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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.28, #2)

Introduction: Vulnerability of the CNS to metabolic and drug-related insults (Chapter 1) by B. D. Kretschmer; R. M. Kostrzewa; M. Herrera-Marschitz (pp. 123-125).

Effect of chronic glutamate administration to pregnant rats during gestation on metabotropic glutamate receptors from mothers and full-term fetuses brain by D. León Navarro; J. L. Albasanz; I. Iglesias; M. A. Ruiz; M. Martín (pp. 127-137).
Chronic glutamate treatment during gestational period caused a significant decrease in total metabotropic glutamate receptors (mGluR) number. Similar results were observed on the steady-state level of mGlu1 receptor detected by immunoblotting assays, suggesting that this is the main receptor subtype modulated by agonist exposure. Furthermore, no variations on mRNA coding mGlu1 receptor were found, suggesting post-transcriptional modulation as a possible mechanism of the lost of receptor detected at the membrane surface. On the other hand, western-blotting to determine level of Gq/11 protein and phospholipase C β1 revealed a significant decrease of both proteins in mothers brain. This decrease was associated with significant variation in glutamate and DHPG-stimulated phospholipase C activity. No significant differences on mGluR transduction pathway components were observed in fetuses brain. These results suggest that glutamate intake during pregnancy causes a down-regulation of different proteins involved in glutamate response mediated by mGluR only in mothers brain without significantly affecting fetuses brain.

Keywords: Keywords: Gestation – Metabotropic glutamate receptors – Down-regulation – Desensitization


A rat model of neurolathyrism: repeated injection of L-β-ODAP induces the paraparesis of the hind legs by K. Kusama-Eguchi; F. Ikegami; T. Kusama; A. Suda; Y. Ogawa; K. Igarashi; K. Watanabe (pp. 139-143).
Neurolathyrisim is a motor neuron disease characterized by spastic paraparesis in the hind legs, and is caused by grass pea, Lathyrus sativus, which contains the excitotoxic amino acid, 3-N-oxalyl-L-2,3-diaminopropanoic acid (L-β-ODAP), an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamatergic receptor agonist. In an attempt to make a useful model of this disease, the CNS distribution and toxicity of L-β-ODAP was studied in rat neonates after parenteral administration. L-β-ODAP was detected in the spinal cord as well as in the pons/medulla oblongata, though only small amounts in the latter. Repeated injection of L-β-ODAP resulted in rats with paraparesis of the legs, though at a low incidence rate of 0.032. These paralyzed rats displayed the severe atrophy of the ventral root of the lumbar cord as well as degenerations of motor neuron. The rats were useful models for the study of motor neuron degeneration in the spinal cord.

Keywords: Keywords: Neurolathyrism – L-β-ODAP – Spinal cord – Excitatory amino acids – Neurotoxin – Lathyrus sativus


Modulation of ventral pallidal dopamine and glutamate release by the intravenous anesthetic propofol studied by in vivo microdialysis by C. Grasshoff; M. Herrera-Marschitz; M. Goiny; B. D. Kretschmer (pp. 145-148).
The intravenous anesthetic propofol is reported to have various psychological side effects as hallucinations, sexual disinhibition, or euphoria. Hedonic and rewarding states like these are modulated by the dopaminergic system in the nucleus accumbens, prefrontal cortex and also in the ventral pallidum and by the glutamatergic system in the neocortex and limbic system. In the present study, propofol was administered either alone or in combination with the GABAA receptor antagonist bicuculline via reverse microdialysis into the ventral pallidum of freely moving rats. Dialysis fractions were taken every 20 min and analyzed for dopamine and glutamate using high performance liquid chromatography. Application of propofol decreased dopamine levels in the ventral pallidum. This effect seems to be mainly mediated through GABAA receptors, since it was compensated by the GABAA receptor antagonist bicuculline. Propofol and propofol plus bicuculline exerted no effect on glutamate release in this brain region. The reduced dopamine release in ventral pallidum was most probably mediated through a GABAergic feedback loop from the ventral pallidum via the nucleus accumbens to the dopaminergic neurons of the ventral tegmental area or by long loop feedback. As an increase but not a decrease of dopamine release in the ventral pallidum is involved in hedonic and rewarding properties, similar symptoms induced by propofol seem to be unrelated to an action of propofol in the ventral pallidum.

