Monique G.M. de Sain-van der Velden

Downy Mildew Resistance in Arabidopsis by Mutation of HOMOSERINE KINASE

Plant disease resistance is commonly triggered by early pathogen recognition and activation of immunity. An alternative form of resistance is mediated by recessive downy mildew resistant 1 (dmr1) alleles in Arabidopsis thaliana. Map-based cloning revealed that DMR1 encodes homoserine kinase (HSK). Six independent dmr1 mutants each carry a different amino acid substitution in the HSK protein. Amino acid analysis revealed that dmr1 mutants contain high levels of homoserine that is undetectable in wild-type plants. Surprisingly, the level of amino acids downstream in the aspartate (Asp) pathway was not reduced in dmr1 mutants. Exogenous homoserine does not directly affect pathogen growth but induces resistance when infiltrated in Arabidopsis. We provide evidence that homoserine accumulation in the chloroplast triggers a novel form of downy mildew resistance that is independent of known immune responses.

Maternal Supplementation With Citrulline Increases Renal Nitric Oxide in Young Spontaneously Hypertensive Rats and Has Long-Term Antihypertensive Effects

NO deficiency is associated with development of hypertension. Defects in the renal citrulline-arginine pathway or arginine reabsorption potentially reduce renal NO in prehypertensive spontaneously hypertensive rats (SHRs). Hence, we investigated genes related to the citrulline-arginine pathway or arginine reabsorption, amino acid pools, and renal NO in 2-week-old prehypertensive SHRs. In addition, because perinatally supporting NO availability reduces blood pressure in SHRs, we supplemented SHR dams during pregnancy and lactation with citrulline, the rate-limiting amino acid for arginine synthesis. In female offspring, gene expression of argininosuccinate synthase (involved in renal arginine synthesis) and renal cationic amino acid Y-transporter (involved in arginine reabsorption) were both decreased in 2-day and 2-week SHRs compared with normotensive WKY, although no abnormalities in amino acid pools were observed. In addition, 2-week-old female SHRs had much less NO in their kidneys (0.46±0.01 versus 0.68±0.05 nmol/g of kidney weight, respectively; P<0.001) but not in their heart. Furthermore, perinatal supplementation with citrulline increased renal NO to 0.59±0.02 nmol/g of kidney weight (P<0.001) at 2 weeks and persistently ameliorated the development of hypertension in females and until 20 weeks in male SHR offspring. Defects in both the renal citrulline-arginine pathway and in arginine reabsorption precede hypertension in SHRs. We propose that the reduced cationic amino acid transporter disables the developing SHR kidney to use arginine reabsorption to compensate for reduced arginine synthesis, resulting in organ-specific NO deficiency. This early renal deficiency and its adverse sequels can be corrected by perinatal citrulline supplementation persistently in female and transiently in male SHRs.

D-serine in the developing human central nervous system.

To elucidate the role of D‐serine in human central nervous system, we analyzed D‐serine, L‐serine, and glycine concentrations in cerebrospinal fluid of healthy children and children with a defective L‐serine biosynthesis (3‐phosphoglycerate dehydrogenase deficiency). Healthy children showed high D‐serine concentrations immediately after birth, both absolutely and relative to glycine and L‐serine, declining to low values at infancy. D‐Serine concentrations were almost undetectable in untreated 3‐phosphoglycerate dehydrogenase–deficient patients. In one patient treated prenatally, D‐serine concentration was nearly normal at birth and the clinical phenotype was normal. These observations suggest a pivotal role for D‐serine in normal and aberrant human brain development. Ann Neurol 2006;60:476–480

Two Mass-Spectrometric Techniques for Quantifying Serine Enantiomers and Glycine in Cerebrospinal Fluid: Potential Confounders and Age-Dependent Ranges

BACKGROUND The recent discovery and specific functions of D-amino acids in humans are bound to lead to the revelation of D-amino acid abnormalities in human disorders. Therefore, high-throughput analysis techniques are warranted to determine D-amino acids in biological fluids in a routine laboratory setting. METHODS We developed 2 chromatographic techniques, a nonchiral derivatization with chiral (chirasil-L-val column) separation in a GC-MS system and a chiral derivatization with Marfeys reagent and LC- MS analysis. We validated the techniques for D-serine, L-serine, and glycine determination in cerebrospinal fluid (CSF), evaluated several confounders, and determined age-dependent human concentration ranges. RESULTS Quantification limits for D-serine, L-serine, and glycine in cerebrospinal fluid were 0.14, 0.44, and 0.14 micromol/L, respectively, for GC-MS and 0.20, 0.41, and 0.14 micromol/L for LC-MS. Within-run imprecision was <3% for both methods, and between-run imprecision was <13%. Comparison of both techniques with Deming regression yielded coefficients of 0.90 (D-serine), 0.92 (L-serine), and 0.96 (glycine). Sample collection, handling, and transport is uncomplicated-there is no rostrocaudal CSF gradient, no effect of storage at 4 degrees C for 1 week before storage at -80 degrees C, and no effect of up to 3 freeze/thaw cycles. Conversely, contamination with erythrocytes increased D-serine, L-serine, and glycine concentrations. CSF concentrations for 145 apparently healthy controls demonstrated markedly and specifically increased (5 to 9 times) D-serine concentrations during early central nervous system development. CONCLUSIONS These 2 clinically applicable analysis techniques will help to unravel pathophysiologic, diagnostic, and therapeutic issues for disorders associated with central nervous system abnormalities, NMDA-receptor dysfunction, and other pathology associated with D-amino acids.

