Exacerbation of NMDA, AMPA, and l-Glutamate Excitotoxicity by the Succinate Dehydrogenase Inhibitor Malonate

Abstract: We report that a subtoxic dose of the succinate dehydrogenase (SDH) inhibitor malonate greatly enhances the neurotoxicity of three different excitatory amino acid agonists: N-methyl-d -aspartate (NMDA), S-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (S-AMPA), and l -glutamate. In male Sprague-Dawley rats, intrastriatal stereotaxic injection of malonate alone (0.6 µmol), NMDA alone (15 nmol), S-AMPA alone (1 nmol), or glutamate alone (0.6 µmol) produced negligible toxicity as assessed by measurement of lesion volume. Coinjection of subtoxic malonate with NMDA produced a large lesion (15.2 ± 1.4 mm³), as did coinjection of malonate with S-AMPA (11.0 ± 1.0 mm³) or glutamate (12.8 ± 0.7 mm³). Administration of the noncompetitive NMDA antagonist MK-801 (5 mg/kg i.p.) completely blocked the toxicity of malonate plus NMDA (0.5 ± 0.3 mm³). This dose of MK-801 had little effect on the lesion produced by malonate plus S-AMPA (9.0 ± 0.7 mm³), but it attenuated the toxicity of malonate plus glutamate by ～40% (7.5 ± 0.9 mm³). Coinjection of the AMPA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)-quinoxaline (NBQX; 2 nmol) had no effect on malonate plus NMDA or malonate plus glutamate toxicity (12.3 ± 1.8 and 14.0 ± 0.9 mm³, respectively) but greatly attenuated malonate plus S-AMPA toxicity (1.5 ± 0.9 mm³). Combination of the two antagonists conferred no additional neuroprotection in any paradigm. These results indicate that metabolic inhibition exacerbates both NMDA receptor- and non-NMDA receptor-mediated excitotoxicity. They also suggest that the NMDA receptor may play a major role in situations of metabolic compromise in vivo, where glutamate is the endogenous agonist. Furthermore, glutamate toxicity under conditions of metabolic compromise may not be mediated entirely by ionotropic glutamate receptors.     

Highly enantioselective oxidation of racemic phenyl-1,2-ethanediol to optically pure (R)-(−)-mandelic acid by a newly isolated Brevibacterium lutescens CCZU12-1

Enantioselective oxidation of racemic phenyl-1,2-ethanediol into (R)-(?)-mandelic acid by a newly isolated Brevibacterium lutescens CCZU12-1 was demonstrated. It was found that optically active (R)-(?)-mandelic acid (e.e.p?>?99.9 %) is produced leaving the other enantiomer (S)-(+)-phenyl-1,2-ethanediol intact. Using fed-batch method, a total of 172.9 mM (R)-(?)-mandelic acid accumulated in the reaction mixture after the seventh feed. Moreover, oxidation of phenyl-1,2-ethanediol using calcium alginate-entrapped resting cells was carried out in the aqueous system, and efficient biocatalyst recycling was achieved as a result of cell immobilization in calcium alginate, with a product-to-biocatalyst ratio of 27.94 g (R)-(?)-mandelic acid g^?1 dry cell weight cell after 16 cycles of repeated use.     

Purification and characterization of an alcohol dehydrogenase from 1,2-propanediol-grownDesulfovibrio strain HDv