Keywords: Keywords: Propofol – Dopamine – Glutamate – Ventral pallidum – Bicuculline


Effects of perinatal asphyxia on cell survival, neuronal phenotype and neurite growth evaluated with organotypic triple cultures by V. Klawitter; P. Morales; S. Johansson; D. Bustamante; M. Goiny; J. Gross; J. Luthman; M. Herrera-Marschitz (pp. 149-155).
The effect of perinatal asphyxia on brain development was studied with organotypic cultures from substantia nigra, neostriatum and neocortex. Asphyxia was induced by immersing foetuses-containing uterine horns removed from ready-to-deliver rats into a water bath for 20 min. Following asphyxia, the pups were nursed by a surrogate dam and sacrificed after three days for preparing organotypic cultures. Non-asphyxiated caesarean-delivered pups were used as controls. Morphological features and cell viability were recorded during in vitro development. At day in vitro (DIV) 24, the cultures were treated for immunocytochemistry using antibodies against the N-methyl-D-aspartate receptor subunit 1 (NR1) and tyrosine hydroxylase (TH).While in vitro survival was similar in cultures from both asphyxiated and control animals, differences were observed when the neuronal phenotype was assessed. Compared to controls, the total number of NR1-positive neurons in substantia nigra, as well as the number of secondary to higher level branching of TH-positive neurites from asphyxiated pups were decreased, illustrating the vulnerability of the dopaminergic systems to perinatal asphyxia.

Keywords: Keywords: Perinatal asphyxia – Basal ganglia – Dopamine – Organotypic cultures – Rat


Peculiarities of L-DOPA treatment of Parkinson’s disease by R. M. Kostrzewa; P. Nowak; J. P. Kostrzewa; R. A. Kostrzewa; R. Brus (pp. 157-164).
L-Dihydroxyphenylalanine (L-DOPA), the anti-parkinsonian drug affording the greatest symptomatic relief of parkinsonian symptoms, is still misunderstood in terms of its neurotoxic potential and the mechanism by which generated dopamine (DA) is able to exert an effect despite the absence of DA innervation of target sites in basal ganglia. This review summaries important aspects and new developments on these themes. On the basis of L-DOPA therapy in animal models of Parkinson’s disease, it appears that L-DOPA is actually neuroprotective, not neurotoxic, as indicated by L-DOPA’s reducing striatal tissue content of the reactive oxygen species, hydroxyl radical (HO•), and by leaving unaltered the extraneuronal in vivo microdialysate level of HO•. In addition, the potential beneficial anti-parkinsonian effect of L-DOPA is actually increased because of the fact that the basal ganglia are largely DA-denervated. That is, from in vivo microdialysis studies it can be clearly demonstrated that extraneuronal in vivo microdialysate DA levels are actually higher in the DA-denervated vs. the intact striatum of rats – owing to the absence of DA transporter (i.e., uptake sites) on the absent DA nerve terminal fibers in parkinsonian brain. In essence, there are fewer pumps removing DA from the extraneuronal pool. Finally, the undesired motor dyskinesias that commonly accompany long-term L-DOPA therapy, can be viewed as an outcome of L-DOPA’s sensitizing DA receptors (D1–D5), an effect easily replicated by repeated DA agonist treatments (especially agonist of the D2 class) in animals, even if the brain is not DA-denervated. The newest findings demonstrate that L-DOPA induces BDNF release from corticostriatal fibers, which in-turn enhances the expression of D3 receptors; and that this effect is associated with motor dyskinesias (and it is blocked by D3 antagonists). The recent evidence on mechanisms and effects of L-DOPA increases our understanding of this benefical anti-parkinsonian drug, and can lead to improvements in L-DOPA effects while providing avenues for reducing or eliminating L-DOPA’s deleterious effects.

