Effects of exogenous glycine betaine and cycloleucine on photosynthetic capacity, amino acid composition, and hormone metabolism in Solanum melongena L.

Although exogenous glycine betaine (GB) and cycloleucine (Cyc) have been reported to affect animal cell metabolism, their effects on plant growth and development have not been studied extensively. Different concentrations of exogenous glycine betaine (20, 40, and 60 mmol L-1) and cycloleucine (10, 20, and 40 mmol L-1), with 0 mmol L-1 as control, were used to investigate the effects of foliar spraying of betaine and cycloleucine on growth, photosynthesis, chlorophyll fluorescence, Calvin cycle pathway, abaxial leaf burr morphology, endogenous hormones, and amino acid content in eggplant. We found that 40 mmol L-1 glycine betaine had the best effect on plant growth and development; it increased the fresh and dry weight of plants, increased the density of abaxial leaf hairs, increased the net photosynthetic rate and Calvin cycle key enzyme activity of leaves, had an elevating effect on chlorophyll fluorescence parameters, increased endogenous indoleacetic acid (IAA) content and decreased abscisic acid (ABA) content, and increased glutamate, serine, aspartate, and phenylalanine contents. However, cycloleucine significantly inhibited plant growth; plant apical dominance disappeared, plant height and dry and fresh weights decreased significantly, the development of abaxial leaf hairs was hindered, the net photosynthetic rate and Calvin cycle key enzyme activities were inhibited, the endogenous hormones IAA and ABA content decreased, and the conversion and utilization of glutamate, arginine, threonine, and glycine were affected. Combined with the experimental results and plant growth phenotypes, 20 mmol L-1 cycloleucine significantly inhibited plant growth. In conclusion, 40 mmol L-1 glycine betaine and 20 mmol L-1 cycloleucine had different regulatory effects on plant growth and development.

Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5'-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover.

Potent mechanism-based inactivators can be rationally designed against pyridoxal 5'-phosphate (PLP)-dependent drug targets, such as ornithine aminotransferase (OAT) or γ-aminobutyric acid aminotransferase (GABA-AT). An important challenge, however, is the lack of selectivity toward other PLP-dependent, off-target enzymes, because of similarities in mechanisms of all PLP-dependent aminotransferase reactions. On the basis of complex crystal structures, we investigate the inactivation mechanism of OAT, a hepatocellular carcinoma target, by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid (FCP), a known inactivator of GABA-AT. A crystal structure of OAT and FCP showed the formation of a ternary adduct. This adduct can be rationalized as occurring via an enamine mechanism of inactivation, similar to that reported for GABA-AT. However, the crystal structure of an off-target, PLP-dependent enzyme, aspartate aminotransferase (Asp-AT), in complex with FCP, along with the results of attempted inhibition assays, suggests that FCP is not an inactivator of Asp-AT, but rather an alternate substrate. Turnover of FCP by Asp-AT is also supported by high-resolution mass spectrometry. Amid existing difficulties in achieving selectivity of inactivation among a large number of PLP-dependent enzymes, the obtained results provide evidence that a desirable selectivity could be achieved, taking advantage of subtle structural and mechanistic differences between a drug-target enzyme and an off-target enzyme, despite their largely similar substrate binding sites and catalytic mechanisms.

Hydrophilic and Cell-Penetrable Pyrrolidinyl Peptide Nucleic Acid via Post-synthetic Modification with Hydrophilic Side Chains.

