MadR mediates acyl CoA-dependent regulation of mycolic acid desaturation in mycobacteria.

Mycobacterium tuberculosis has a lipid-rich cell envelope that is remodeled throughout infection to enable adaptation within the host. Few transcriptional regulators have been characterized that coordinate synthesis of mycolic acids, the major cell wall lipids of mycobacteria. Here, we show that the mycolic acid desaturase regulator (MadR), a transcriptional repressor of the mycolate desaturase genes desA1 and desA2, controls mycolic acid desaturation and biosynthesis in response to cell envelope stress. A madR-null mutant of M. smegmatis exhibited traits of an impaired cell wall with an altered outer mycomembrane, accumulation of a desaturated α-mycolate, susceptibility to antimycobacterials, and cell surface disruption. Transcriptomic profiling showed that enriched lipid metabolism genes that were significantly down-regulated upon madR deletion included acyl-coenzyme A (aceyl-CoA) dehydrogenases, implicating it in the indirect control of ß-oxidation pathways. Electromobility shift assays and binding affinities suggest a unique acyl-CoA pool-sensing mechanism, whereby MadR is able to bind a range of acyl-CoAs, including those with unsaturated as well as saturated acyl chains. MadR repression of desA1/desA2 is relieved upon binding of saturated acyl-CoAs of chain length C16 to C24, while no impact is observed upon binding of shorter chain and unsaturated acyl-CoAs. We propose this mechanism of regulation as distinct to other mycolic acid and fatty acid synthesis regulators and place MadR as the key regulatory checkpoint that coordinates mycolic acid remodeling during infection in response to host-derived cell surface perturbation.

D-cysteine is an endogenous regulator of neural progenitor cell dynamics in the mammalian brain.

d-amino acids are increasingly recognized as important signaling molecules in the mammalian central nervous system. However, the d-stereoisomer of the amino acid with the fastest spontaneous racemization ratein vitro in vitro, cysteine, has not been examined in mammals. Using chiral high-performance liquid chromatography and a stereospecific luciferase assay, we identify endogenous d-cysteine in the mammalian brain. We identify serine racemase (SR), which generates the N-methyl-d-aspartate (NMDA) glutamate receptor coagonist d-serine, as a candidate biosynthetic enzyme for d-cysteine. d-cysteine is enriched more than 20-fold in the embryonic mouse brain compared with the adult brain. d-cysteine reduces the proliferation of cultured mouse embryonic neural progenitor cells (NPCs) by ∼50%, effects not shared with d-serine or l-cysteine. The antiproliferative effect of d-cysteine is mediated by the transcription factors FoxO1 and FoxO3a. The selective influence of d-cysteine on NPC proliferation is reflected in overgrowth and aberrant lamination of the cerebral cortex in neonatal SR knockout mice. Finally, we perform an unbiased screen for d-cysteine-binding proteins in NPCs by immunoprecipitation with a d-cysteine-specific antibody followed by mass spectrometry. This approach identifies myristoylated alanine-rich C-kinase substrate (MARCKS) as a putative d-cysteine-binding protein. Together, these results establish endogenous mammalian d-cysteine and implicate it as a physiologic regulator of NPC homeostasis in the developing brain.

The NMDA receptor activation by d-serine and glycine is controlled by an astrocytic Phgdh-dependent serine shuttle.

Astrocytes express the 3-phosphoglycerate dehydrogenase (Phgdh) enzyme required for the synthesis of l-serine from glucose. Astrocytic l-serine was proposed to regulate NMDAR activity by shuttling to neurons to sustain d-serine production, but this hypothesis remains untested. We now report that inhibition of astrocytic Phgdh suppressed the de novo synthesis of l-and d-serine and reduced the NMDAR synaptic potentials and long-term potentiation (LTP) at the Schaffer collaterals-CA1 synapse. Likewise, enzymatic removal of extracellular l-serine impaired LTP, supporting an l-serine shuttle mechanism between glia and neurons in generating the NMDAR coagonist d-serine. Moreover, deletion of serine racemase (SR) in glutamatergic neurons abrogated d-serine synthesis to the same extent as Phgdh inhibition, suggesting that neurons are the predominant source of the newly synthesized d-serine. We also found that the synaptic NMDAR activation in adult SR-knockout (KO) mice requires Phgdh-derived glycine, despite the sharp decline in the postnatal glycine levels as a result of the emergence of the glycine cleavage system. Unexpectedly, we also discovered that glycine regulates d-serine metabolism by a dual mechanism. The first consists of tonic inhibition of SR by intracellular glycine observed in vitro, primary cultures, and in vivo microdialysis. The second involves a transient glycine-induce d-serine release through the Asc-1 transporter, an effect abolished in Asc-1 KO mice and diminished by deleting SR in glutamatergic neurons. Our observations suggest that glycine is a multifaceted regulator of d-serine metabolism and implicate both d-serine and glycine in mediating NMDAR synaptic activation at the mature hippocampus through a Phgdh-dependent shuttle mechanism.