The sulfate-reducing bacterimDesulfovibrio strain HDv (DSM 6830) grew faster on (S)- and on (R, S)-1,2-propanediol (μ_max 0.053 h⁻) than on (R)-propanediol (0.017 h⁻¹) and ethanol (0.027 h⁻¹). From (R, S)-1,2-propanediol-grown cells, an alcohol dehydrogenase was purified. The enzyme was oxygen-labile, NAD-dependent, and decameric; the subunit mol. mass was 48 kDa. The N-terminal amino acid sequence indicated similarity to alcohol dehydrogenases belonging to family III of NAD-dependent alcohol dehydrogenases, the first 21 N-terminal amino acids being identical to those of theDesulfovibrio gigas alcohol dehydrogenase. Best substrates were ethanol and propanol (K _m of 0.48 and 0.33 mM, respectively). (R, S)-1,2-Propanediol was a relatively poor substrate for the enzyme, but activities in cell extracts were high enough to account for the growth rate. The enzyme showed a preference for (S)-1,2-propanediol over (R)-1,2-propanediol. Antibodies raised against the alcohol dehydrogenase ofD. gigas showed cross-reactivity with the alcohol dehydrogenase ofDesulfovibrio strain HDv and with cell extracts of six other ethanol-grown sulfate-reducing bacteria.     

Modulation of the levels of NMDA receptor subunit mRNA and the bindings of [3H]MK-801 in rat brain by chronic infusion of subtoxic dose of MK-801

The effects of continuous infusion of NMDA receptor antagonist MK-801 on the modulation of NMDA receptor subunits NR1, NR2A, NR2B, and NR2C were investigated by using in situ hybridization study. Differential assembly of NMDA receptor subunits determines their functional characteristics. Continuous intracerebroventricular (i.c.v.) infusion with MK-801 (1 pmol/10 l/h) for 7 days resulted in significant modulations in the NR1, NR2A, and NR2B mRNA levels without producing stereotypic motor syndromes. The levels of NR1 mRNA were significantly increased (9-20%) in the cerebral cortex, striatum, septum, and CA1 of hippocampus in MK-801-infused rats. The levels of NR2A mRNA were significantly decreased (11-16%) in the CA3 and dentate gyrus of hippocampus in MK-801-infused rats. In contrast to NR2A, NR2B subunit mRNA levels were increased (10-14%) in the cerebral cortex, caudate putamen, and thalamus. However, no changes of NR2C subunits in cerebellar granule layer were observed. Using quantitative ligand autoradiography, the binding of NMDA receptor ligand [³H]MK-801 was increased (12-25%) significantly in almost all brain regions except in the thalamus and cerebellum after 7 days infusion with MK-801. These results suggest that region-specific changes of NMDA receptor subunit mRNA and [³H]MK-801 binding are involved in the MK-801-infused adult rats.     

Metabotropic Glutamate Receptor in C6BU-1 Glioma Cell Has NMDA Receptor-Ion Channel Complex-Like Properties and Interacts with Serotonin2 Receptor-Stimulated Signal Transduction

Abstract: We found in cultured glioma (C6BU-1) cells that excitatory amino acids (EAAs) such as glutamate, N-methyl-d -aspartate (NMDA), aspartate, and metabotropic glutamate receptor agonist trans-(±)-1-amino-1,3-cyclopentanedicarboxylate caused an increase in the inositol 1,4,5-trisphosphate formation and the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the absence of extracellular Mg²⁺ and Ca²⁺. Pertussis toxin treatment abolished this glutamate-induced [Ca²⁺]_i increase. Various antagonists against NMDA receptor-ion channel complex, such as Mg²⁺, d -2-amino-5-phosphonovalerate (d -APV), HA-966, and MK-801, also inhibited the increase in [Ca²⁺]_i induced by glutamate. These results indicate that these metabotropic EAA receptors coupled to pertussis toxin-susceptible GTP-binding protein and phospholipase C system in C6BU-1 glioma cells have the pharmacological properties of NMDA receptor-ion channel complexes. We also found that in the presence of Mg²⁺ these metabotropic receptors resemble the NMDA receptor-ion channel complex interacted with 5-hydroxytryptamine₂ (5-HT₂) receptor signaling. EAAs inhibited 5-HT₂ receptor-mediated intracellular Ca²⁺ mobilization and inositol 1,4,5-trisphosphate formation in a concentration-dependent manner. The inhibitory effect of glutamate was reversed by various NMDA receptor antagonists (d -APV, MK-801, phencyclidine, and HA-966), but l -APV failed to block the inhibitory effect of glutamate. The same result was observed in the absence of extracellular Ca²⁺. In addition, this inhibitory effect on 5-HT₂ receptor-mediated signal transduction was abolished by treatment of C6BU-1 cells with pertussis toxin, whereas 5-HT₂ receptor-mediated [Ca²⁺]_i increase was not abolished by pertussis toxin treatment. We can, therefore, conclude that the inhibitory effect of glutamate is not a result of the influx of Ca²⁺ through the ion channel and that it operates via metabotropic glutamate receptors, having NMDA receptor-ion channel complex-like properties and being coupled with pertussis toxin-sensitive GTP-binding protein and phospholipase C.     