Peptide nucleic acid (PNA) is a nucleic acid mimic in which the deoxyribose-phosphate was replaced by a peptide-like backbone. The absence of negative charge in the PNA backbone leads to several unique behaviors including a stronger binding and salt independency of the PNA-DNA duplex stability. However, PNA possesses poor aqueous solubility and cannot directly penetrate cell membranes. These are major obstacles that limit in vivo applications of PNA. In previous strategies, the PNA can be conjugated to macromolecular carriers or modified with positively charged side chains such as guanidinium groups to improve the aqueous solubility and cell permeability. In general, a preformed modified PNA monomer was required. In this study, a new approach for post-synthetic modification of PNA backbone with one or more hydrophilic groups was proposed. The PNA used in this study was the conformationally constrained pyrrolidinyl PNA with prolyl-2-aminocyclopentanecarboxylic acid dipeptide backbone (acpcPNA) that shows several advantages over the conventional PNA. The aldehyde modifiers carrying different linkers (alkylene and oligo(ethylene glycol)) and end groups (-OH, -NH2, and guanidinium) were synthesized and attached to the backbone of modified acpcPNA by reductive alkylation. The hybrids between the modified acpcPNAs and DNA exhibited comparable or superior thermal stability with base-pairing specificity similar to those of unmodified acpcPNA. Moreover, the modified apcPNAs also showed the improvement of aqueous solubility (10-20 folds compared to unmodified PNA) and readily penetrate cell membranes without requiring any special delivery agents. This study not only demonstrates the practicality of the proposed post-synthetic modification approach for PNA modification, which could be readily applied to other systems, but also opens up opportunities for using pyrrolidinyl PNA in various applications such as intracellular RNA sensing, specific gene detection, and antisense and antigene therapy.

Biodistribution and human dosimetry of enantiomer-1 of the synthetic leucine analog anti-1-amino-2-fluorocyclopentyl-1-carboxylic acid.

INTRODUCTION: The enantiomerically enriched (ee=90%, enantiomer 1) synthetic amino acid (R,S)-anti-1-amino-2-fluorocyclopentyl-1-carboxylic acid (anti-2-[(18)F]FACPC-1) accumulates in malignant cells by elevated transport through the sodium-independent system-L (leucine preferring) amino acid transporter. The purpose of this study was to evaluate in vivo uptake and single-dose toxicity of anti-2-[(18)F]FACPC-1 in animals as well as the individual organ and whole-body dose in humans. METHODS: A DU145 xenograft rodent model was used to measure anti-2-[(18)F]FACPC-1 uptake at 15, 30 and 60 min post-injection. Animals were sacrificed and organs harvested to measure the percent injected activity per organ and to calculate residence time. Anti-2-[(18)F]FACPC-1 toxicity was assessed using a single microdose (37-74 MBq/kg) in nonhuman primates. Their vital signs were monitored for 2 h post-injection for drug-related effects. Human biodistribution studies were collected by sequential whole-body PET/CT scans on six healthy volunteers (three male and three female) for 120 min following a single 247±61 MBq bolus injection of anti-2-[(18)F]FACPC-1. Estimates of radiation dose from anti-2-[(18)F]FACPC-1 to the human body were calculated using recommendations of the MIRD committee and MIRDOSE 3.0 software. RESULTS: High anti-2-[(18)F]FACPC-1 residence time was observed in the pancreas of the rodent model compared to the human data. No abnormal treatment-related observations were made in the nonhuman primate toxicity studies. Human venous blood showed no metabolites of anti-2-[(18)F]FACPC-1 in the first 60 min post-injection. All volunteers showed initially high uptake in the kidneys followed by a rapid washout phase. The estimated effective dose equivalent was 0.0196 mSv/MBq. CONCLUSION: Anti-2-[(18)F]FACPC-1 showed low background uptake in the brain, thoracic and abdominal cavities of humans, suggesting a possible use for detecting malignant tissues in these regions.

Hepatoprotective effects of S-adenosyl-L-methionine against alcohol- and cytochrome P450 2E1-induced liver injury.

S-adenosyl-L-methionine (SAM) acts as a methyl donor for methylation reactions and participates in the synthesis of glutathione. SAM is also a key metabolite that regulates hepatocyte growth, differentiation and death. Hepatic SAM levels are decreased in animal models of alcohol liver injury and in patients with alcohol liver disease or viral cirrhosis. This review describes the protection by SAM against alcohol and cytochrome P450 2E1-dependent cytotoxicity both in vitro and in vivo and evaluates mechanisms for this protection.

Modulation of endotoxin stimulated interleukin-6 production in monocytes and Kupffer cells by S-adenosylmethionine (SAMe).