Keywords: Keywords: L-DOPA – Dopamine – Parkinson’s disease – Volume transmission – Basal ganglia – Striatum

Introduction: Signaling in neurodegeneration (Chapter 2) by A. Novelli; R. A. R. Tasker (pp. 165-167).

Genomics and variation of ionotropic glutamate receptors: implications for neuroplasticity by R. H. Lipsky; X. Jiang; K. Xu; A. J. Marko; K. M. Neyer; T. R. Anderson; A. M. Marini (pp. 169-175).
We used two approaches to identify sequence variants in ionotropic glutamate receptor (IGR) genes: high-throughput screening and resequencing techniques, and “information mining” of public (e.g. dbSNP, ENSEMBL) and private (i.e. Celera Discovery System) sequence databases. Each of the 16 known IGRs is represented in these databases, their positions on a canonical physical map are established. Comparisons of mouse, rat, and human sequences revealed substantial conservation among these genes, which are located on different chromosomes but found within syntenic groups of genes. The IGRs are members of a phylogenetically ancient gene family, sharing similarities with glutamate-like receptors in plants. Parsimony analysis of amino acid sequences groups the IGRs into three distinct clades based on ligand-binding specificity and structural features, such as the channel pore and membrane spanning domains. A collection of 38 variants with amino acid changes was obtained by combining screening, resequencing, and informatics approaches for several of the IGR genes. This represents only a fraction of the sequence variation across these genes, but in fact these may constitute a large fraction of the common polymorphisms at these genes and these polymorphisms are a starting point for understanding the role of these variants in function.Genetically influenced human neurobehavioral phenotypes are likely to be linked to IGR genetic variants. Because ionotropic glutamate receptor activation leads to calcium entry, which is fundamental in brain development and in forms of synaptic plasticity essential for learning and memory and is essential for neuronal survival, it is likely that sequence variants in IGR genes may have profound functional roles in neuronal activation and survival mechanisms.

Keywords: Keywords: Ionotropic glutamate receptor genes – Sequence variant – Genomics – Polymorphism – Neuroplasticity


Calcium accumulation in neurites and cell bodies of rat cerebellar granule cells in culture: effects on GABAA receptor function by A. Cupello; A. Esposito; C. Marchetti; F. Pellistri; M. Robello (pp. 177-182).
Accumulation of calcium in rat cerebellar granule cells in culture was studied by two photon laser scanning microscopy. Depolarizations by high extracellular potassium induced short-lived increases in calcium in both cell bodies and neurites. However, although the increase in neurites subsided completely after the initial peak, in cell bodies there was a persistent plateau until the high potassium stimulus was removed. On the contrary, the calcium signal due to NMDA receptors activation was persistent in both cell bodies and neurites and remained until the agonist was present.The nature of these calcium signals provides an interpretation key for the effects of NMDA receptors activation on GABAA receptors. In particular, the persistent calcium increase in neurites may explain the decrease in GABA activated chloride currents which are related to activation of dendritic/synaptic GABAA receptors.

Keywords: Keywords: Two photon microscopy – Intracellular calcium – NMDA receptors – GABAA receptors – Cerebellar granules


Nefopam inhibits calcium influx, cGMP formation, and NMDA receptor-dependent neurotoxicity following activation of voltage sensitive calcium channels by A. Novelli; R. Díaz-Trelles; A. Groppetti; M. T. Fernández-Sánchez (pp. 183-191).
Nefopam hydrochloride is a potent non sedative benzoxazocine analgesic that possesses a profile distinct from that of anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanism remains unclear. We have investigated the actions of nefopam on voltage sensitive calcium channels and calcium-mediated pathways. We found that nefopam prevented N-methyl-D-aspartate (NMDA)-mediated excitotoxicity following stimulation of L-type voltage sensitive calcium channels by the specific agonist BayK8644. Nefopam protection was concentration-dependent. 47 μM nefopam provided 50% protection while full neuroprotection was achieved at 100 μM nefopam. Neuroprotection was associated with a 73% reduction in the BayK8644-induced increase in intracellular calcium concentration. Nefopam also inhibited intracellular cGMP formation following BayK8644 in a concentration-dependent manner, 100 μM nefopam providing full inhibition of cGMP synthesis and 58 μM allowing 50% cGMP formation. Nefopam reduced NMDA receptor-mediated cGMP formation resulting from the release of glutamate following activation of channels by BayK8644. Finally, we also showed that nefopam effectively reduced cGMP formation following stimulation of cultures with domoic acid, while not providing neuroprotection against domoic acid. Thus, the novel action of nefopam we report here may be important both for its central analgesic effects and for its potential therapeutic use in neurological and neuropsychiatric disorders involving an excessive glutamate release.