Nickel-pincer cofactor biosynthesis involves LarB-catalyzed pyridinium carboxylation and LarE-dependent sacrificial sulfur insertion.

The lactate racemase enzyme (LarA) of Lactobacillus plantarum harbors a (SCS)Ni(II) pincer complex derived from nicotinic acid. Synthesis of the enzyme-bound cofactor requires LarB, LarC, and LarE, which are widely distributed in microorganisms. The functions of the accessory proteins are unknown, but the LarB C terminus resembles aminoimidazole ribonucleotide carboxylase/mutase, LarC binds Ni and could act in Ni delivery or storage, and LarE is a putative ATP-using enzyme of the pyrophosphatase-loop superfamily. Here, we show that LarB carboxylates the pyridinium ring of nicotinic acid adenine dinucleotide (NaAD) and cleaves the phosphoanhydride bond to release AMP. The resulting biscarboxylic acid intermediate is transformed into a bisthiocarboxylic acid species by two single-turnover reactions in which sacrificial desulfurization of LarE converts its conserved Cys176 into dehydroalanine. Our results identify a previously unidentified metabolic pathway from NaAD using unprecedented carboxylase and sulfur transferase reactions to form the organic component of the (SCS)Ni(II) pincer cofactor of LarA. In species where larA is absent, this pathway could be used to generate a pincer complex in other enzymes.

AMACR overexpression as a poor prognostic factor in patients with nasopharyngeal carcinoma.

The molecular prognostic adjunct in patients with nasopharyngeal carcinomas (NPCs) still remains obscured. Through data mining from published transcriptomic database, alpha-methylacyl-CoA racemase (AMACR) was first identified as a differentially upregulated gene in NPC tissues, which is a key enzyme for isometric conversion of fatty acids entering the ß-oxidation. Given the roles of AMACR in prognostication and frontline therapeutic regimen of common carcinomas, such as prostate cancer, we explored AMACR immunoexpression status and its clinical significance in NPC patients. AMACR immunohistochemistry was retrospectively performed and analyzed using H-score for biopsy specimens from 124 NPC patients who received standard treatment without distant metastasis at initial diagnosis. Those cases with H-score larger than the median value were construed as featuring AMACR overexpression. The findings were correlated with the clinicopathological variables, disease-specific survival (DSS), distant metastasis-free survival (DMFS), and local recurrence-free survival (LRFS). Endogenous AMACR protein expressions were assessed by real-time reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting in NPC cells and non-neoplastic mucosal cells. AMACR overexpression was significantly associated with increment of primary tumor status (P = 0.009) and univariately predictive of adverse outcomes for DSS, DMFS, and LRFS. In the multivariate comparison, AMACR overexpression still remained prognostically independent to portend worse DSS (P = 0.006, hazard ratio = 2.129), DMFS (P = 0.001, hazard ratio = 2.795), and LRFS (P = 0.041, hazard ratio = 2.009), together with advanced American Joint of Cancer Committee (AJCC) stages III-IV. Compared with non-neoplastic cells, both HONE1 and TW01 NPC cells demonstrated markedly increased AMACR expression. AMACR overexpression was identified as an important prognosticator and a potential therapeutic target in the future.

Role of AMACR (α-methylacyl-CoA racemase) and MFE-1 (peroxisomal multifunctional enzyme-1) in bile acid synthesis in mice.