Interleukin-6 (IL-6) is a multifunctional cytokine having primarily anti-apoptotic and anti-inflammatory effects. Recent reports have documented that IL-6 plays a key role in liver regeneration. Intracellular deficiency of S-adenosylmethionine (SAMe) is a hallmark of toxin-induced liver injury. Although the administration of exogenous SAMe attenuates liver injury, its mechanisms of action are not fully understood. Here we investigated the effects of exogenous SAMe on IL-6 production in monocytes and Kupffer cells. RAW 264.7 cells, a murine monocyte cell line, and isolated rat Kupffer cells were stimulated with lipopolysaccharide (LPS) in the absence or presence of exogenous SAMe. IL-6 production was assayed by ELISA and intracellular SAMe concentrations were measured by HPLC. We have found that exogenous SAMe administration enhanced both IL-6 protein production and gene expression in LPS-stimulated monocytes and Kupffer cells. Cycloleucine (CL), an inhibitor for extrahepatic methionine adenosyltransferases (MAT), inhibited LPS-stimulated IL-6 production. The enhancement of LPS-stimulated IL-6 production by SAMe was inhibited by ZM241385, a specific antagonist of adenosine (A2) receptor. Our results demonstrate that SAMe administration may exert its anti-inflammatory and hepatoprotective effects, at least in part, by enhancing LPS-stimulated IL-6 production.

Assessment of treatment response by autoradiography with (14)C-aminocyclopentane carboxylic acid, (67)Ga-DTPA, and (18)F-FDG in a herpes simplex virus thymidine kinase/ganciclovir brain tumor model.

UNLABELLED: Assessments of herpes simplex virus 1 thymidine kinase (HSV-tk)/ganciclovir (GCV) treatment response, early in the course of therapy, are important in the evaluation and clinical management of patients. This study addresses whether imaging amino acid transport, glucose utilization, and passive vascular permeability provides an early indication of treatment response and can predict long-term outcome. METHODS: Fischer 344 rats with intracerebral HSV-tk transduced RG2TK+ xenografts were studied. GCV-treated (50 mg/kg twice daily) and saline-treated control animals were compared; triple-label quantitative autoradiography was performed 3 d after initiating treatment, and long-term survival was determined. Autoradiograms of (18)F-FDG, (67)Ga-diethylenetriaminepentaacetic acid ((67)Ga-DTPA), and (14)C-aminocyclopentane carboxylic acid ((14)C-ACPC) were obtained; measurements of (14)C-ACPC and (67)Ga-DTPA plasma clearance (K(1)), (14)C-ACPC transport ( partial differential K(1)), relative glucose utililization (R), and normalized radioactivity (% dose/g) were obtained in tumor and brain tissues. Adjacent sections were stained to detect apoptotic cells, microvessels, and type L neutral amino acid transporter in tumor and normal brain. RESULTS: GCV treatment reduced partial differential K(1) and % dose/g of (14)C-ACPC in RG2TK+ xenografts to approximately 30% of that in nontreated animals (from 34 +/- 9 [mean +/- SD] to 9.5 +/- 2.7 microL/min/g and from 0.28 +/- 0.09 to 0.11 +/- 0.04 % dose/g, respectively). GCV had a significant but substantially smaller effect than toxicity on glucose utilization and little or no effect on passive vascular permeability of RG2TK+ xenografts. These differences could not be explained by differences in plasma amino acid or glucose concentration at the time of the study. Histology revealed a large fraction of dead tumor cells and only a sparse distribution of apoptotic cells in GCV-treated tumors. Many CD34-positive endothelial cells in GCV-treated tumors showed only weak or marginal LAT1 staining, whereas CD98 staining remained unchanged. Survival was significantly increased by GCV treatment from 18 +/- 4 to 56 +/- 17 d. CONCLUSION: (14)C-ACPC influx, K(1)(ACPC), facilitated transport, partial differential K(1)(ACPC), and % dose/g (ACPC) are good indicators of early treatment response after HSV-tk/GCV gene therapy. The parametric images and changes in K(1)(ACPC), partial differential K(1)(ACPC), and % dose/g (ACPC) are substantial and are better than the corresponding measures obtained in the same animals and in the same tissue (tumor) regions with (67)Ga-DTPA and (18)F-FDG. Amino acid transport imaging may be a good surrogate paradigm to monitor treatment response of brain tumors.

Acamprosate inhibits the binding and neurotoxic effects of trans-ACPD, suggesting a novel site of action at metabotropic glutamate receptors.