Allosteric Regulation of the N-Methyl-d-Aspartate Receptor-Linked Ion Channel Complex and Effects of Ethanol in Ethanol-Withdrawal Seizure-Prone and -Resistant Mice

Abstract: The effects of ethanol, glycine, and spermidine on the specific binding of [³H]MK-801 were characterized in Triton-treated membranes prepared from the hippocampus and cortex of ethanol-withdrawal seizure-prone (WSP) and -resistant (WSR) mice. Glycine, an allosteric agonist at the NMDA receptor-linked ion channel complex, caused an increase in specific [³H]MK-801 binding to hippocampal membrane preparations. There were no significant differences in EC₅₀ values between the selected lines for the effect of glycine (WSP, 391.7 ± 48.4 nM; WSR, 313.4 ± 77 nM) in the presence of 10 µM NMDA or in the maximal response to the agonist (WSP, 1.75 ± 0.26 pmol/mg of protein; WSR, 1.67 ± 0.22 pmol/mg of protein). The EC₅₀ values for the spermidine-induced increase in [³H]MK-801 binding in membranes from hippocampus in the absence (WSP, 11.7 ± 0.83 µM; WSR, 9.98 ± 1.29 µM) or in the presence of 10 µM glycine and 10 µM NMDA (WSP, 2.1 ± 0.35 µM; WSR, 2.37 ± 0.42 µM) also did not differ. Similar results were obtained in cortical membranes. Saturation isotherms indicated that there was no difference in the density of [³H]MK-801 binding sites, or in their affinity for the radioligand, between the mouse lines. In addition, administration of ethanol by inhalation (24 h) to WSP and WSR mice did not cause an increase in the density of [³H]MK-801 binding sites, and there was no difference in the density or affinity of binding sites between the mouse lines. Withdrawal from ethanol (6 h), which causes an increase in the severity of handling-induced convulsions in WSP mice, also did not alter the binding site density or affinity for radioligand. The results suggest that the characteristics of the NMDA receptor-linked ion channel complex in the tissue preparations described here do not differ in WSP and WSR mice. Thus, genetic differences in seizure susceptibility during ethanol withdrawal can be dissociated from the total density of hippocampal or cortex NMDA receptors under activating conditions.     

Manganese(II) complexes of di-2-pyridinylmethylene-1,2-diimine di-Schiff base ligands: Structures and reactivity

Manganese(II) complexes [Mn(L)X₂] were prepared and characterized, where L is a neutral di-Schiff base ligand incorporating pyridylimine donor arms, including (1R,2R)-N,N′-bis(2-pyridylmethylidene)-1,2-diphenylethylenediimine (L¹), (1R,2R)-N,N′-bis(6-methyl-2-pyridylmethylidene)-1,2-cyclohexyldiimine (L²), or (1R,2R)-, (1S,2S)- or racemic N,N′-bis(2-pyridylmethylidene)-1,2-cyclohexyldiimine (L³), and X⁻ =  or Cl⁻. Product complexes were structurally characterized, specifically including [Mn(R,R-L¹)(NCCH₃)₃](ClO₄)₂, [Mn(R,R-L²)(OH₂)₂](ClO₄)₂ and racemic [Mn(L³)Cl₂]. The first of these complexes features a heptacoordinate ligand field in a distorted pentagonal bipyramid, and the latter two are hexacoordinate, but retain equatorially monovacant pentagonal bipyramidal structures. Complexes [Mn(L³)X₂] (X⁻ = Cl⁻, ) were reacted with the primary phosphine FcCH₂PH₂ (Fc = -C₅H₄FeC₅H₅), H₂O and ethyldiazoacetate (EDA). The first two substrates prompted reactivity at a single ligand imine bond, resulting in hydrophosphination and hydrolysis, respectively. Complexes of the derivative ligands were also structurally characterized. Evidence for EDA activation was obtained by electrospray ionization mass spectrometry, but catalytic carbene transfer was not obtained.     