Keywords: Keywords: Nefopam – Voltage sensitive calcium channels – Excitotoxicity – cGMP – Cultured cerebellar neurons


NMDA receptor involvement in the effects of low dose domoic acid in neonatal rats by R. A. R. Tasker; M. A. Perry; T. A. Doucette; C. L. Ryan (pp. 193-196).
We have previously reported that neonatal rats display enhanced sensitivity to domoic acid relative to adults, and that perinatal injections of low doses of domoic acid alter early associational learning in the newborn rat. The current study was designed to further investigate the effects of low dose domoic acid on neonatal odour conditioning and to determine if the observed effects are due in part to an action on NMDA receptors. Groups of rat pups were conditioned to a novel odour on postnatal day (PND) 8, injected with 20 μg/kg domoic acid either alone, or in combination with the NMDA antagonist CPP (or appropriate controls), daily from day 8–14, reexposed to the conditioning odour or a novel odour on day 9, and tested for odour preference on day 13 using a standard 3-choice paradigm. Results indicated that rats treated with domoic acid spent significantly more time over the conditioning odour than did saline-treated rats when tested on PND 13. This effect was antagonized by concomitant injection of CPP, indicating an involvement of NMDA receptors in the actions of DOM in this paradigm. Rats injected with either saline or CPP alone showed the opposite effect, i.e. a preference for the alternate odour. The results indicate that a very low dose of DOM produces a conditioned odour preference in neonatal rats and that this effect is due in part to NMDA receptor involvement, thereby emphasizing a role for both kainate and NMDA glutamate receptors in implicit memory.

Keywords: Keywords: Domoic acid – Kainate receptors – NMDA receptors – Odour conditioning – Postnatal development


Increased DNA breaks and up-regulation of both G1 and G2 checkpoint genes p21WAF1/CIP1 and 14-3-3 σ in circulating leukocytes of glaucoma patients and vasospastic individuals by H. Moenkemann; J. Flammer; K. Wunderlich; W. Breipohl; H. H. Schild; O. Golubnitschaja (pp. 199-205).
Objective: Vascular disorder leading to local ischemia/reperfusion has been shown to play an important role in the glaucomatous damage. A decreased expression level of XPGC-gene has been found in circulating leukocytes of normal-tension glaucoma patients. Although decreased activity of XPGC-gene leads to insufficient DNA-repair, no leukopenia has been observed in glaucoma. Molecular mechanisms ensuring cell survival have not been elucidated yet for glaucoma with vascular disorder.Material and methods: Using the ex vivo optical imaging method of alkaline “comet assay” comparative quantification of DNA breaks was performed in circulating leukocytes of non-glaucomatous non-vasospastic and vasospastic individuals as well as both normal-tension and high-tension glaucoma patients. Relative expression levels of the anti-apoptotic factors P21WAF1/CIP1 and 14-3-3 σ were investigated in all groups tested.Results and conclusions: The quantification of P21WAF1/CIP1 showed the highest expression rates in high-tension glaucoma patients which were significantly higher than those in all other groups tested. The highest expression rates of 14-3-3 σ were found in both groups of glaucoma patients. These expression levels correlated well with DNA breaks measured. Since the expression of P21WAF1/CIP1 in leukocytes was shown to be crucial for their survival under stress conditions, we suppose further that the up-regulation of this gene is the key event in the survival mechanisms of leukocytes in glaucoma accompanied with vascular disorder. The p21WAF1/CIP1 gene should be further taken into consideration as a potential marker, the up-regulation of which in circulating leukocytes of vasospastic individuals may indicate an increased risk for the developing glaucoma.