BACKGROUND: Several reported effects of acamprosate within the glutamatergic system could result from interactions with metabotropic glutamate receptors (mGluRs). The following experiments were performed to determine whether acamprosate could compete with trnas-ACPD (+/--1-aminocyclopentane-trans-1,3-dicarboxylic acid, an equimolecular mixture of 1S, 3R and 1R, 3S-ACPD and an agonist at both group I and group II mGluRs) sensitive binding sites and protect against trans-ACPD-induced neurotoxicity in organotypic hippocampal slice cultures. METHODS: A P2 membrane preparation of cortices, cerebellums, and hippocampi of adult, male Sprague Dawley rats was used to determine the abilities of N-methyl-D-aspartic acid (NMDA) and trans-ACPD to displace [3H]glutamate in both the absence and the presence of the sodium salt of acamprosate (sodium mono N-acetyl homotaurine or Na-acamprosate). A comparison of the effects of 100 microM guanosine 5'-triphosphate on unlabeled glutamate, trans-ACPD, and Na-acamprosate was performed in the same paradigm. For the neurotoxicity studies, organotypic hippocampal slice cultures from male and female 8-day-old neonatal rats were exposed to either 500 microM -ACPD or 50 microM NMDA for 24 hr in normal culture medium containing serum on day 20 in vitro. The effects of Na-acamprosate and 2-methyl-6-(2-phenylethenyl)pyridine (SIB-1893), a noncompetitive antagonist at metabotropic type 5 receptors (mGluR5s), were assessed by determining differences in propidium iodide uptake as compared with neurotoxic challenges alone. RESULTS: Na-acamprosate displaced 31% of [3H]glutamate but did not compete with NMDA for [3H]glutamate binding sites. Na-acamprosate displayed total competition with trans-ACPD. The presence of 100 microM guanosine 5'-triphosphate differentially altered the displacing capabilities of the two mGluR agonists, unlabeled glutamate and trans-ACPD, as compared with Na-acamprosate. Na-acamprosate (200-1000 microM) and SIB-1893 (20-500 microM) both were neuroprotective against trans-ACPD induced neurotoxicity that likely results from mGluR potentiation of NMDARs. In turn, Na-acamprosate and SIB-1893 had no direct effects on NMDA-induced neurotoxicity. CONCLUSIONS: Na-acamprosate demonstrates the binding and functional characteristics that are consistent with a group I mGluR antagonist. The functional similarities between Na-acamprosate and SIB-1893 support an interaction of Na-acamprosate at mGluR5s. The neuroprotective properties of acamprosate and possibly its ability to reduce craving in alcohol-dependent patients may result from its alterations in glutamatergic transmission through mGluRs.

Cycloleucine fluxes during rat vasa recta and loop microinfusions in vivo and loop microperfusions in vitro.

Amino acids are apparently recycled between loops of Henle and vasa recta in rat papilla in vivo. To examine this process in the absence of metabolism, we performed continuous microinfusions of rat renal papillary ascending thin limbs (ATLs) and vasa recta in vivo, and microperflusions of isolated rat renal papillary descending thin limbs (DTLs) and ATLs in vitro using the nonmetabolizable, synthetic, neutral amino acid cycloleucine. Like naturally occurring amino acids, approximately = 25% of radiolabeled cycloleucine microinfused into ATLs in vivo was reabsorbed by a process that was not saturable or inhibitable. Also, like naturally occurring amino acids, approximately = 47% (relative to inulin) of radiolabeled cycloleucine microinfused into ascending vasa recta in vivo was transferred directly into ipsilateral tubular structures (probably DTLs) by a saturable and inhibitable process. In DTLs perfused in vitro, unidirectional bath-to-lumen fluxes (Jbl) tended to exceed unidirectional lumen-to-bath fluxes (Jlb), whereas in ATLs perfused in vitro Jlb tended to exceed Jbl, but the differences were not statistically significant. Moreover, none of the unidirectional fluxes was saturable or inhibitable, an observation compatible with apparent reabsorption from ATLs in vivo but incompatible with apparent movement from vasa recta to DTLs in vivo. These in vitro observations are like those made previously for the naturally occurring neutral amino acid L-alanine. The lack of saturation and inhibition, like the previous data on L-alanine, suggest that transepithelial movement of amino acids in thin limbs of Henle's loop may occur via a paracellular route and that regulation of amino acid movement in vivo may involve vasa recta, not DTLs. They also suggest that cycloleucine is a good nonmetabolizable surrogate for the study of neutral amino acid transport in the kidney.

