Brain-specific Phgdh deletion reveals a pivotal role for L-serine biosynthesis in controlling the level of D-serine, an N-methyl-D-aspartate receptor co-agonist, in adult brain.

In mammalian brain, D-serine is synthesized from L-serine by serine racemase, and it functions as an obligatory co-agonist at the glycine modulatory site of N-methyl-D-aspartate (NMDA)-selective glutamate receptors. Although diminution in D-serine level has been implicated in NMDA receptor hypofunction, which is thought to occur in schizophrenia, the source of the precursor L-serine and its role in D-serine metabolism in adult brain have yet to be determined. We investigated whether L-serine synthesized in brain via the phosphorylated pathway is essential for D-serine synthesis by generating mice with a conditional deletion of D-3-phosphoglycerate dehydrogenase (Phgdh; EC 1.1.1.95). This enzyme catalyzes the first step in L-serine synthesis via the phosphorylated pathway. HPLC analysis of serine enantiomers demonstrated that both L- and D-serine levels were markedly decreased in the cerebral cortex and hippocampus of conditional knock-out mice, whereas the serine deficiency did not alter protein expression levels of serine racemase and NMDA receptor subunits in these regions. The present study provides definitive proof that L-serine-synthesized endogenously via the phosphorylated pathway is a key rate-limiting factor for maintaining steady-state levels of D-serine in adult brain. Furthermore, NMDA-evoked transcription of Arc, an immediate early gene, was diminished in the hippocampus of conditional knock-out mice. Thus, this study demonstrates that in mature neuronal circuits L-serine availability determines the rate of D-serine synthesis in the forebrain and controls NMDA receptor function at least in the hippocampus.

Clinical profile and visual outcome of intraocular inflammation associated with cat-scratch disease in Japanese patients.

PURPOSE: To evaluate intraocular inflammation in Japanese patients with cat-scratch disease (CSD). STUDY DESIGN: Retrospective clinical chart review. PATIENTS AND METHODS: The cases of 15 consecutive patients (19 affected eyes) in Kochi Prefecture, Japan who were serologically positive for Bartonella henselae or Bartonella quintana infection in association with intraocular inflammation were reviewed. The clinical manifestations, ocular complications, and treatment modalities were recorded. The clinical charts and photographic records were also reviewed for evidence of optic disc lesions, macular star, foci of chorioretinitis, and other findings. RESULTS: Thirteen patients reported fever before or at the time of the initial presentation. Ten of 11 patients with decreased visual acuity manifested neuroretinitis, and the remaining patient showed retinochoroiditis with macular involvement. One patient with a visual field defect manifested branch retinal artery occlusion. Three patients without visual disturbance presented with fever of unknown cause. Discrete white retinal or retinochoroidal lesions were the most common findings (84% of eyes, 87% of patients), followed by retinal hemorrhage (63% of eyes, 80% of patients), optic disc lesions (63% of eyes, 73% of patients), serous retinal detachment (53% of eyes, 67% of patients), and macular star (47% of eyes, 60% of patients). CONCLUSION: White retinal or retinochoroidal foci were the most common ocular posterior segment manifestations of CSD in this patient population. A diagnosis of CSD should be suspected in patients with fever and chorioretinal white spots, and the absence of neuroretinitis or macular star does not exclude the possibility of intraocular inflammation in CSD.

A Chiral [3]Rotaxane Comprising Achiral Bis-macrocyclic and Dumbbell-Shaped Components.

In this study, we synthesized a molecularly chiral [3]rotaxane comprising a calix-bis-crown ether (as the macrocyclic component) and two unsymmetrical dialkylammonium salts (as dumbbell-shaped components) without any chirality in any of the individual components. Chiral high-performance liquid chromatography was used to separate the enantiomers, which were characterized by circular dichroism spectroscopy. Density functional theory calculations gave an insight into the absolute configuration of each [3]rotaxane.

Distribution of retinal cone photoreceptor oil droplets, and identification of associated carotenoids in crow (Corvus macrorhynchos).