Substrate specificity and stereospecificity of limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis DCL14; an enzyme showing sequential and enantioconvergent substrate conversion

Limonene-1,2-epoxide hydrolase (LEH) from Rhodococcus erythropolis DCL14, an enzyme involved in the limonene degradation pathway of this microlorganism, has a narrow substrate specificity. Of the compounds tested, the natural substrate, limonene-1,2-epoxide, and several alicyclic and 2-methyl-1,2-epoxides (e.g. 1-methylcyclohexene oxide and indene oxide), were substrates for the enzyme. When LEH was incubated with a diastereomeric mixture of limonene-1,2-epoxide, the sequential hydrolysis of first the (1R,2S)- and then the (1S,2R)-isomer was observed. The hydrolysis of (4R)- and (4S)-limonene-1,2-epoxide resulted in, respectively, (1S,2S,4R)- and (1R,2R,4S)-limonene-1,2-diol as the sole product with a diastereomeric excess of over 98%. With all other substrates, LEH showed moderate to low enantioselectivities (E ratios between 34 and 3).     

Absolute stereochemistry of (+)-trans-1,2-dihydroxyacenaphthene,a mammalian metabolite of acenaphthylene

A sample of (+)-trans-1,2-dihydroxyacenaphthene, a mammalian metabolite of acenaphthylene, was prepared by stereoselective partial hydrolysis of the corresponding synthetic racemic diacetate using the mold, Rhizopus nigricans. The absolute stereochemistry of the trans-diol was established as (1R,2R) by conversion to the known dimethyl (2S,3S)diacetoxysuccinate.     

On the role of NMDA receptors in blood pressure regulation in spontaneously hypertensive rats (SHR)

Summary In the present study the binding of [³H]MK-801 to glutamatergic receptors of the NMDA type was compared in spontaneously hypertensive (SHR) and normotensive (WKY) rats in various brain structures (including nucleus tractus solitarii) by quantitative receptor autoradiography. Additionally, blood pressure changes after treatment with the NMDA antagonist MK-801 were studied in both strains. There were no differences between SHR and WKY rats either in the level of [³H]MK-801 binding or in the hypertensive reaction to MK-801.     

Effects of Excitatory Amino Acids on Cerebral Oxygen Consumption and Blood Flow in Rat

This investigation tested the importance of excitatory amino acids' effects on regional cerebral O₂ consumption and the concomitant changes in cerebral blood flow (rCBF) in isoflurane anesthetized rats. In the glutamate or N-methyl-D-aspartate (NMDA) groups, 10^–2 M glutamate or NMDA was topically applied to the right cortex and the left cortex was used as a control. One mg/kg dizocilpine maleate (MK-801), a non-competitive NMDA receptor antagonist, was administered (iv) to the MK-801 group and saline was given to the control group. Cortical rCBF was determined using ¹⁴C-iodoantipyrine and regional O₂ extraction was measured microspectrophotometrically. Cerebral O₂ consumption increased 77% after glutamate (contralateral cortex: 9.0 ± 1.1 ml O₂/min/100 g, glutamate treated cortex: 15.9 ± 3.9), while a 46% increase was observed with the same concentration of NMDA (contralateral cortex: 9.8 ± 2.0, NMDA treated cortex: 14.3 ± 5.5). After MK-801, the O₂ consumption decreased to 37% of the control value (control cortex: 7.0 ± 1.3, MK-801 treated cortex: 2.6 ± 3.9). MK-801 significantly decreased cerebral O₂ extraction from 7.1 ± 1.3 ml O₂/100 ml (control cortex) to 5.3 ± 0.6 (MK-801 treated cortex). However, there was no significant difference in cerebral O₂ extraction between treated and contralateral cortex in either the glutamate or NMDA groups. The increase in O₂ consumption caused by glutamate or NMDA was coupled with increased rCBF. Glutamate increased rCBF from 95 ± 5 ml/min/100 g (contralateral cortex) to 165 ± 31 (treated cortex), while NMDA increased rCBF from 114 ± 12 (contralateral cortex) to 178 ± 60 (treated cortex). MK-801 decreased O₂ consumption with a lesser decrease of rCBF. The rCBF was 48 ± 9 in the MK-801 treated cortex and 99 ± 22 in the control cortex. Some substances produced by the activation of NMDA receptors may be related to the coupling of cerebral metabolism and blood flow, since after blockade of NMDA receptors with MK-801, this relationship is uncoupled. These findings suggest that glutamatergic processes have a major effect on cerebral O₂ consumption and that this is at least partly due to NMDA receptors.     