Uptake and transport by the ovine placenta of neutral nonmetabolizable amino acids with different transport system affinities.

Placental uptake and transport of three nonmetabolizable amino acids with different reactivities for transport systems were studied in sheep under normal physiologic conditions. Methylaminoisobutyric acid (MeAIB), which has specific affinity for the sodium-dependent A system transporters, demonstrated placental concentrative uptake from the uterine and the umbilical circulations, but virtually no transport from mother to fetus. By contrast, aminoisobutyric acid (AIB) and aminocyclopentane-1-carboxylic acid (ACP), which have affinity for both sodium-dependent and sodium-independent transporters, demonstrated both concentrative uptake and transport from mother to fetus. ACP transport rate to the fetus was approximately twice the AIB transport rate. It is concluded that a neutral amino acid which interacts almost exclusively with the weakly reversible system A transporters may be transported rapidly into the placenta and may attain high concentrations within this organ but cannot escape from placenta to fetus down its own concentration gradient because the exit route is controlled by reversible amino acid transporters at the fetal surface of the placenta. Conversely, high affinity for reversible Na-independent transporters may be a necessary condition for the rapid transport of an amino acid from placenta to fetus.

Molecular and pharmacological characterization of recombinant rat/mice N-methyl-D-aspartate receptor subtypes in the yeast Saccharomyces cerevisiae.

The genes encoding the ionotropic N-methyl-D-aspartate (NMDA) receptor subunits NR1a, NR2B and NR2D were cloned in the multi-copy yeast-Escherichia coli shuttle vectors pMBO1 and pMB02. The protease-deficient yeast Saccharomyces cerevisiae c13-ABYS-86 (leu-, ura-, his-) was transformed with the recombinant plasmids pMBNR1a (leu+), pMBNR1a/pMBNR2D (ura+), pMBNR1a/pMBNR2D/ pMBNR2B (his+) or pMBNR1a/pMBNR2A/pMBNR2B, respectively, and was used to express the different NMDA receptor subunit genes. Western-blotting analysis with the specific NMDA receptor antibodies showed a clear but differently strong expression of the recombinant receptor proteins which were found to be only partially glycosylated in the cell membranes of the recombinant yeast strains. By immunofluorescence microscopy using the specific subunit antibodies and fluorescence-labeled secondary antibodies, the distinctly expressed NR1a and NR2D subunits could be located in the plasma membrane of the transformed yeast cells. Pharmacological characterization of crude membrane preparations of the recombinant yeast cells expressing 1-3 NMDA receptor subunits showed saturable binding of the glycine antagonist [3H]MDL105,519 with different Kd values of 56.88+/-5.38 nM (NR1a), 1365.11+/-76 nM (NR1a/NR2D), 22.97+/-3.37 nM for NR1a/NR2B/NR2D and 7.4+/-1.2 nM for NR1a/NR2A/NR2B. The bound capacities were 13.07+/-0.92 (NRla), 14.63+/-0.50 (NR1a/NR2D), 12.85+/-1.68 (NR1a/NR2B/NR2D) and 8.3+/-0.7 (NR1a/NR2A/NR2B) pmol/mg membrane protein. The [3H]MDL105,519 binding was inhibited by the glycine antagonist 5,7-dichlorokynurenate (DCKA), ethyl-2-carboxy-4.6-dichloro-3-indoleacetate (ECDI) and itself, but not by glycine, D-serine and 1-amino-cyclopropanecarboxylic acid (ACPC). Each of these recombinant receptor proteins consisting both of NR1 and NR2 subunits also showed a specific binding site for the NMDA agonist glutamate when using L-[3H]glutamate as a radioligand. Analysis of saturation experiments revealed that this ligand binds to a specific site with Kd values of 536+/-43, 688+/-60, and 856+/-48 nM for NR1a/NR2B, NR1a/NR2D, and NR1a/NR2B/NR2D respectively.

Predictive study by molecular modeling to promote specific probes of glutamate receptors, using methylated cyclic glutamic acid derivatives (trans- and cis-ACPD). Comparison with specific agonists.