Cholesterol is catabolized to bile acids by peroxisomal ß-oxidation in which the side chain of C27-bile acid intermediates is shortened by three carbon atoms to form mature C24-bile acids. Knockout mouse models deficient in AMACR (α-methylacyl-CoA racemase) or MFE-2 (peroxisomal multifunctional enzyme type 2), in which this ß-oxidation pathway is prevented, display a residual C24-bile acid pool which, although greatly reduced, implies the existence of alternative pathways of bile acid synthesis. One alternative pathway could involve Mfe-1 (peroxisomal multifunctional enzyme type 1) either with or without Amacr. To test this hypothesis, we generated a double knockout mouse model lacking both Amacr and Mfe-1 activities and studied the bile acid profiles in wild-type, Mfe-1 and Amacr single knockout mouse line and Mfe-1 and Amacr double knockout mouse lines. The total bile acid pool was decreased in Mfe-1-/- mice compared with wild-type and the levels of mature C24-bile acids were reduced in the double knockout mice when compared with Amacr-deficient mice. These results indicate that Mfe-1 can contribute to the synthesis of mature bile acids in both Amacr-dependent and Amacr-independent pathways.

Decreased levels of free D-aspartic acid in the forebrain of serine racemase (Srr) knock-out mice.

d-Serine, an endogenous co-agonist of the N-methyl-d-aspartate (NMDA) receptor is synthesized from l-serine by serine racemase (SRR). A previous study of Srr knockout (Srr-KO) mice showed that levels of d-serine in forebrain regions, such as frontal cortex, hippocampus, and striatum, but not cerebellum, of mutant mice are significantly lower than those of wild-type (WT) mice, suggesting that SRR is responsible for d-serine production in the forebrain. In this study, we attempted to determine whether SRR affects the level of other amino acids in brain tissue. We found that tissue levels of d-aspartic acid in the forebrains (frontal cortex, hippocampus and striatum) of Srr-KO mice were significantly lower than in WT mice, whereas levels of d-aspartic acid in the cerebellum were not altered. Levels of d-alanine, l-alanine, l-aspartic acid, taurine, asparagine, arginine, threonine, γ-amino butyric acid (GABA) and methionine, remained the same in frontal cortex, hippocampus, striatum and cerebellum of WT and mutant mice. Furthermore, no differences in d-aspartate oxidase (DDO) activity were detected in the forebrains of WT and Srr-KO mice. These results suggest that SRR and/or d-serine may be involved in the production of d-aspartic acid in mouse forebrains, although further detailed studies will be necessary to confirm this finding.

Hyposmolality differentially and spatiotemporally modulates levels of glutamine synthetase and serine racemase in rat supraoptic nucleus.

Prolonged hyposmotic challenge (HOC) has a dual effect on vasopressin (VP) secretion [Yagil and Sladek (1990) Am J Physiol 258(2 Pt 2):R492-R500]. We describe an electrophysiological correlate of this phenomenon, whereby in vitro HOC transiently reduced the firing activity of VP neurons within the supraoptic nucleus of brain slices, which was followed by a rebound increase of their activity; this was paralleled by changes in the level of proteins relevant to astroglia-neuronal interactions. Hence, in vitro HOC transiently (at 5 min) increased the level of astrocyte-specific glial fibrillary acidic protein (GFAP), which then declined to control or base level (at 20 min); this was blocked by the gliotoxin L-aminoadipic acid, but not by tetanus toxin, which was used to inhibit neurotransmission. Similarly, in vivo HOC led to changes in GFAP level, which after an early increase (10 min) returned to normal (30 min). Immunoassays revealed that neuronal, but not astrocytic, expression of serine racemase (SR) was increased at the late stage of HOC in vivo, whereas at an early stage there was a transient increase in level of the astrocyte-specific glutamine synthetase (GS). Furthermore, there was an increased molecular association between GFAP and GS at 10 min, whereas SR increased its association with the neuronal nuclear antigen NeuN at 30 min. These results suggest that the dual effect of HOC on VP neuronal secretion/activity could be related to metabolic/signaling changes in astrocytes (glutamate-glutamine conversion) and neurons (D-serine synthesis/ammonia production), which may account for the rebound in VP neuronal activity, presumably by promoting the activation of neuronal glutamate receptors.

Paradoxical roles of serine racemase and D-serine in the G93A mSOD1 mouse model of amyotrophic lateral sclerosis.