Keywords: Keywords: Glaucoma – Vascular disorder – Circulating leukocytes – Molecular imaging ex vivo – DNA damage – Survival factors P21WAF1/CIP1 and 14-3-3 σ


An enhanced expression of ABC 1 transporter in circulating leukocytes as a potential molecular marker for the diagnostics of glaucoma by K. Yeghiazaryan; J. Flammer; K. Wunderlich; H. H. Schild; S. Orgul; O. Golubnitschaja (pp. 207-211).
Objective: Glaucoma is a neurodegenerative disease. Since vascular dysregulation is supposed to be a risk factor for the development of glaucomatous damage, the preventive treatment might slow down the disease development. The efficiency of the therapeutic treatment depends particularly on a drug efflux pump regulated by ABC transporters. ABC 1 is also known to participate on the vascular regulation. This study was focused on the comparative analysis of ABC 1 expression levels in circulating leukocytes of non-glaucomatous individuals and glaucoma patients.Results and conclusions: The expression rates of ABC 1 were significantly increased in leukocytes of glaucoma patients compared to non-glaucomatous individuals. The expression level of ABC 1 was, furthermore, highly homogeneous in glaucoma patients. In contrast, these expression levels in non-glaucomatous individuals were extremely heterogeneous. This transporter acts as the energy-dependent unidirectional transmembrane cholesterol efflux pump and can export a wide range of hydrophobic drugs. Additionally an observed enhanced ABC 1 expression in circulating leukocytes may be implicated in the vascular regulation mechanisms of glaucoma. We proposed the enhanced expression of ABC 1 in leukocytes as a potential marker for the diagnostics and ex vivo molecular monitoring of glaucoma.

Keywords: Keywords: Glaucoma – Vascular dysregulation – ABC transporter – Circulating leukocytes – Molecular monitoring ex vivo – Diagnostics


Expression of ABC-1 transporter is elevated in human glioma cells under irradiation and temozolomide treatment by D. Trog; H. Moenkemann; N. Haertel; H. Schüller; O. Golubnitschaja (pp. 213-219).
Objective: Chemo-therapeutic treatment of glioma patients has minor success. Little is known about mechanisms of a pronounced resistance of gliomas towards actual therapies, yet. ABC-1 belongs to the group of transporters known to be involved in the export of hydrophobic substances and vascular regulation. This study investigates an effect of both temozolomide (TMZ) treatment and/or irradiation on the expression of the ABC-1 transporter in human U87-MG glioma cells.Material and methods: In parallel experiments U87-MG cells underwent either irradiation (RT), chemo-treatment (CT) using TMZ, and combined chemo/radiation-treatment (CT/RT). After each treatment the cells were incubated either 2 or 24 hours at 37°C and counted before protein analysis using Western-Blot technique.Results and conclusions: An exponential growth of cellular density was observed for both untreated and irradiated cells being, however, about 2-times slower in irradiated compared to untreated cells. In contrast the density increase of chemo-treated cells as well as that of cells, which underwent the combined CT/RT treatment was of linear nature. ABC-1 expression was detected in untreated as well as treated cells. Increasing cell density and all kinds of treatment resulted in a considerably enhanced ABC-1 expression. CT treatment resulted in highly up-regulated ABC-1 expression especially in non-confluent cultures compared to untreated cells. Irradiation had a comparable or even higher inducible effect on the ABC-1 expression rates depending, however, on cell density. The highest expression rates were observed in cultures with high cellular density 2 hours after application of the combined treatment. Strong up-regulation of ABC-1 expression under both irradiation and chemo-treatment might be a clue to multidrug and irradiation cross-resistance mechanisms of malignant glioma cells converting the ABC-1 transporter into an attractive pharmacological target for a clinical breakthrough in the therapy of malignant gliomas.