Two classes of glutamate receptors (metabotropic and ionotropic) and their subclasses (groups I-III and N-methyl-D-aspartic acid (NMDA), kainic acid (KA)), respectively, are characterized by the binding of a L-glutamate moiety in a specific conformation. The conformations may be grouped by the two backbone torsion angles, chi1 [alpha-CO2-C(2)-C(3)-C4)] and chi2 [+NC(2)-C(3)-C(4)-gamma-CO2] and by the two characteristic distances between the potentially active functional groups, alpha-N+-gamma-CO2 (d1) and alpha-CO2-gamma-CO2 (d2). The conformational preferences of 2,3,4-methyl(a and b)-cis and trans-1-aminocyclopentane-1,3-dicarboxylate are discussed in the light of the physical features known for specific metabotropic (groups I-II) and specific ionotropic (NMDA, KA) agonists, respectively. The spatial orientation of the perceived functional groups was elucidated in cyclic derivatives which contain an embedded L-glutamate moiety in a particularly restricted conformation (relative to the C(2)-C(3)-C(4) bond) using a combination of NMR experimental results and mechanics and dynamics calculations. One important conclusion of the study is that a single glutamate receptor is privileged for each theoretical model considered by molecular dynamics. This study showed clearly what would be conformational preferences of cyclic glutamate derivatives following the geometrical isomerism of the methyl group.

Imaging experimental brain tumors with 1-aminocyclopentane carboxylic acid and alpha-aminoisobutyric acid: comparison to fluorodeoxyglucose and diethylenetriaminepentaacetic acid in morphologically defined tumor regions.

The goal of this study was to evaluate the differences and define the advantages of imaging experimental brain tumors in rats with two nonmetabolized amino acids, 1-aminocyclopentane carboxylic (ACPC) acid and alpha-aminoisobutyric (AIB) acid compared with imaging with fluorodeoxyglucose (FDG) or the gallium-diethylenetriaminepentaacetic acid chelate (Ga-DTPA). 1-aminocyclopentane carboxylic acid, AIB, and FDG autoradiograms were obtained 60 minutes after intravenous injection to simulate positron emission tomography (PET) imaging, whereas the Ga-DTPA autoradiograms were obtained 5 or 10 minutes after injection to simulate gadolinium (Gd)-DTPA-enhanced magnetic resonance (MR) images. Three experimental tumors were studied (C6, RG2, and Walker 256) to provide a range of tumor types. Triple-label quantitative autoradiography was performed, and parametric images of the apparent distribution volume (Va, mL/g) for ACPC or AIB, relative glucose metabolism (R, micromol/100 g/min), vascular permeability to Ga-DTPA (K1, microL/min/g), and histology were obtained from the same tissue section. The four images were registered in an image array processor, and regions of interest in tumor and contralateral brain were defined on morphologic criteria (histology) and were transferred to the autoradiographic images. A comparative analysis of all measured values was performed. The location and morphologic characteristics of the tumor had an effect on the images and measurements of Va, R, and K1. Meningeal extensions of all three tumors consistently had the highest amino acid uptake (Va) and vascular permeability (K1) values, and subcortical portions of the tumors usually had the lowest values. Va and R (FDG) values generally were higher in tumor regions with high-cell density and lower in regions with low-cell density. Tumor areas identified as "impending" necrosis on morphologic criteria consistently had high R values, but little or no change in Va or K1. Tumor necrosis was seen consistently only in the larger Walker 256 tumors; low values of R and Va for AIB (less for ACPC) were measured in the necrotic-appearing regions, whereas K1 was not different from the mean tumor value. The highest correlations were observed between vascular permeability (K1 for Ga-DTPA) and Va for AIB in all three tumors; little or no correlation between vascular permeability and R was observed. The advantages of ACPC and AIB imaging were most convincingly demonstrated in C6 gliomas and in Walker 256 tumors. 1-aminocyclopentane was substantially better than FDG or Ga-DTPA for identifying tumor infiltration of adjacent brain tissue beyond the macroscopic border of the tumor; ACPC also may be useful for identifying low-grade tumors with an intact blood-brain barrier. Contrast-enhancing regions of the tumors were visualized more clearly with AIB than with FDG or Ga-DTPA; viable and necrotic-appearing tumor regions could be distinguished more readily with AIB than with FDG. [11C]-labeled ACPC and AIB are likely to have similar advantages for imaging human brain tumors with PET.