Yield of additional metabolic studies in neurodevelopmental disorders.

The timing and yield of metabolic studies for patients with neurodevelopmental disorders is a matter of continuing debate. We determined the yield of additional or repeated metabolic studies in patients with neurodevelopmental disorders. Patients referred to a tertiary diagnostic center for patients with unexplained neurodevelopmental disorders were included. Initial metabolic studies had been performed in most patients (87%) before referral. Additional/repeated metabolic studies were individually tailored. Twelve metabolic diseases of 433 patients studied (2.8%) were diagnosed, despite normal initial metabolic studies before referral. Specific metabolic investigations lead to a greater diagnostic yield in patients with neurodevelopmental disorders. Ann Neurol 2008

Enhanced conversion of triglyceride-rich lipoproteins and increased low-density lipoprotein removal in LPLS447X carriers

Objective—Lipoprotein lipase (LPL) exerts 2 principal actions, comprising enzymatic hydrolysis of triglyceride-rich lipoproteins (TRLs) and nonenzymatic ligand capacity for enhancing lipoprotein removal. The common LPLS447X variant has been associated with cardiovascular protection, for which the mechanism is unknown. We therefore evaluated enzymatic and nonenzymatic consequences of this LPL variant on TRL metabolism. Methods and Results—TRL apolipoprotein B100 (apoB100) metabolism was determined in 5 homozygous LPLS447X carriers and 5 controls. Subjects were continuously fed and received infusion of stable isotope l-[1-13C]-valine. Results were analyzed by SAAMII modeling. Also, preheparin and postheparin LPL concentration and activity were measured. Compared with controls, carriers presented increased very low–density lipoprotein 1 (VLDL1) to VLDL2 apoB100 flux (P=0.04), increased VLDL2 to intermediate-density lipoprotein (IDL) apoB100 flux (P=0.02), increased IDL to low-density lipoprotein (LDL) apoB100 flux (P=0.049), as well as an increased LDL clearance (P=0.04). Additionally, IDL apoB100 synthesis was attenuated (P=0.05). Preheparin LPL concentration was 4-fold higher compared with controls (P=0.01), and a correlation was observed between preheparin LPL concentration and LDL clearance (r2=0.92; P=0.01). Conclusions—Enhanced TRL conversion and enhanced LDL removal combined with increased preheparin LPL concentration suggest increased enzymatic consequences as well as increased nonenzymatic consequences of LPL in LPLS447X carriers, which might both contribute to the cardiovascular benefit of this LPL variant.

Cerebrospinal fluid D-serine and glycine concentrations are unaltered and unaffected by olanzapine therapy in male schizophrenic patients.

N-Methyl D-aspartate (NMDA)-receptor hypofunction has been implicated in the pathophysiology of schizophrenia and D-serine and glycine add-on therapy to antipsychotics has shown beneficial effects in schizophrenic patients. Nevertheless, previous studies have not shown consistently altered D-serine concentrations in cerebrospinal fluid (CSF) of schizophrenic patients. To confirm and extend these results, CSF concentrations of both endogenous NMDA-receptor co-agonists d-serine and glycine and their common precursor L-serine were analyzed simultaneously in 17 healthy controls and 19 schizophrenic patients before and 6 weeks after daily olanzapine (10 mg) treatment. CSF D-serine, L-serine and glycine concentrations and their relative ratios were similar between schizophrenic patients and controls and no differences were observed before and after olanzapine therapy. Thus, the NMDA-receptor hypofunction hypothesis in schizophrenia is not explained by olanzapine therapy-dependent absolute or relative decreases in CSF D-serine and glycine concentrations in this series of male patients, thereby not providing convenient markers for the disorder.