The topography of cone oil droplets and their carotenoids were investigated in the retina of jungle crow (Corvus macrorhynchos). Fresh retina was sampled for the study of retinal cone oil droplets, and extracted retinal carotenoids were saponified using methods adapted from a recent study, then identified with reverse-phase high-performance liquid chromatography (HPLC). To assess the effects of saponification conditions on carotenoid recovery from crow retina, we varied base concentration and total time of saponification across a wide range of conditions, and again used HPLC to compare carotenoid concentrations. Based on colors, at least four types of oil droplets were recognized, i.e., red, orange, green, and translucent, across the retina. With an average of 91,202 /mm(2), density gradually declines in an eccentric manner from optic disc. In retina, the density and size of droplets are inversely related. In the peripheral zone, oil droplets were significantly larger than those of the central area. The proportion of orange oil droplets (33%) was higher in the central area, whereas green was predominant in other areas. Three types of carotenoid (astaxanthin, galloxanthin and lutein), together with one unknown carotenoid, were recovered from the crow retina; astaxanthin was the dominant carotenoid among them. The recovery of carotenoids was affected by saponification conditions. Astaxanthin was well recovered in weak alkali (0.06 M KOH), in contrast, xanthophyllic carotenoids were best recovered in strong alkali (0.6 M KOH) after 12 h of saponification at freeze temperature.

Distinct role of growth hormone on epilepsy progression in a model of temporal lobe epilepsy.

Temporal lobe epilepsy is a common form of pharmacoresistant epilepsy, in which epileptogenic foci propagate to other regions of the brain from the area of the initial insult. The present study focused on epileptogenesis, that is, the development of the first foci inducing seizures in amygdala-kindled mice, a model of temporal lobe epilepsy, to find the molecular process promoting the formation of epileptogenic networks. The expression of growth hormone (GH) was up-regulated along neural circuits during the epileptogenesis, while there was no difference in the pituitary gland. The up-regulation was associated with increased phosphorylation/activation of signal transducer and activator of transcription 5 and expression of the Serum Response Element-regulated genes, FBJ osteosarcoma oncogene, early growth response 1, and Jun-B oncogene, suggesting that expression of GH leads to GH signaling in the hippocampus and cortex. Furthermore, the administration of the hormone into the hippocampus markedly enhanced the progression of kindling. The administration of an inhibitor of its secretion into the hippocampus elicited a delay in the progression. Our results demonstrate directly that regulation via growth hormone has a robust impact in epileptogenesis.

Impaired neurogenesis in embryonic spinal cord of Phgdh knockout mice, a serine deficiency disorder model.

Mutations in the d-3-phosphoglycerate dehydrogenase (PHGDH; EC 1.1.1.95) gene, which encodes an enzyme involved in de novol-serine biosynthesis, are shown to cause human serine deficiency disorder. This disorder has been characterized by severe neurological symptoms including congenital microcephaly and psychomotor retardation. Our previous work demonstrated that targeted disruption of mouse Phgdh leads to a marked decrease in serine and glycine, severe growth retardation of the central nervous system, and lethality after embryonic day 13.5. To clarify how a serine deficiency causes neurodevelopmental defects, we characterized changes in metabolites, gene expression and morphological alterations in the spinal cord of Phgdh knockout mice. BeadChip microarray analysis revealed significant dysregulation of genes involved in the cell cycle. Ingenuity Pathway Analysis also revealed a significant perturbation of regulatory networks that operate in the cell cycle progression. Moreover, morphological examinations of the knockout spinal cord demonstrated a marked deficit in dorsal horn neurons. Radial glia cells, native neural stem/progenitor cells, accumulated in the dorsal ventricular zone, but they did not proceed to a G(0)-like quiescent state. The present integrative study provides in vivo evidence that normal cell cycle progression and subsequent neurogenesis of radial glia cells are severely impaired by serine deficiency.

Carotenoid production in Bacillus subtilis achieved by metabolic engineering.

The carotenoid synthetic genes, crtM and crtN, derived from Staphylococcus aureus, were introduced into B. subtilis, resulting in yellow pigmentation. Absorption maxima of pigments and MALDI-TOF mass spectrometry demonstrated that the pigmented strain accumulated two C(30) carotenoids, 4,4'-diapolycopene and 4,4'-diaponeurosporene. A survival test using H(2)O(2) revealed that the pigmented strain was more resistant to oxidative stress than the strain harboring an empty-vector. These findings indicate that B. subtilis can produce carotenoids, and the strain accumulating the carotenoids, CarotenoBacillus, will become a basal host for production of C(30) carotenoids and evaluation of their antioxidative effects.

Novel carotenoid-based biosensor for simple visual detection of arsenite: characterization and preliminary evaluation for environmental application.