Free amino acids reflect impact of selenite-dependent stress on primary metabolism in rat lens.

PURPOSE: A decrease in phase separation temperature, prior to nuclear cataract, has been correlated with elevated free amino acid content. Hence, we determined how selenite-induced stress alters free amino acid pools in the rat lens, following a single subcutaneous dose of sodium selenite (30 nmol g-1 body weight) in 10- to 14-day-old Sprague Dawley rats. RESULT: Oxidative stress was evident in lenses 24 h after rats were treated with selenite. Glutathione content was decreased by 60% in the lens cortex and nucleus; the flux of glucose through the pentose phosphate pathway was increased; and glycerol-3-phosphate content was elevated. Amino acid transport, evaluated as 14C-cycloleucine uptake, was not altered, although 14C-glutamine was oxidized at a slower rate. Lenses from treated animals displayed, among the free amino acids, increased glutamine, proline, serine, glycine and the branched chain amino acids, while aspartate, glutamate, and taurine were less. CONCLUSIONS: A systemic delivery of sodium selenite caused oxidative stress in the rat lens. Direct effects on primary metabolism altered free amino acid pools that may contribute to transient and permanent changes in lens transparency.

Mice lacking metabotropic glutamate receptor 5 show impaired learning and reduced CA1 long-term potentiation (LTP) but normal CA3 LTP.

Class I metabotropic glutamate receptors (mGluRs) have been postulated to play a role in synaptic plasticity. To test the involvement of one member of this class, we have recently generated mutant mice that express no mGluR5 but normal levels of other glutamate receptors. The CNS revealed normal development of gross anatomical features. To examine synaptic functions we measured evoked field EPSPs in the hippocampal slice. Measures of presynaptic function, such as paired pulse facilitation in mutant CA1 neurons, were normal. The response of mutant CA1 neurons to low concentrations of (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD) was missing, which suggests that mGluR5 may be the primary high affinity ACPD receptor in these neurons. Long-term potentiation (LTP) in mGluR5 mutants was significantly reduced in the NMDA receptor (NMDAR)-dependent pathways such as the CA1 region and dentate gyrus of the hippocampus, whereas LTP remained intact in the mossy fiber synapses on the CA3 region, an NMDAR-independent pathway. Some of the difference in CA1 LTP could lie at the level of expression, because the reduction of LTP in the mutants was no longer observed 20 min after tetanus in the presence of 2-amino-5-phosphonopentanoate. We propose that mGluR5 plays a key regulatory role in NMDAR-dependent LTP. These mutant mice were also impaired in the acquisition and use of spatial information in both the Morris water maze and contextual information in the fear-conditioning test. This is consistent with the hypothesis that LTP in the CA1 region may underlie spatial learning and memory.

Characterization of metabotropic glutamate receptors in rat C6 glioma cells.

Metabotropic glutamate receptors in rat C6 glioma cells have been characterized by pharmacological and kinetic binding experiments, using both L-[3H]glutamate and [3H(+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid ([3H](+/-)-trans-ACPD) radioligands. Saturation experiments revealed a single binding site with a Kd = 1250 +/- 101 nM and Bmax = 12.1 +/- 1.8 pmol/mg protein when the assays were performed with L-[3H]glutamate as radioligand in the presence of AMPA, kainate, NMDA and DL-threo-beta-hydroxyaspartic acid. When [3H](+/-)-trans-ACPD was used as radioligand, the kinetic parameters obtained were Kd = 2605 +/- 1042 nM and Bmax = 13.66 +/- 5.01 pmol/mg protein. Pharmacological characterization indicated that specific binding of L-[3H]glutamate was sensitive to different agonists of mGlu receptors, showing a rank order of affinity L-glutamate > L-quisqualic acid > (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) > ibotenic acid >>> (2S, 'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I). Specific binding of L-[3H]glutamate to mGlu receptors is regulated by guanine nucleotides. Guanylyl imidodiphosphate (Gpp(NH)p) causes an affinity shift on the L-glutamate dose-response curve, increasing the IC50 value. These results support the evidence that metabotropic glutamate receptors are present in rat C6 glioma cells and they are coupled to a G-protein.

