therapeutic avenues for CP and related demyelinating diseases.

Differentiation of oligodendrocyte precursor cells (OPCs) is the most important event for the myelination of central nervous system (CNS) axons during development and remyelination in demyelinating diseases, while the underlying molecular mechanisms remain largely unknown. Here we show that NMDA receptor (NMDAR) is a functional regulator of OPCs differentiation and remyelination. First, GluN1, GluN2A, and GluN2B subunits are expressed in oligodendrocyte lineage cells (OLs) in vitro and in vivo by immunostaining and Western blot analysis. Second, in a purified rat OPC culture system, NMDARs specially mediate OPCs differentiation by enhancing myelin proteins expression and the processes branching at the immature to mature oligodendrocyte transition analyzed by a serial of developmental stage-specific antigens. Moreover, pharmacological NMDAR antagonists or specific knockdown of GluN1 by RNA interference in OPCs prevents the differentiation induced by NMDA. NMDA can activate the mammalian target of rapamycin (mTOR) signal in OPCs and the pro-differentiation effect of NMDA is obstructed by the mTOR inhibitor rapamycin, suggesting NMDAR exerts its effect through mTOR-dependent mechanism. Furthermore, NMDA increases numbers of myelin segments in DRG-OPC cocultures. Finally, NMDAR specific antagonist MK801 delays remyelination in the cuprizone model examined by LFB-PAS, immunofluorescence and electron microscopy. This effect appears to result from inhibiting OPCs differentiation as more NG2(+) OPCs but less GST-π(+) mature oligodendrocytes are observed. Together, these results indicate that NMDAR plays a critical role in the regulation of OPCs differentiation in vitro and remyelination in cuprizone model which may provide potential target for the treatment of demyelination disease.

Acute challenges with psychostimulants such as amphetamine affect impulsive behavior in both animals and humans. With regard to amphetamine, it is important to unravel how this drug affects impulsivity since it is not only a widely abused recreational drug but also regularly prescribed to ameliorate maladaptive impulsivity. Therefore, we studied the effects of amphetamine in two rat models of impulsivity, the five-choice serial reaction time task and the delayed-reward task, providing measures of inhibitory control and impulsive choice, respectively. We focused on the role of opioid receptor activation in amphetamine-induced impulsivity as there is ample evidence indicating an important role for endogenous opioids in several behavioral and neurochemical effects of amphetamine. Results showed that amphetamine-induced inhibitory control deficits were dose-dependently attenuated by the preferential μ-opioid receptor antagonist naloxone, but not by the selective δ-opioid receptor antagonist naltrindole or κ-opioid receptor antagonist nor-BNI (nor-binaltorphimine dihydrochloride). In contrast, naloxone did not affect amphetamine-induced improvements in impulsive decision making. Naloxone also completely prevented inhibitory control deficits induced by GBR 12909 [1-(2-[bis(4-fluorophenyl)methoxy] ethyl)-4-(3-phenylpropyl)piperazine dihydrochloride], a selective dopamine transporter inhibitor. Intracranial infusions of naloxone, the selective μ-opioid receptor antagonist CTAP (H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2)), morphine, and the selective μ-opioid receptor agonist DAMGO ([D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin acetate salt) revealed that μ-opioid receptor activation in the shell rather than the core subregion of the nucleus accumbens (NAc) modulates inhibitory control and subserves the effect of amphetamine thereon. Together, these results indicate an important role for NAc shell μ-opioid receptors in the regulation of inhibitory control, probably via an interaction between these receptors and the mesolimbic dopamine system.

NG2 (nerve/glia antigen-2) is a type I transmembrane glycoprotein and also known as chondroitin sulfate proteoglycan 4. In the parenchyma of the central nervous system, NG2-expressing (NG2(+) ) cells have been identified as a novel type of glia with a strong potential to generate oligodendrocytes (OLs) in the developing white matter. However, the differentiation potential of NG2 glia remained controversial, largely attributable to shortcomings of transgenic mouse models used for fate mapping. To minimize these restrictions and to more faithfully mimic the endogenous NG2 expression in vivo, we generated a mouse line in which the open reading frame of the tamoxifen-inducible form of the Cre DNA recombinase (CreERT2) was inserted into the NG2 locus by homologous recombination. Results from this novel mouse line demonstrate that at different developmental stages of the brain, NG2(+) cells either stayed as NG2 glia or differentiated into OLs during the whole life span. Interestingly, when Cre activity was induced at embryonic stages, a significant number of reporter(+) astrocytes could be detected in the gray matter after birth. However, in other brain regions, such as olfactory bulb, brain stem, and cerebellum, all of the NG2 glia was restricted to the OL lineage. In addition, tamoxifen-sensitive and NG2 gene locus-dependent gene recombination could be detected in a small, but persistent population of cortical NeuN(+) neurons starting from the second postnatal week.

