A compound heterozygote case of isolated sulfite oxidase deficiency

We report an isolated sulfite oxidase deficiency in the first child boy of a non-consanguineous Caucasian family. He's a compound heterozygote for the sulfite oxidase gene, presenting low cystine, undetectable homocysteine and normal uric acid blood concentrations and undetectable sulfite oxidase activity in his cultured fibroblasts. Both mutations are not reported yet. The clinical presentation was typical and severe, with generalized status epilepticus and premature death

Distribution of rat brain XA binding sites.

<p>Autoradiogram of sagittal sections of rat brain (lateral 1.90 mm, according to Paxinos and Watson (1998)), showing in <b>A</b> the distribution of XA binding sites as a pseudo-color image (dark brown/red: +++; brown: ++; yellow: +). 1: cerebellum; 2: cortex; 3: hippocampus; 4: olfactory tracts; 5: striatum; 6: thalamus; 7: mesencephalic dopaminergic nuclei (A₉/A₁₀). In the presence of an excess of non radioactive XA (1 mM), a very faint image can be detected on autoradiographies (<b>B</b>).</p

Xanthurenic Acid Binds to Neuronal G-Protein-Coupled Receptors That Secondarily Activate Cationic Channels in the Cell Line NCB-20

<div><p>Xanthurenic acid (XA) is a metabolite of the tryptophan oxidation pathway through kynurenine and 3-hydroxykynurenine. XA was until now considered as a detoxification compound and dead-end product reducing accumulation of reactive radical species. Apart from a specific role for XA in the signaling cascade resulting in gamete maturation in mosquitoes, nothing was known about its functions in other species including mammals. Based upon XA distribution, transport, accumulation and release in the rat brain, we have recently suggested that XA may potentially be involved in neurotransmission/neuromodulation, assuming that neurons presumably express specific XA receptors. Recently, it has been shown that XA could act as a positive allosteric ligand for class II metabotropic glutamate receptors. This finding reinforces the proposed signaling role of XA in brain. Our present results provide several lines of evidence in favor of the existence of specific receptors for XA in the brain. First, binding experiments combined with autoradiography and time-course analysis led to the characterization of XA binding sites in the rat brain. Second, specific kinetic and pharmacological properties exhibited by these binding sites are in favor of G-protein-coupled receptors (GPCR). Finally, in patch-clamp and calcium imaging experiments using NCB-20 cells that do not express glutamate-induced calcium signals, XA elicited specific responses involving activation of cationic channels and increases in intracellular Ca²⁺ concentration. Altogether, these results suggest that XA, acting through a GPCR-induced cationic channel modulatory mechanism, may exert excitatory functions in various brain neuronal pathways.</p> </div

Saturation curve showing specific binding of [³H]XA displaced by 1 mM non-radioactive XA on crude synaptosomal membranes from rat brain.

<p>TB: total binding; NSB: non-specific binding; SB: specific binding. Non-linear regression lines with GraphPad Prism Program; R² = 0.78. Each point is the mean±SD of three experiments made in triplicate at each concentration. Kd = 0.74 µM; Bmax = 7.5 pmoles/mg protein.</p

Pharmacological characteristics of XA responses recorded in the cell-attached mode from differentiated NCB-20 cells.

<p><b>A</b> and <b>B</b>: Agonistic action of XT-21 and NCS-482, and inhibition by NCS-486 of XA, XT-21 and NCS-482 responses. Note the absence of effect of NCS-486 when applied alone (<b>B</b>). Horizontal bars represent periods of drug application as indicated. <b>C</b>: Statistical data of mean response amplitude (± SEM) obtained with XA, XT-21 and NCS-482 (6, 7 and 4 cells respectively) applied at concentrations as indicated. <b>D</b>: Inhibition of the agonist-induced responses by NCS-486 20 µM (12 cells). The mean of agonist responses (control) was set at 100%.</p

Structural representation of XA and selected analogs and their IC₅₀ of [³H]XA binding displacement.

<p>XA analogs were selected for their agonist (NCS-482 and XT-21) or antagonist (NCS-486) properties.</p

Quantitative autoradiographic densities of binding sites in brain regions.

<p>Results are mean±SD of the quantitative optical density in the autoradiographic trace (Biocom program) by reference to standard autoradiographic scales (Amersham). Results are mean of 8 to 10 measures in each brain region.</p><p>The limit of the signal detection is about 50 fmoles/mg equivalent tissue.</p

Reversal potential of XA-induced responses in the cell-attached configuration.

<p>The membrane patch was recorded in the NMDG-Cl condition and stimulated by a ramp potential protocol (−70 to 90 mV, 600 ms duration) at a frequency of 0.2 Hz. <b>A</b>: Membrane patch current traces recorded at different membrane potentials (Vm) as indicated in the figure. The colored vertical lines 1 to 5 indicate the different steps of the Er variations. The dashed lines represent the zero current level for each current trace. <b>B</b>: Time evolution of the Er value obtained by the ramp potential stimulation protocol during the XA response given in <b>A</b> (control current was subtracted). <b>C</b>: Frequency histogram of the Er values obtained during the XA response. The distribution was best fitted by the sum of 5 Gaussians with parameter values of: mean Er = −23.2, −9.6, 0.8, 12.6 and 20.3 mV, standard deviation σ = 6.3, 2.1, 3.6, 2.1 and 2.3 and amplitude = 3.0, 5.6, 8.4, 23.8 and 20.4 respectively. <b>D</b>: I–V relationship of the XA-induced current recorded at the times indicated by numbers 1 to 5 in <b>A</b>. The reversal potential for each I–V curve is indicated by dashed vertical lines (1 to 5). <b>E</b>: Mean data (± SEM) of the reversal potential classes (Er1 to Er5) obtained from the different XA responses. In parenthesis are given the number of time a given Er class was observed overall the XA responses. ***: p<0.001.</p

Effects of amino acid neurotransmitters, selected XA-related analogues and compounds of the kynurenine pathway on XA binding sites.

<p>Each compound was tested at 200 µM concentration in the presence of radioactive XA. Displacement of radioactive XA induced by 200 µM XA was arbitrarily set at 100%. Result values are the mean of 3 experiments performed in triplicate±SEM.</p

XA-induced intracellular Ca²⁺ increase in differentiated NCB-20 cells. A

<p>: Time course of intracellular Ca²⁺ increase detected by fluo-4 probe. The presence of XA 10 µM induced an increase of fluorescence intensity. <b>B</b>: Dose-effect of XA-induced response. Data point distribution was biphasic and could be described by activation and partial inactivating phases (green and dark red curves respectively obtained by fitting with the Hill’s equation using optimal parameters as indicated in the figure). <b>C</b>: NCS-486 antagonism of the XA-induced cellular calcium response. NCS-486 (100 µM) applied alone or in combination with XA 25 µM did not modify significantly the intracellular Ca²⁺ concentration. After 5 min of washout of the antagonist, XA 25 µM induced a typical increase of fluorescence intensity. <b>D</b>: Statistical data of the fluorescence intensity obtained in the presence or absence of XA and/or NCS-486. Results are means±SD obtained from 15 to 50 cells in each condition. Horizontal bar indicates the period of drug application. ***: p<0.001.</p