D-serine is an endogenous neurotransmitter that binds to the NMDA receptor, thereby increasing the affinity for glutamate, and the potential for excitotoxicity. The primary source of D-serine in vivo is enzymatic racemization by serine racemase (SR). Regulation of D-serine in vivo is poorly understood, but is thought to involve a combination of controlled production, synaptic reuptake by transporters, and intracellular degradation by D-amino acid oxidase (DAO). However, SR itself possesses a well-characterized eliminase activity, which effectively degrades D-serine as well. D-serine is increased two-fold in spinal cords of G93A Cu,Zn-superoxide dismutase (SOD1) mice--the standard model of amyotrophic lateral sclerosis (ALS). ALS mice with SR disruption show earlier symptom onset, but survive longer (progression phase is slowed), in an SR-dependent manner. Paradoxically, administration of D-serine to ALS mice dramatically lowers cord levels of D-serine, leading to changes in the onset and survival very similar to SR deletion. D-serine treatment also increases cord levels of the alanine-serine-cysteine transporter 1 (Asc-1). Although the mechanism by which SOD1 mutations increases D-serine is not known, these results strongly suggest that SR and D-serine are fundamentally involved in both the pre-symptomatic and progression phases of disease, and offer a direct link between mutant SOD1 and a glial-derived toxic mediator.

Serine racemase deletion abolishes light-evoked NMDA receptor currents in retinal ganglion cells.

Glycine and/or D-serine are obligatory coagonists of the N-methyl-D-aspartate receptor (NMDAR). Serine racemase, the D-serine-synthesizing enzyme, is expressed by astrocytes and Müller cells of the retina, but little is known about its role in retinal signalling. In this study, we utilize a serine racemase knockout (SRKO) mouse to explore the contribution of D-serine to inner-retinal function. Retinal tissue levels of D-serine in SRKO mice are reduced by 85%. Whole-cell recordings from SRKO retinal ganglion cells showed markedly reduced coagonist occupancy of NMDARs and consequently a dramatic reduction in the NMDAR component of light-evoked responses. NMDAR currents in SRKOs could be rescued by applying exogenous coagonist, but SRKO ganglion cells still displayed lower NMDA/AMPA receptor ratios than wild-type (WT) controls when the coagonist site was saturated. Despite having abnormalities in synaptic glutamatergic transmission, SRKO mice displayed no obvious signs of visual impairment in behavioural testing. These findings raise interesting questions about the role of D-serine in inner-retinal function and development.

Protein kinase C activity regulates D-serine availability in the brain.

D-serine is a co-agonist of NMDA receptor (NMDAR) and plays important roles in synaptic plasticity mechanisms. Serine racemase (SR) is a brain-enriched enzyme that converts L-serine to D-serine. SR interacts with the protein interacting with C-kinase 1 (PICK1), which is known to direct protein kinase C (PKC) to its targets in cells. Here, we investigated whether PKC activity regulates SR activity and D-serine availability in the brain. In vitro, PKC phosphorylated SR and decreased its activity. PKC activation increased SR phosphorylation in serine residues and reduced D-serine levels in astrocyte and neuronal cultures. Conversely, PKC inhibition decreased basal SR phosphorylation and increased cellular D-serine levels. In vivo modulation of PKC activity regulated both SR phosphorylation and D-serine levels in rat frontal cortex. Finally, rats that completed an object recognition task showed decreased SR phosphorylation and increased D-serine/total serine ratios, which was markedly correlated with decreased PKC activity in both cortex and hippocampus. Results indicate that PKC phosphorylates SR in serine residues and regulates D-serine availability in the brain. This interaction may be relevant for the regulation of physiological and pathological mechanisms linked to NMDAR function.

Discordant behavioral effects of psychotomimetic drugs in mice with altered NMDA receptor function.