Tumour growth and fetal uptake of amino acids in the pregnant rat.

The aim of the present investigation was to determine the effects of maternal tumour burden on fetal growth and to relate them to amino acid availability to the fetus. A fast-growing tumour, the Yoshida AH-130 ascites hepatoma, was inoculated into rats during pregnancy. Late pregnant rats bearing a rapidly-growing tumour presented a normal conceptus mass while the tumour cell content was unaffected by gestation. In addition, no changes were found in fetal uptake of amino acids as measured by the fetal accumulation of [14C]aminoisobutyrate and [14C] cycloleucine. However, increased alanine and leucine concentrations in the fetal circulation of the tumour-bearing rats suggest an enhanced fetal amino acid availability which does not seem to be the result of changes in placental or fetal relative blood flow, as indicated by the tissue accumulation of [14C]DDT, which were actually lower in the tumour-bearing rats. It may be suggested that tumour burden induces changes in placental amino acid transport systems.

Selective photodynamic inactivation of a multidrug transporter by a cationic photosensitising agent.

We have characterised sites of photodamage catalysed by the cationic photosensitiser tetrabromorhodamine 123, using P388 murine leukaemia cells and a subline (P388/ADR) which has a multidrug resistance phenotype and hyperexpresses mdr1 mRNA for P-glycoprotein. Fluorescence emission spectra were consistent with sensitiser localisation in hydrophobic regions of the P388 cell, and in more aqueous loci in P388/ADR. Subsequent irradiation resulted in photodamage to the P388 cells, resulting in loss of viability. In contrast, P388/ADR cells were unaffected except for an irreversible inhibition of P-glycoprotein, leading to enhanced accumulation of daunorubicin and rhodamine 123 and a corresponding increase in daunorubicin cytotoxicity. These results are consistent with the premise that substrates for P-glycoprotein are confined to membrane loci associated with the transporter, and indicate a very limited migration of cytotoxic photo-products in a cellular environment.

Glutamate modulates [Ca2+]i and gonadotropin-releasing hormone secretion in immortalized hypothalamic GT1-7 neurons.

Glutamate and its receptors are present in the hypothalamus and have been proposed to participate in neuroendocrine regulation, including the control of GnRH secretion. To address the mechanism of glutamate action, we measured [Ca2+]i, inositol phosphate, and secretory responses to glutamate receptor subtype agonists and antagonists in the immortalized GT1-7 cell line of GnRH-secreting hypothalamic neurons. Glutamate, N-methyl-D-aspartate (NMDA), kainate, and trans-(+/-)-1-amino-(1S,3R)-cyclopentanedicarboxylic acid increased GnRH secretion. In monolayer cultures of GT1-7 cells, L- but not D-glutamate induced a moderate, concentration-dependent rise in [Ca2+]i. The action of glutamate on [Ca2+]i was mimicked by NMDA, alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA), and kainate. Responses to NMDA were potentiated by the coagonist, glycine, and were inhibited by an antagonist of the glycine site on the NMDA receptor, 5,7-dichlorokynurenic acid (DCKA). NMDA-induced [Ca2+]i responses were also inhibited by Mg2+ and by the NMDA receptor antagonist, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,1 0-imine hydrogen maleate (MK-801), but not by the AMPA/kainate antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In contrast, responses to AMPA and kainate were inhibited by CNQX but not by Mg2+, DCKA, or MK-801. Responses to glutamate were more inhibited by MK-801 plus CNQX than by either antagonist alone. All [Ca2+]i responses were nearly abolished in Ca(2+)-free solution. None of the agonists stimulated inositol phosphate formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Co-administration of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid and arachidonic acid potentiates synaptic transmission in rat hippocampal slices.

Perfusion of the 1S,3R isomer of trans-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD, 50 microM), or arachidonic acid (10 microM), for 5 min produced only depression of the field excitatory postsynaptic potential recorded in the CA1 region of rat hippocampal slices from which the CA3 region had been removed. However, perfusion of t-ACPD and arachidonic acid in combination induced a rapid potentiation of the response which in 4/6 slices was maintained for at least 90 min.