Pharmacological characterization of the N-methyl-d-aspartate (NMDA) receptor-channel in rodent and dog brain and rat spinal cord using [3H]MK-801 binding

The biochemical and pharmacological properties of [³H]MK-801 binding to the N-methyl-d-aspartate (NMDA) receptor-channel in homogenates of mouse, guinea pig and dog brain, dog cerebral cortex and rat spinal cord were determined using radioligand binding techniques. Specific [³H]MK-801 binding increased linearily with increasing tissue concentration and in general represented 80–93% of the total binding at 6–8 nM radioligand concentration. [³H]MK-801 interacted with brain and spinal homogenates with high affinity. The dissociation constants (K _d ) for all tissues studied were similar ranging between 7.9 and 11.9 nM, whereas the maximum number of binding sites (B_max) showed a wide, tissue-dependent range (0.1–6.75 pmol/mg protein). The rank order of tissue enrichment was found to be as follows: mouse brain>>dog cerebral cortex>>dog brain>> guinea pig brain>>rat spinal cord. Specific [³H]MK-801 binding in rodent and dog brain, dog cerebral cortex and rat spinal cord exhibited a similar pharmacological profile 9correlation coefficients=0.93–0.99). The rank order of potency of unlabelled compounds competing for [³H]MK-801 binding was: (+)MK-801>(–)MK-801>phencyclidine>(–)cyclazocine>>(+)cyclazocine ketamine>(+)N-allyl-N-normetazocine>(–)N-allyl-N-normetazocine>(–)pentazocine>(+)pentazocine. NMDA, Kainate, quisqualate and several other compounds failed to inhibit [³H]MK-801 binding at 100 M. In modulation studies conducted on extensively washed dog cortex membranes, Mg²⁺ ions stimulated [³H]MK-801 binding at 10 M-1 mM (EC₅₀=91.5 M) and then inhibited the binding from 1 mM to 10 mM (IC₅₀=3.1 mM). Glycine stimulated [³H]MK-801 binding at 30 nM-1 mM (EC₅₀=256 nM). In contrast, Zn²⁺ ions inhibited the binding of [³H]MK-801 binding site exhibited similar pharmacological and biochemical properties. These data appear to suggest that the pharmacological profile of the NMDA-receptor-channel is species and tissue independent.     