The present study investigates the presence of vitamin D receptor (VDR) in cells of the rat oligodendrocyte (OL) lineage. VDR transcripts were detected by in situ hybridization in a fraction of rat OL in secondary cultures. The VDR protein was shown to be co-localized in cells that are also recognized by an anti-myelin basic protein (MBP) antibody. Likewise, in vivo, VDR-positive cells were found in the brain white matter, such as the internal capsule of the striatum or the corpus callosum but also in the spinal cord. At least part of these positive cells in vivo correspond to OL, since they were co-stained by an anti-carbonic anhydrase II antiserum. Northern blot analyses of the CG-4 OL cell line demonstrated that the VDR transcripts are already found in the O-2A precursors. There was a two-fold increase in the relative abundance of these transcripts in differentiated OL or in type-2 astrocytes. 1, 25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] increased the pool of transcripts encoding its own receptor, the VDR. The hormone also enhanced the abundance of the mRNA of the nerve growth factor (NGF) and of its low-affinity receptor, the p75(NTR) protein. By contrast, the hormone had no effect on the levels of MBP or proteolipid protein (PLP) mRNA. This finding suggests that unlike retinoic acid (RA) or thyroid hormone, 1,25-(OH)(2)D(3) has no regulatory action on the synthesis of myelin proteins.

Children with specific reading-comprehension difficulties can read accurately, but they have poor comprehension. In a randomized controlled trial, we examined the efficacy of three interventions designed to improve such children's reading comprehension: text-comprehension (TC) training, oral-language (OL) training, and TC and OL training combined (COM). Children were assessed preintervention, midintervention, postintervention, and at an 11-month follow-up. All intervention groups made significant improvements in reading comprehension relative to an untreated control group. Although these gains were maintained at follow-up in the TC and COM groups, the OL group made greater gains than the other groups did between the end of the intervention and follow-up. The OL and COM groups also demonstrated significant improvements in expressive vocabulary compared with the control group, and this was a mediator of the improved reading comprehension of the OL and COM groups. We conclude that specific reading-comprehension difficulties reflect (at least partly) underlying oral-language weaknesses that can be effectively ameliorated by suitable teaching.

OBJECTIVE

To estimate flavonoid intake in the Australian population.

METHODS

Flavonoid consumption was estimated from 24-hour recall data and apparent consumption data using US Department of Agriculture flavonoid composition data.

METHODS

The National Nutrition Survey 1995 assessed dietary intake (24-hour recall) in a representative sample (n=13,858) of the Australian population aged 2 years and over.

RESULTS

Analysis of the 24-hour recall data indicated an average adult intake (>18 years) of 454 mg day(-1) (92% being flavan-3-ols). Apple was the highest quercetin source until age 16-18 years, after which onion became an increasingly important prominent source. Variations in hesperetin consumption reflected orange intake. Apple, apricot and grapes were the major sources of epicatechin and catechin for children, but subsided as wine consumption increased in adulthood. Wine was the main source of malvidin. Naringenin intake remained static as a percentage of total flavonoid intake until age 19-24 years, corresponding to orange intake, and then increased with age from 19-24 years, corresponding to grapefruit intake. Apparent dietary flavonoid consumption was 351 mg person(-1) day(-1), of which 75% were flavan-3-ols. Black tea was the major flavonoid source (predominantly flavan-3-ols) representing 70% of total intake. Hesperetin and naringenin were the next most highly consumed flavonoids, reflecting orange intake. Both 24-hour recall and apparent consumption data indicated that apigenin intake was markedly higher in Australia than reported in either the USA or Denmark, presumably due to differences in consumption data for leaf and stalk vegetables and parsley.

CONCLUSIONS

Tea was the major dietary flavonoid source in Australia. Flavonoid consumption profiles and flavonoid sources varied according to age. More consistent methodologies, survey tools validated for specific flavonoid intakes and enhanced local flavonoid content data for foods would facilitate better international comparisons of flavonoid intake.

Oligodendrocytes (OLs) fail to regenerate myelin destroyed by the immune attack in multiple sclerosis (MS) and lesion areas are eventually largely occupied by astrocytic scar tissue. Loss of OLs in MS does not account for the limited myelin repair as lesions contain a considerable number of OL precursor cells (OPC). Activation of the Notch pathway has been shown to provide inhibitory signals for OPC and to hamper their ability to produce myelin during CNS development. Here we show that gamma-secretase inhibition of Notch signaling within OL of CNS of SJL/J mice with experimental autoimmune encephalomyelitis (EAE) significantly enhanced clinical recovery and in the CNS, promoted remyelination and reduced axonal damage. Functional assays confirmed decreased Notch signaling in inhibitor-treated groups. Therefore, gamma-secretase inhibition led to an environment more conducive to myelin repair and axonal survival. Our results suggest that manipulation of the environment associated with Notch activation in the mature CNS provides a promising therapeutic target in MS.

GABAA and GABAB receptor agonists and antagonists were administered locally in the striatum of intact and kainic acid lesioned rats. (+/-)-Baclofen, a GABAB receptor agonist, significantly decreased the level of extracellular dopamine in the striatum of intact rats. (+/-)-Phaclofen, a GABAB receptor antagonist, increased the level of extracellular dopamine in the striatum of intact rats and to a lesser extent in the striatum after kainic acid lesion. Pregnanolone (5 beta-pregnan-3 alpha-ol-20-one), a positive allosteric modulator of the GABAA receptor, significantly decreased the level of extracellular dopamine in intact rats. (-)-Bicuculline, a GABAB receptor antagonist, increased the level of extracellular dopamine in the striatum of intact rats, but failed to increase the level of extracellular dopamine after kainic acid lesion. The release of extracellular dopamine, due to infusion of phaclofen or bicuculline, was totally suppressed by tetrodotoxin. These results support a direct influence of GABA on the dopaminergic terminals via presynaptic GABAB receptors, while the effects via the GABAA receptor seem to be postsynaptic and mediated by striatal interneurons or the striatonigral feedback loop.