A novel whole-cell arsenite biosensor was developed using the photosynthetic bacterium Rhodopseudomonas palustris no. 7 and characterized. A sensor plasmid containing the operator-promoter region of the ars operon and arsR gene from Escherichia coli and the crtI gene from R. palustris no. 7 was introduced into a blue-green mutant with crtI deleted, R. palustris no. 711. The biosensor changed color in response to arsenite, and the change was obvious to the naked eye after 24 h without further manipulation. Real-time reverse transcription-PCR showed that the crtI mRNA was induced 3-fold at 3 h and 2.5-fold at 6 h after addition of 50 microg/liter arsenite compared with the no-arsenite control, and consistent with this, the relative levels of lycopene and rhodopin also increased compared with the control. Colorimetric analysis of the bacteria showed that the hue angle had clearly shifted from green-yellow toward red in an arsenic dose-dependent manner at 24 h after arsenite addition. This obvious shift occurred irrespective of the culture conditions before arsenite was added, indicating that the color change of the biosensor is stable in water samples containing various concentrations of dissolved oxygen. Finally, assays using samples prepared in various types of mineral water indicated that this biosensor could be used to screen groundwater samples for the presence of arsenite in a variety of locations, even where electricity is not available.

Functional substitution of the transient membrane-anchor domain in Escherichia coli FtsY with an N-terminal hydrophobic segment of Streptomyces lividans FtsY.

FtsY is a signal recognition particle receptor in Escherichia coli that mediates the targeting of integral membrane proteins to translocons by interacting with both signal recognition particle (SRP)-nascent polypeptide-ribosome complexes and the cytoplasmic membrane. Genes encoding the N-terminal segments of Streptomyces lividans FtsY were fused to a gene encoding the E. coli FtsY NG domain (truncated versions of FtsY lacking the transient membrane-anchor domain at the N-terminus), introduced into a conditional ftsY-deletion mutant of E. coli, and expressed in trans to produce chimeric FtsY proteins. Under FtsY-depleted conditions, strains producing chimeric proteins including 34 N-terminal hydrophobic residues grew whereas strains producing chimeric proteins without these 34 residues did not. A strain producing the chimeric protein comprising the 34 residues and NG domain processed beta-lactamase, suggesting that the SRP-dependent membrane integration of leader peptidase was restored in this strain. These results suggest that the N-terminal hydrophobic segment of FtsY in this Gram-positive bacterium is responsible for its interaction with the cytoplasmic membrane.

Formation of [2]- and [3]Rotaxanes through Bridging under Kinetic and Thermodynamic Control.

An efficient synthesis of a doubly stranded [3]rotaxane has been developed through bridging of a pseudo[3]rotaxane featuring two axle components. Reversible azine formation was effective as the bridging reaction. Kinetic and thermodynamic conditions provided the [2]- and [3]rotaxanes, respectively.

Mouse 3-phosphoglycerate dehydrogenase gene: genomic organization, chromosomal localization, and promoter analysis.

d-3-Phosphoglycerate dehydrogenase (Phgdh; EC 1.1.1.95) is the first committed enzyme of l-serine biosynthesis in the phosphorylated pathway. We have recently demonstrated that, in developing and mature brain, expression of Phgdh is highly regulated in a cell lineage-specific manner, mainly in neuroepithelial stem cells, radial glia, and astrocytes (J. Neurosci. 21 (2001) 7691; Arch. Histol. Cytol. 66 (2003) 109). To gain insight into the regulatory mechanism of Phgdh expression, we have isolated a mouse genomic clone that contains the entire mouse Phgdh gene. Structural analysis demonstrated that the Phgdh gene spans approximately 27 kilobases (kb) in length and comprises 12 exons with 11 intervening introns. Using fluorescent in situ hybridization (FISH), we mapped the gene to mouse chromosome 3, region F2-F3. Analysis of a 1.8 kb fragment of the 5'-flanking region showed that the classical TATA-box motif near transcription initiation sites was absent. Instead, a GC-rich proximal region containing a potential Sp1 recognition sequence was present; this region is conserved in mouse, rat, and human counterparts. Transient transfection analysis revealed that the cis-acting elements necessary for basal transcription of Phgdh are contained within the -196/+4 proximal sequence of the promoter, in which the conserved Sp1 recognition sites play an important role for basal promoter activity.

Acyclic carotenoid and cyclic apocarotenoid cleavage by an orthologue of lignostilbene-α,ß-dioxygenase in Rhodopseudomonas palustris.