RATIONALE: Enhancement of N-methyl-D: -aspartate receptor (NMDAR) activity through its glycine modulatory site (GMS) is a novel therapeutic approach in schizophrenia. Brain concentrations of endogenous GMS agonist D: -serine and antagonist N-acetyl-aspartylglutamate are regulated by serine racemase (SR) and glutamic acid decarboxylase 2 (GCP2), respectively. Using mice genetically, under-expressing these enzymes may clarify the role of NMDAR-mediated neurotransmission in schizophrenia. OBJECTIVES: We investigated the behavioral effects of two psychotomimetic drugs, the noncompetitive NMDAR antagonist, phencyclidine (PCP; 0, 1.0, 3.0, or 6.0 mg/kg), and the indirect dopamine receptor agonist, amphetamine (AMPH; 0, 1.0, 2.0, or 4.0 mg/kg), in SR -/- and GCP2 -/+ mice. Outcome measures were locomotor activity and prepulse inhibition (PPI) of the acoustic startle reflex. Acute effects of an exogenous GMS antagonist, gavestinel (0, 3.0, or 10.0 mg/kg), on PCP-induced behaviors were examined in wild-type mice for comparison to the mutants with reduced GMS activity. RESULTS: PCP-induced hyperactivity was increased in GCP2 -/+ mice, and PCP-enhanced startle reactivity was increased in SR -/- mice. PCP disruption of PPI was unaffected in either mutant. In contrast, gavestinel attenuated PCP-induced PPI disruption without effect on baseline PPI or locomotor activity. AMPH effects were similar to controls in both mutant strains. CONCLUSIONS: The results of the PCP experiments demonstrate that convergence of pharmacological and genetic manipulations at NMDARs may confound the predictive validity of these preclinical assays for the effects of GMS activation in schizophrenia. The AMPH data provide additional evidence that hyperdopaminergia in schizophrenia may be distinct from NMDAR hypofunction.

Serine racemase deletion disrupts memory for order and alters cortical dendritic morphology.

There is substantial evidence implicating N-methyl-D-aspartate receptors (NMDARs) in memory and cognition. It has also been suggested that NMDAR hypofunction might underlie the cognitive deficits observed in schizophrenia as morphological changes, including alterations in the dendritic architecture of pyramidal neurons in the prefrontal cortex (PFC), have been reported in the schizophrenic brain post mortem. Here, we used a genetic model of NMDAR hypofunction, a serine racemase knockout (SR-/-) mouse in which the first coding exon of the mouse SR gene has been deleted, to explore the role of D-serine in regulating cognitive functions as well as dendritic architecture. SR-/- mice exhibited a significantly disrupted representation of the order of events in distinct experiences as showed by object recognition and odor sequence tests; however, SR-/- animals were unimpaired in the detection of novel objects and in spatial displacement, and showed intact relational memory in a test of transitive inference. In addition, SR-/- mice exhibited normal sociability and preference for social novelty. Neurons in the medial PFC of SR-/- mice displayed reductions in the complexity, total length and spine density of apical dendrites. These findings show that D-serine is important for specific aspects of cognition, as well as in regulating dendritic morphology of pyramidal neurons in the medial PFC (mPFC). Moreover, they suggest that NMDAR hypofunction might, in part, be responsible for the cognitive deficits and synaptic changes associated with schizophrenia, and highlight this signaling pathway as a potential target for therapeutic intervention.

Potential roles of D-serine and serine racemase in experimental temporal lobe epilepsy.

To confirm the roles of D-serinergic gliotransmission in epilepsy, we investigated the relationship between spatiotemporally specific glial responses and the D-serine/serine racemase system in mesial temporal structures following status epilepticus (SE). In control animals, D-serine and serine racemase immunoreactivities were detected mainly in astrocytes. After SE, D-serine and serine racemase immunoreactivities were increased in astrocytes. Double-immunofluorescence study revealed that up-regulation of serine racemase immunoreactivity was relevant not to D-serine immunoreactivity but to nestin or vimentin immunoreactivity. Neither D-serine nor serine racemase was found in naïve or reactive microglia. In addition, phosphorylated N-methyl-D-aspartate (NMDA) receptor subunit 1 (pNR1-Ser896) immunoreactivity in the hippocampus was increased compared with controls. Increased D-serine immunoreactivity showed direct correlation with the phosphorylation of Ser896 of NR1. Given the findings of our previous study, these findings suggest that D-serine and serine racemase in astrocytes may play roles in neuronal hyperexcitability via a cooperative activation of NMDA receptors. Furthermore, serine racemase may be involved in migration and differentiation of immature astrocytes, which is relevant to reactive astrogliosis.

FolX and FolM are essential for tetrahydromonapterin synthesis in Escherichia coli and Pseudomonas aeruginosa.