Regional Profile of Developmental Changes in the Sensitivity of the N-Methyl-D-Aspartate Receptor to Polyamines

Abstract: The NMDA receptor exhibits increased sensitivity to stimulation during early development compared with the adult. In this study, we examined modulation of the NMDA receptor by polyamines during development to see if it correlates with differences in the functional responsiveness of the NMDA receptor. [³H]MK-801 binding was measured in discrete brain regions in the presence and absence of polyamines in 3-, 7-, 15-, 25-, and 60-day-old Sprague-Dawley rats. [³H]MK-801 binding increased between postnatal days 3 and 15, with adult levels of binding being reached between days 15 and 25. Spermidine (75 μM) caused maximal stimulation of [³H]MK-801 binding during early development, ranging from 250% in the thalamus to 450% in the caudate putamen at postnatal day 3. This effect gradually declined to levels seen in the adult by postnatal days 15–25. During all developmental stages, the stimulation seen was greater in the caudate putamen compared with the hippocampus. Diethylenetriamine (1 μM) exhibited similar developmental and regional heterogeneity in its effects on [³H]MK-801 binding, producing substantial stimulation of binding in the neonate, but not in the adult. The EC₅₀ and E_max values for the stimulatory effect of spermidine were significantly higher at day 7 compared with the adult. Unlike spermidine and diethylenetriamine, there was no regional variation in the effects of the putative “polyamine site” inverse agonist 1,10-diaminodecane at any age and only a slightly attenuated inhibition at postnatal day 3 compared with the adult. This lack of complementarity in the regional and developmental profiles of spermidine and diethylenetriamine, on the one hand, and 1,10-diaminodecane, on the other, suggests that their effects on [³H]MK-801 binding are mediated at different sites. The altered sensitivity of the NMDA receptor to polyamines during development could reflect the expression of molecular variants with different sensitivities to modulation by polyamines.     

Polyamines Modulate the Binding of [3H]MK-801 to the Solubilized N-Methyl-D-Aspartate Receptor

Spermine and spermidine enhance the binding of [³H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a, d]cyclohepten-5,10-imine ([³H]MK-801) to N-methyl-D-aspartate (NMDA) receptors in membranes prepared from rat brain. These polyamines also enhance binding of [³H]MK-801 to NMDA receptors that have been solubilized with deoxycholate. Other polyamines selectively antagonize this effect, a finding indicating that the polyamine recognition site retains pharmacological and structural specificity after solubilization. In the presence of spermidine, an increase in the affinity of the solubilized NMDA receptor for [³H]MK-801 is observed. However, the rates of both association and dissociation of [³H]MK-801 binding to solubilized NMDA receptors are accelerated when assays are carried out in the presence of spermidine. When kinetic data are transformed, pseudo-first-order association and first-order dissociation plots are nonlinear in the presence of spermidine, an observation indicating a complex binding mechanism. Effects of spermidine on solubilized NMDA receptors are similar to effects previously described in studies of membrane-bound receptors. The data indicate that polyamines interact with a specific recognition site that remains associated with other components of the NMDA receptor complex after detergent solubilization.     

Rhodococcus erythropolis DCL14 Contains a Novel Degradation Pathway for Limonene

Strain DCL14, which is able to grow on limonene as a sole source of carbon and energy, was isolated from a freshwater sediment sample. This organism was identified as a strain of Rhodococcus erythropolis by chemotaxonomic and genetic studies. R. erythropolis DCL14 also assimilated the terpenes limonene-1,2-epoxide, limonene-1,2-diol, carveol, carvone, and (−)-menthol, while perillyl alcohol was not utilized as a carbon and energy source. Induction tests with cells grown on limonene revealed that the oxygen consumption rates with limonene-1,2-epoxide, limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and carveol were high. Limonene-induced cells of R. erythropolis DCL14 contained the following four novel enzymatic activities involved in the limonene degradation pathway of this microorganism: a flavin adenine dinucleotide- and NADH-dependent limonene 1,2-monooxygenase activity, a cofactor-independent limonene-1,2-epoxide hydrolase activity, a dichlorophenolindophenol-dependent limonene-1,2-diol dehydrogenase activity, and an NADPH-dependent 1-hydroxy-2-oxolimonene 1,2-monooxygenase activity. Product accumulation studies showed that (1S,2S,4R)-limonene-1,2-diol, (1S,4R)-1-hydroxy-2-oxolimonene, and (3R)-3-isopropenyl-6-oxoheptanoate were intermediates in the (4R)-limonene degradation pathway. The opposite enantiomers [(1R,2R,4S)-limonene-1,2-diol, (1R,4S)-1-hydroxy-2-oxolimonene, and (3S)-3-isopropenyl-6-oxoheptanoate] were found in the (4S)-limonene degradation pathway, while accumulation of (1R,2S,4S)-limonene-1,2-diol from (4S)-limonene was also observed. These results show that R. erythropolis DCL14 metabolizes both enantiomers of limonene via a novel degradation pathway that starts with epoxidation at the 1,2 double bond forming limonene-1,2-epoxide. This epoxide is subsequently converted to limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and 7-hydroxy-4-isopropenyl-7-methyl-2-oxo-oxepanone. This lactone spontaneously rearranges to form 3-isopropenyl-6-oxoheptanoate. In the presence of coenzyme A and ATP this acid is converted further, and this finding, together with the high levels of isocitrate lyase activity in extracts of limonene-grown cells, suggests that further degradation takes place via the β-oxidation pathway.     