Carotenoid cleavage oxygenases catalyse formation of apocarotenoids and the precursors of phytohormones, abscisic acid and strigolactones through oxidative cleavage at specific double bonds of carotenoids. A gene encoding a presumed bacterial oxygenase homologous to lignostilbene-α,ß-dioxygenases has been found in the genome of Rhodopseudomonas palustris. By analysing apocarotenoids in recombinant Escherichia coli strains, it was found that the presumed oxygenase catalyses the 15,15' double bond cleavage of lycopene and neurosporene. Cell lysate containing the recombinant protein cleaved all-trans-ß-apo-8'-carotenal at the 15,15' double bond into retinal and apo-8',15'-apocarotene-dial. These data demonstrate for the first time that the orthologue of lignostilbene-α,ß-dioxygenase found in the carotenogenic phototrophic bacterium has the 15,15' double bond cleavage activity towards both the acyclic carotenoids and cyclic apocarotenoid.

Monitoring of environmental arsenic by cultures of the photosynthetic bacterial sensor illuminated with a near-infrared light emitting diode array.

Recombinant Rhodopseudomonas palustris, harboring the carotenoid-metabolizing gene crtI (CrtIBS), and whose color changes from greenish yellow to red in response to inorganic As(III), was cultured in transparent microplate wells illuminated with a light emitting diode (LED) array. The cells were seen to grow better under near-infrared light, when compared with cells illuminated with blue or green LEDs. The absorbance ratio of 525 to 425 nm after cultivation for 24 h, which reflects red carotenoid accumulation, increased with an increase in As(III) concentrations. The detection limit of cultures illuminated with near-infrared LED was 5 microgram/l, which was equivalent to that of cultures in test tubes illuminated with an incandescent lamp. A near-infrared LED array, in combination with a microplate, enabled the simultaneous handling of multiple cultures, including CrtIBS and a control strain, for normalization by the illumination of those with equal photon flux densities. Thus, the introduction of a near-infrared LED array to the assay is advantageous for the monitoring of arsenic in natural water samples that may contain a number of unknown factors and, therefore, need normalization of the reporter event.

Application of fluorescent protein-tagged trans factors and immobilized cis elements to monitoring of toxic metals based on in vitro protein-DNA interactions.

Environmental toxic metals cause serious global public health problems. On-site monitoring protects people from exposure to such harmful elements. In this study, the bacterial transcriptional switches were applied to monitoring of toxic metals. ArsR and CadC, trans factors of Escherichia coli and Staphylococcus aureus, were fused to GFP. The fusion proteins, ArsR-GFP and CadC-GFP, associated with cis elements, P(ars)-O(ars) and P(cad)-O(cad), respectively and dissociated from those upon recognition of As(III) or Pb/Cd. Cell lysates containing ArsR-GFP were pre-incubated with As(III) standard solutions for 15 min and loaded into P(ars)-O(ars)-immobilized microplate wells. Cell lysates containing CadC-GFP were pre-incubated with Pb or Cd solutions and loaded into P(cad)-O(cad)-immobilized wells. The cell lysates were incubated for 15 min and removed from the wells. Fluorescence intensity in the wells dose-dependently decreased in response to As(III) up to 200 µg/l or Pb/Cd up to 100 µg/l. Detection limits were 10 µg/l for As(III) 10 µg/l for Cd, and 20 µg/l for Pb with a microplate fluororeader, whereas 5.0 µg/l for As(III), 1.0 µg/l for Cd, and 10 µg/l for Pb with a handheld fluorometer. This method was available to detect Pb/Cd or As(III) in water containing soil extracts. This is the first demonstration of a simple and rapid fluorometry to detect analytes based on in vitro interaction between a cis element and a trans factor.

Sensitive fluorescent microplate bioassay using recombinant Escherichia coli with multiple promoter-reporter units in tandem for detection of arsenic.

Genetically modified bacterial biosensors can detect specific environmental compounds. Here, we attempted to establish a fluorescent microplate method to detect arsenic using recombinant Escherichia coli cells transformed with plasmids harboring three tandem copies of the ars promoter/operator-the gene for green fluorescent protein (gfp). In the biosensors, one copy of arsR, whose transcription is autoregulated by the ars promoter/operator and ArsR in the genome of E. coli, was placed in trans in another plasmid under the control of isopropyl-1-thio-beta-D-galactopyranoside-inducible promoter. First, this manipulation enabled regulation of the arsR expression at an adequate level. Second, the copy number of reporter unit also affected signal and noise. When the plasmid harboring three copies of the reporter unit was used, the signal-to-noise ratio doubled and the detection limit decreased from 20 to 7.5 microg L(-1) As(III), compared to the use of the plasmid harboring one copy of the ars promoter/operator-arsR-gfp. Thus, segregation of arsR from the ars promoter/operator-gfp using two plasmids is effective in regulating the signal-to-noise ratio and the detection limit with the different functions.