Tetrahydromonapterin is a major pterin in Escherichia coli and is hypothesized to be the cofactor for phenylalanine hydroxylase (PhhA) in Pseudomonas aeruginosa, but neither its biosynthetic origin nor its cofactor role has been clearly demonstrated. A comparative genomics analysis implicated the enigmatic folX and folM genes in tetrahydromonapterin synthesis via their phyletic distribution and chromosomal clustering patterns. folX encodes dihydroneopterin triphosphate epimerase, which interconverts dihydroneopterin triphosphate and dihydromonapterin triphosphate. folM encodes an unusual short-chain dehydrogenase/reductase known to have dihydrofolate and dihydrobiopterin reductase activity. The roles of FolX and FolM were tested experimentally first in E. coli, which lacks PhhA and in which the expression of P. aeruginosa PhhA plus the recycling enzyme pterin 4a-carbinolamine dehydratase, PhhB, rescues tyrosine auxotrophy. This rescue was abrogated by deleting folX or folM and restored by expressing the deleted gene from a plasmid. The folX deletion selectively eliminated tetrahydromonapterin production, which far exceeded folate production. Purified FolM showed high, NADPH-dependent dihydromonapterin reductase activity. These results were substantiated in P. aeruginosa by deleting tyrA (making PhhA the sole source of tyrosine) and folX. The DeltatyrA strain was, as expected, prototrophic for tyrosine, whereas the DeltatyrA DeltafolX strain was auxotrophic. As in E. coli, the folX deletant lacked tetrahydromonapterin. Collectively, these data establish that tetrahydromonapterin formation requires both FolX and FolM, that tetrahydromonapterin is the physiological cofactor for PhhA, and that tetrahydromonapterin can outrank folate as an end product of pterin biosynthesis.

Serine racemase suppresses chondrogenic differentiation in cartilage in a Sox9-dependent manner.

Serine racemase (SR) is responsible for the biosynthesis of D-serine (D-Ser), an endogenous co-agonist for N-methyl-D-aspartate (NMDA) receptors, from L-serine (L-Ser) in the central nervous system. In the present study, we investigated the role of SR in the regulation of chondrogenic differentiation in cartilage. On in situ hybridization analysis of tibia from neonatal rats, SR mRNA was ubiquitously expressed in all cell layers of proliferating to hypertrophic chondrocytes. In the pre-chondrogenic cell line ATDC5 cells, mRNA expression was seen with SR irrespective of the cellular maturity, with no mRNA expression of the NR1 subunit essential for the heteromeric assembly of functional NMDA receptor channels. In ATDC5 cells stably overexpressing SR, significant inhibition was found with the maturation-dependent temporal increases in Alcian blue staining, alkaline phosphatase (ALP) activity and mRNA expression of type II and type X collagens. Stable overexpression of SR significantly impaired the sry-type HMG box 9 (Sox9) transcriptinal activity in ATDC5 cells, while Sox9 transcriptional activity was significantly inhibited in COS7 cells with co-introduction of SR and Sox9. However, no significant inhibition was seen with Sox9 transcriptional activity in COS7 cells co-introduced of either SR(K56G) defective of D-Ser formation ability or 3-phosphoglycerate dehydrogenase essential for D-Ser biosynthesis. The co-introduction of SR with Sox9 significantly decreased the Sox9 protein level with that of Sox9 mRNA being unchanged. These results suggest that SR may negatively regulate cellular differentiation through the inhibition of Sox9 transcriptional activity in chondrocytes.

[Role of D-serine in the mammalian brain].

D-Serine has recently been identified as a major gliotransmitter in the mammal central nervous system (CNS). The distribution of D-serine is analogous to the N-methyl-D-aspartate (NMDA)-type glutamate receptors in the brain. D-Serine is as potent as glycine as a coagonist at the glycine-binding site of NMDA receptors. Thus, D-serine has been considered as an endogenous ligand of the NMDA receptors in the brain. D-Serine is synthesized by serine racemase (SR) from L-serine. Both D-serine and SR have been enriched to astrocytes which are the dynamic partners of neurons at synapses and participate in controlling synaptic transmission, synaptic plasticity and synaptogenesis. The present review highlights the most recent findings on the molecular mechanisms of controlling D-serine metabolism in the CNS, the physiological role of D-serine in synaptic plasticity, and the pathological relevance of D-serine to schizophrenia, excitotoxicity- and neuroinflammation-induced neuronal death as well as neuropathic pain. Finally, as we have recently established SR knockout mouse strain with pure C57BL/6 genetic background, this novel mouse model will contribute the analysis of physiological and pathophysiological role of D-serine in vivo.