Characterisation of [3H]MK-801 Binding and its Cooperative Modulation by PIG Brain Membranes

Abstract

Cooperative modulation of [³H]MK-801 binding to extensively washed pig cortical brain membranes in the presence of various concentrations of L-glutamate, glycine, spermine, CPP and DCKA was evaluated in association experiments. In saturation experiments [³H]MK-801 labelled a homogeneous population of binding sites with a K_d-value of 1.26 ± 0.18 nmol 1^?1 and a B_max-value of 2130 ± 200 fmol/mg protein. The pharmacological profile of this site was further evaluated in competition experiments with known NMDA receptor channel blockers. In nonequilibrium binding experiments EC₅₀-values of reference compounds acting at the L-glutamate, at the glycine, and at the polyamine site, were determined by increasing or decreasing [³H]MK-801 binding. Ifenprodil reduced [³H]MK-801 binding in a biphasic manner. All the data obtained are in agreement with results from [³H]MK-801 binding to rodent as well as human brain membranes. This study therefore strongly suggests, that pig cortical membranes are a suitable alternative to rodent brain membranes, and an acceptable substitute for human brain membranes in [³H]MK-801 binding experiments.     

Isolation of a bacterium assimilating (R)-3-chloro-1,2-propanediol and production of (S)-3-chloro-1,2-propanediol using microbial resolution

A bacterium capable of assimilating 3-chloro-1,2-propanediol was isolated from soil by enrichment culture. The strain was identified as Alcaligenes sp. by taxonomic studies. The crude extracts of the cells had dehalogenating activities and converted various halohydrins to the corresponding epoxides. 3-Chloro-1,2-propanediol was degraded stereospecifically by the strain, liberating chloride ion. The residual isomer was found to be the (S)-form (99.4% enantiomeric excess). (S)-3-Chloro-1,2-propanediol was obtained from the racemate by use of this strain in 38% yield, and (S)-glycidol (99.4% enantiomeric excess) was subsequently synthesized from the obtained (S)-3-chloro-1,2-propanediol by alkaline treatment.     

Pathologically Activated Neuroprotection via Uncompetitive Blockade of N-Methyl-d-aspartate Receptors with Fast Off-rate by Novel Multifunctional Dimer Bis(propyl)-cognitin