Inactivation of the 3-phosphoglycerate dehydrogenase gene in mice: changes in gene expression and associated regulatory networks resulting from serine deficiency.

D-3-Phosphoglycerate dehydrogenase (Phgdh) is a necessary enzyme for de novo L-serine biosynthesis. Mutations in the human PHGDH cause serine deficiency disorders characterized by severe neurological symptoms including congenital microcephaly and psychomotor retardation. We showed previously that targeted disruption of Phgdh in mice causes overall growth retardation with severe brain microcephaly and leads to embryonic lethality. Here, amino acid analysis of Phgdh knockout (KO) mouse embryos demonstrates that free serine and glycine concentrations are decreased markedly in head samples, reflecting the metabolic changes of serine deficiency found in human patients. To understand the pathogenesis of serine deficiency disorders at the molecular level, we have exploited this animal model to identify altered gene expression patterns using a microarray technology. Comparative microarray analysis of the Phgdh KO and wild-type head at gestational day 13.5 revealed an upregulation of genes involved in transfer RNA aminoacylation, amino acid metabolism, amino acid transport, transcriptional regulation, and translation, and a downregulation of genes involved in transcription in neuronal progenitors and muscle and cartilage development. A computational network analysis software was used to construct transcriptional regulatory networks operative in the Phgdh KO embryos in vivo. These observations suggest that Phgdh inactivation alters transcriptional programs in several regulatory networks.

Evaluation of colors in green mutants isolated from purple bacteria as a host for colorimetric whole-cell biosensors.

The change in carotenoid-based bacterial color from yellow to red can be applied to whole-cell biosensors. We generated several green mutants to emphasize the color change in such biosensors. The blue-green crtI-deleted mutant, Rhodopseudomonas palustris no.711, accumulated the colorless carotenoid precursor, phytoene. Green Rhodovulum sulfidophilum M31 accumulated neurosporene, a downstream product of phytoene. Another green mutant, Rhodobacter sphaeroides Ga, accumulated neurosporene and chloroxanthin, which are both downstream products of phytoene. All green mutants accumulated bacteriochlorophyll a. Photosynthetic membrane obtained from the green mutants all exhibited decreased absorption of wavelength range at 510-570 nm. Therefore, these indicate that the greenish bacterial colors were mainly caused by the existence of bacteriochlorophyll a and the changes in carotenoid composition in photosynthetic membrane. The colors of the green mutants and their wild-type strains were plotted in the CIE-L*a*b* color space, and the color difference (DeltaE*ab) values between a green mutant and its wild type were calculated. DeltaE*ab values were higher in the green mutants than in Rdv. sulfidophilum CDM2, the yellowish host strain of reported biosensors. These data indicate that change in bacterial color from green to red is more distinguishable than that from yellow to red as a reporter signal of carotenoid-based whole-cell biosensors.

Targeted disruption of the mouse 3-phosphoglycerate dehydrogenase gene causes severe neurodevelopmental defects and results in embryonic lethality.

D-3-Phosphoglycerate dehydrogenase (Phgdh; EC 1.1.1.95) is the first committed enzyme of L-serine biosynthesis in the phosphorylated pathway. To determine the physiological importance of Phgdh-dependent L-serine biosynthesis in vivo, we generated Phgdh-deficient mice using targeted gene disruption in embryonic stem cells. The absence of Phgdh led to a drastic reduction of L-serine metabolites such as phosphatidyl-L-serine and sphingolipids. Phgdh null embryos have small bodies with abnormalities in selected tissues and died after days post-coitum 13.5. Striking abnormalities were evident in the central nervous system in which the Phgdh null mutation culminated in hypoplasia of the telencephalon, diencephalon, and mesencephalon; in particular, the olfactory bulbs, ganglionic eminence, and cerebellum appeared as indistinct structures. These observations demonstrate that the Phgdh-dependent phosphorylated pathway is essential for normal embryonic development, especially for brain morphogenesis.