Propionyl-CoA and adenosylcobalamin metabolism in Caenorhabditis elegans: evidence for a role of methylmalonyl-CoA epimerase in intermediary metabolism.

We have utilized Caenorhabditis elegans to study human methylmalonic acidemia. Using bioinformatics, a full complement of mammalian homologues for the conversion of propionyl-CoA to succinyl-CoA in the genome of C. elegans, including propionyl-CoA carboxylase subunits A and B (pcca-1, pccb-1), methylmalonic acidemia cobalamin A complementation group (mmaa-1), co(I)balamin adenosyltransferase (mmab-1), MMACHC (cblc-1), methylmalonyl-CoA epimerase (mce-1) and methylmalonyl-CoA mutase (mmcm-1) were identified. To verify predictions that the entire intracellular adenosylcobalamin metabolic pathway existed and was functional, the kinetic properties of the C. elegans mmcm-1 were examined. RNA interference against mmcm-1, mmab-1, mmaa-1 in the presence of propionic acid revealed a chemical phenotype of increased methylmalonic acid; deletion mutants of mmcm-1, mmab-1 and mce-1 displayed reduced 1-[(14)C]-propionate incorporation into macromolecules. The mutants produced increased amounts of methylmalonic acid in the culture medium, proving that a functional block in the pathway caused metabolite accumulation. Lentiviral delivery of the C. elegans mmcm-1 into fibroblasts derived from a patient with mut(o) class methylmalonic acidemia could partially restore propionate flux. The C. elegans mce-1 deletion mutant demonstrates for the first time that a lesion at the epimerase step of methylmalonyl-CoA metabolism can functionally impair flux through the methylmalonyl-CoA mutase pathway and suggests that malfunction of MCEE may cause methylmalonic acidemia in humans. The C. elegans system we describe represents the first lower metazoan model organism of mammalian propionate spectrum disorders and demonstrates that mass spectrometry can be employed to study a small molecule chemical phenotype in C. elegans RNAi and deletion mutants.

Galactomannan hydrolysis and mannose metabolism in Cellvibrio mixtus.

Galactomannan hydrolysis results from the concerted action of microbial endo-mannanases, manosidases and alpha-galactosidases and is a mechanism of intrinsic biological importance. Here we report the identification of a gene cluster in the aerobic soil bacterium Cellvibrio mixtus encoding enzymes involved in the degradation of this polymeric substrate. The family 27 alpha-galactosidase, termed CmAga27A, preferentially hydrolyse galactose containing polysaccharides. In addition, we have characterized an enzyme with epimerase activity, which might be responsible for the conversion of mannose into glucose. The role of the identified enzymes in the hydrolysis of galactomannan by aerobic bacteria is discussed.

A variant of the alpha-methyl-acyl-CoA racemase gene created by a deletion in exon 5 and its expression in prostate cancer.

BACKGROUND: Alpha-methylacyl-CoA racemase (AMACR) is a mitochondrial and peroxisomal enzyme that is overexpressed in prostate cancer. Alternatively spliced variants of AMACR have recently been reported, however, their role in prostate cancer pathogenesis is unclear. METHODS: Using PCR techniques we have identified a novel variant of AMACR. RESULTS: This transcript arises by an alternative splicing event in the 5th exon of the gene whereby a 749 base sequence is deleted causing a shift in the reading frame. The protein encoded by this transcript has a predicted molecular weight of 43,833 kDa and a pI of 7.01 and therefore differs in size and physical characteristics from the main form of AMACR. The carboxyl terminus of this variant does not contain the peroxisomal targeting signal found in the main form of AMACR. Using real time PCR it was demonstrated that this transcript also occurs in normal prostate tissue and is elevated in prostate cancer. Coordinate expression of this transcript with the other forms of AMACR was shown. This transcript was expressed as a FLAG fusion protein in Cos-7 cells and probed with relevant antibodies. CONCLUSION: A deletion event in exon 5 of the AMACR gene creates a novel transcript that is coordinately expressed with the other forms of AMACR but with different biochemical characteristics. (c) 2005 Wiley-Liss, Inc.