Uncompetitive N-methyl-d-aspartate (NMDA) receptor antagonists with fast off-rate (UFO) may represent promising drug candidates for various neurodegenerative disorders. In this study, we report that bis(propyl)-cognitin, a novel dimeric acetylcholinesterase inhibitor and γ-aminobutyric acid subtype A receptor antagonist, is such an antagonist of NMDA receptors. In cultured rat hippocampal neurons, we demonstrated that bis(propyl)-cognitin voltage-dependently, selectively, and moderately inhibited NMDA-activated currents. The inhibitory effects of bis(propyl)-cognitin increased with the rise in NMDA and glycine concentrations. Kinetics analysis showed that the inhibition was of fast onset and offset with an off-rate time constant of 1.9 s. Molecular docking simulations showed moderate hydrophobic interaction between bis(propyl)-cognitin and the MK-801 binding region in the ion channel pore of the NMDA receptor. Bis(propyl)-cognitin was further found to compete with [³H]MK-801 with a K_i value of 0.27 μm, and the mutation of NR1(N616R) significantly reduced its inhibitory potency. Under glutamate-mediated pathological conditions, bis(propyl)-cognitin, in contrast to bis(heptyl)-cognitin, prevented excitotoxicity with increasing effectiveness against escalating levels of glutamate and much more effectively protected against middle cerebral artery occlusion-induced brain damage than did memantine. More interestingly, under NMDA receptor-mediated physiological conditions, bis(propyl)-cognitin enhanced long-term potentiation in hippocampal slices, whereas MK-801 reduced and memantine did not alter this process. These results suggest that bis(propyl)-cognitin is a UFO antagonist of NMDA receptors with moderate affinity, which may provide a pathologically activated therapy for various neurodegenerative disorders associated with NMDA receptor dysregulation.     

Biosynthesis of pisatin: Experiments with enantiomeric precursors

Feeding experiments in cupric chloride-treated Pisum sativum pods and seedlings have demonstrated the preferential incorporation of (+)-(6aS,11aS)-[³H]maackiain over (?)-(6aR, 11aR)-[¹⁴C]maackiain into (+)-(6aR, 11aR)-pisatin, establishing that the 6a-hydroxylation of pterocarpans proceeds with retention of configuration. (+)- (6aR,11aR)-6a-hydroxymaackiain was similarly incorporated much better than (?)-(6aS,11aS)-6a- hydroxymaackiain. Where (?)-isomers were incorporated, optical activity measurements on the pisatin produced indicated significant synthesis of (?)-pisatin as well as the normal (+)-pisatin. 7,2′-Dihydroxy-4′,5′- methylenedioxyisoflav-3-ene and both enantiomers of 7,2′-dihydroxy-4′,5′-methylenedioxyisoflavan were poor precursors of pisatin.     

A Metabotropic-Like Flux-Independent NMDA Receptor Regulates Ca2+ Exit from Endoplasmic Reticulum and Mitochondrial Membrane Potential in Cultured Astrocytes

Astrocytes were long thought to be only structural cells in the CNS; however, their functional properties support their role in information processing and cognition. The ionotropic glutamate N-methyl D-aspartate (NMDA) receptor (NMDAR) is critical for CNS functions, but its expression and function in astrocytes is still a matter of research and debate. Here, we report immunofluorescence (IF) labeling in rat cultured cortical astrocytes (rCCA) of all NMDAR subunits, with phenotypes suggesting their intracellular transport, and their mRNA were detected by qRT-PCR. IF and Western Blot revealed GluN1 full-length synthesis, subunit critical for NMDAR assembly and transport, and its plasma membrane localization. Functionally, we found an iCa²⁺ rise after NMDA treatment in Fluo-4-AM labeled rCCA, an effect blocked by the NMDAR competitive inhibitors D(-)-2-amino-5-phosphonopentanoic acid (APV) and Kynurenic acid (KYNA) and dependent upon GluN1 expression as evidenced by siRNA knock down. Surprisingly, the iCa²⁺ rise was not blocked by MK-801, an NMDAR channel blocker, or by extracellular Ca²⁺ depletion, indicating flux-independent NMDAR function. In contrast, the IP₃ receptor (IP₃R) inhibitor XestosponginC did block this response, whereas a Ryanodine Receptor inhibitor did so only partially. Furthermore, tyrosine kinase inhibition with genistein enhanced the NMDA elicited iCa²⁺ rise to levels comparable to those reached by the gliotransmitter ATP, but with different population dynamics. Finally, NMDA depleted the rCCA mitochondrial membrane potential (mΔψ) measured with JC-1. Our results demonstrate that rCCA express NMDAR subunits which assemble into functional receptors that mediate a metabotropic-like, non-canonical, flux-independent iCa²⁺ increase.