Proline and COMT status affect visual connectivity in children with 22q11.2 deletion syndrome

Background Individuals with the 22q11.2 deletion syndrome (22q11DS) are at increased risk for schizophrenia and Autism Spectrum Disorders (ASDs). Given the prevalence of visual processing deficits in these three disorders, a causal relationship between genes in the deleted region of chromosome 22 and visual processing is likely. Therefore, 22q11DS may represent a unique model to understand the neurobiology of visual processing deficits related with ASD and psychosis. Methodology We measured Event-Related Potentials (ERPs) during a texture segregation task in 58 children with 22q11DS and 100 age-matched controls. The C1 component was used to index afferent activity of visual cortex area V1; the texture negativity wave provided a measure for the integrity of recurrent connections in the visual cortical system. COMT genotype and plasma proline levels were assessed in 22q11DS individuals. Principal Findings Children with 22q11DS showed enhanced feedforward activity starting from 70 ms after visual presentation. ERP activity related to visual feedback activity was reduced in the 22q11DS group, which was seen as less texture negativity around 150 ms post presentation. Within the 22q11DS group we further demonstrated an association between high plasma proline levels and aberrant feedback/feedforward ratios, which was moderated by the COMT 158 genotype. Conclusions These findings confirm the presence of early visual processing deficits in 22q11DS. We discuss these in terms of dysfunctional synaptic plasticity in early visual processing areas, possibly associated with deviant dopaminergic and glutamatergic transmission. As such, our findings may serve as a promising biomarker related to the development of schizophrenia among 22q11DS individuals.

Vitamin B6 Vitamer Concentrations in Cerebrospinal Fluid Differ Between Preterm and Term Newborn Infants

BACKGROUND AND OBJECTIVE: Vitamin B6 plays a pivotal role in brain development and functioning. Differences in vitamin B6 homeostasis between preterm and term newborn infants have been reported. The authors sought to investigate whether B6 vitamers in cerebrospinal fluid (CSF) of preterm and term newborn infants are different. METHODS: B6 vitamer concentrations were determined in 69 CSF samples of 36 newborn infants (26 born preterm and 10 born term) by ultra performance liquid chromatography-tandem mass spectrometry. CSF samples, taken from a subcutaneous intraventricular reservoir, were bedside frozen and protected from light. RESULTS: Concentrations of pyridoxal (PL), pyridoxal phosphate (PLP), pyridoxic acid (PA), and pyridoxamine (PM) in preterm newborns (postmenstrual age 30–37 weeks) were at least twice as high as in older newborns (postmenstrual age ≥42 weeks). Pyridoxine and pyridoxamine phosphate concentrations were below limits of quantification in all newborns. In CSF of 2 very preterm newborns (postmenstrual age <30 weeks), significant amounts of pyridoxine were present besides high concentrations of PL, PA, and PM, whereas PLP concentrations were relatively low. B6 vitamers in CSF were positively correlated, especially PA, PLP, and PL. CONCLUSIONS: In CSF of newborn infants, PL, PLP, PA, and PM are present, and concentrations are strongly dependent on postmenstrual age. Our results indicate that vitamin B6 homeostasis in brain differs between preterm and term newborns. These results should be taken into account for diagnosis and treatment of epilepsy and vitamin B6 deficiency in newborn infants.

Diagnostic Value of Urinary Mevalonic Acid Excretion in Patients with a Clinical Suspicion of Mevalonate Kinase Deficiency (MKD).

OBJECTIVE In patients suffering from mevalonate kinase deficiency (MKD), the reduced enzyme activity leads to an accumulation of mevalonic acid which is excreted in the urine. This study aims to evaluate the diagnostic value of urinary mevalonic acid measurement in patients with a clinical suspicion of mevalonate kinase deficiency. METHODS In this single-center, retrospective analysis, all patients in whom both measurement of mevalonic acid and genetic testing had been performed in the preceding 17 years have been included. The presence of two pathogenic MVK mutations or demonstration of decreased enzyme activity was considered to be the gold standard for the diagnosis of MKD. RESULTS Sixty-one patients were included in this study. Thirteen of them harbored two MVK mutations; twelve of them showed elevated levels of mevalonic acid. Forty-eight patients did not harbor any MVK mutations, yet five of them excreted increased amounts of mevalonic acid. This corresponds to a sensitivity of 92%, a specificity of 90%, a positive predictive value of 71%, and a negative predictive value of 98%. The positive likelihood ratio is 10 and the negative likelihood ratio is 0.09. CONCLUSION MKD seems very unlikely in patients with a normal mevalonic acid excretion, but it cannot be excluded completely. Further, a positive urinary mevalonic acid excretion still requires MVK analysis to confirm the diagnosis of MKD. Therefore, detection of urinary mevalonic acid should not be mandatory before genetic testing. However, as long as genetic testing is not widely available and affordable, measurement of urinary mevalonic acid is a fair way to select patients for MVK gene analysis or enzyme assay.