Keywords: Keywords: Glioma – Multidrug resistance – ABC-1 – Differential gene expression – Irradiation – Temozolomide treatment


Combined analysis of biochemical parameters in serum and differential gene expression in circulating leukocytes may serve as an ex vivo monitoring system to estimate risk factors for complications in Diabetes mellitus by M. Kapalla; K. Yeghiazaryan; M. Hricová; H. Moenkemann; A. Pírek; H. H. Schild; O. Golubnitschaja (pp. 221-227).
Objective: Oxidative stress plays a crucial role in the development of complications in Diabetes mellitus (DM). Individual sensitivity against stress, however, varies among DM-patients and results, therefore, in differential severity of consequent complications. To allow more complex interpretation of a delicate antioxidant/free radicals balance and its effect on cellular functions in individual DM-patients, we analysed a correlation between total antioxidant status (TAS), antioxidant gap (AtxGap), level of free radicals (FR), routine clinical biochemical parameters in blood and differential gene expression in circulating leukocytes of DM-patients versus non-diabetic individuals.Results and conclusions: Positive correlation was found between TAS and creatinine (p=0.05), AtxGap and iron (p=0.025), and between AtxGap and anti-streptolysin O (p=0.025). Whereas no correlation was found between FR and any of the routine clinical parameters tested, a negative correlation was observed between AtxGap and glucose content (p=0.025) and between AtxGap and γ-glutamyltransferase (p=0.05). An increased content of FR was shown to influence significantly an expression of selected stress responsible genes in leukocytes. Transcription levels of NF-kappaB, XRCC1 and 90-kDa heat-shock protein A were increased in all DM-patients compared to non-diabetic individuals. In contrast, an expression of XIAP and cytochrome P450 reductase was up-regulated in patients with decreased levels of both FR and TAS and increased body mass index. This differential expression of the stress responsible genes might be further considered as a potential risk factor for diverse DM-complications helping also in reliable monitoring of supplemental antioxidant therapy and more complex interpretation of delicate antioxidant/free radicals balance.

Keywords: Keywords: Molecular monitoring ex vivo – Diabetic complications – Risk factors – Free radicals/antioxidant status – Circulating leukocytes – Differential gene expression


Molecular imaging system for possible prediction of active retinopathy in patients with Diabetes mellitus by O. Golubnitschaja; A. Jaksche; H. Moenkemann; K. Yeghiazaryan; S. E. Karl; D. Trog; H. H. Schild; K. U. Löffler (pp. 229-237).
Objective: The deposition of advanced glycation end products is enhanced in Diabetes mellitus (DM) and has been linked to diabetic complications such as a microvascular disease. Glycated proteins have receptors on mononuclear blood cells (MBCs) and have been shown to generate reactive oxygen species altering gene expression and modifying cellular targets, such as endothelial cells. Retinal angiopathy is a frequently observed microvascular complication in DM-patients. Because of the central role of activated MBCs, we hypothesised a functional link between specific alterations in gene expression of MBCs, an increased activity of matrix proteases in serum, and the extent of retinal angiopathy in DM.Material and methods: An appearance and proliferation index of diabetic retinopathy was evaluated in 38 DM-patients using fluorescein angiography. Alterations of gene transcription levels in MBCs were investigated using hybridisation of individual mRNA-pools to Atlas Array with a concomitant quantification of specific cDNAs by “Real-Time”-PCR. The activity of matrix metalloproteinases MMP-2 and MMP-9 in individual serum samples was measured by zymography combined with densitometric imaging system.Results and conclusions: Hybridisation to Atlas Array of mRNA-pools isolated from MBCs revealed an enhanced expression of recoverin in DM-patients compared to the control group. “Real-Time”-PCR showed the highest recoverin levels in the DM-subgroup with a high proliferation index. MMP-2 activity was highly increased in 36% of all patients, and in 44, 44, and 19% of patients with proliferative retinopathy, advanced proliferative retinopathy, and no detectable proliferation respectively. In those 3 groups MMP-9 activity was highly increased in 56, 67, and 31% of patients respectively, and in 44% of all DM-patients. In contrast to patients with active proliferation, the simultaneous high activation of all three genes was not observed in patients without active proliferation. The ex vivo molecular imaging system developed in this work may be helpful for the prediction of active proliferative retinopathy in DM.

Keywords: Keywords: Diabetic retinopathy – Molecular Imaging – Differential gene expression – Recoverin – Metalloproteinases – Smoking
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