Biomedical subjects

A Cozzi

Publications and source records attributed to A Cozzi.

At least 19 recordsLinked to original sources

PubMed2003

Assessment of ozone visible symptoms in the field: perspectives of quality control.

The second UN/ECE ICP-Forests Intercalibration Course on the Assessment of Ozone Injury on European Tree Species was carried out in August 2001 at Lattecaldo (Canton Ticino, CH) and Moggio (Lombardy, I). Forty-eight experts from several European countries participated in the exercises and assessed visible symptoms of ozone injury both in open-top chambers (OTC) (Lattecaldo) and under open field (Moggio) conditions. Evaluation of the results indicated a large variability among the teams and call for adequate training of the observers prior to symptom assessment for quality assurance purposes. Highest variability was found for the species developing unclear symptoms which could be confused with senescence processes; such species should not be used in the field. The authors provide suggestions to improve the reliability of the ozone injury assessment on forest plant species.

Air Pollutants↗

PubMed2002

Acute cerebral focal ischaemia alters the adrenergic and NANC responses in the bisected rat vas deferens.

1. Disturbances of the autonomic nervous system are common in right hemisphere stroke patients, including a marked decline in male sexual functions. There is a lack of information on the influence of stroke on male secondary sex organs such as the vas deferens. 2. This study investigates the effect of right brain focal ischaemia on the adrenergic and purinergic responses in isolated epididymal and prostatic portions of rat vas deferens. 3. In both epididymal and prostatic portions the concentration-response curves to noradrenaline are flattened resulting in a reduction (up to 67 - 76%) of the maximum contractile response in the tissue from ischaemic rats compared to the controls. In the prostatic portion from ischaemic rats the concentration-response curve to alpha,beta-methylene ATP was also depressed. 4. The first purinergic and the second delayed adrenergic phase to single pulse was not modified by brain ischaemia. In contrast both phasic and tonic components of the electrically induced contractions by trains of stimuli at high frequencies (2 - 30 Hz) were significantly depressed in the epididymal and prostatic portions from ischaemic rats. 5. These results demonstrate an autonomic imbalance at the level of male sexual secondary organs which may contribute to sexual impairment after stroke.

Acute Disease↗

PubMed2001

Kynurenine 3-mono-oxygenase activity and neurotoxic kynurenine metabolites increase in the spinal cord of rats with experimental allergic encephalomyelitis.

Kynurenine 3-mono-oxygenase, one of the key enzymes of the "kynurenine pathway", catalyses the formation of 3-hydroxykynurenine and may direct the neo-synthesis of quinolinic and kynurenic acids. While 3-hydroxykynurenine and quinolinic acid have neurotoxic properties, kynurenic acid antagonizes excitotoxic neuronal death. Here we report that the expression and activity of kynurenine 3-mono-oxygenase significantly increased in the spinal cord of rats with experimental allergic encephalopathy, an experimental model of multiple sclerosis. As a consequence of this increase, the spinal cord content of 3-hydroxykynurenine and quinolinic acid reached neurotoxic levels. We also report that systemic administration of Ro 61-8048, a selective kynurenine 3-mono-oxygenase inhibitor, reduced the increase of both 3-hydroxykynurenine and quinolinic acid, and caused accumulation of kynurenic acid. In the brain and spinal cord of the controls, kynurenine 3-mono-oxygenase immunoreactivity was located in granules (probably mitochondria) present in the cytoplasm of both neurons and astroglial cells. In the spinal cord of rats with experimental allergic encephalopathy, however, cells with a very intense kynurenine 3-mono-oxygenase immunoreactivity, also able to express class II major histocompatibility complex and inducible nitric oxide synthase, were found in perivascular, subependymal and subpial locations. These cells (most probably macrophages) were responsible for the large increase in 3-hydroxykynurenine and quinolinic acid found in the spinal cords of affected animals. The results show that cells of the immune system are responsible for the increased formation of 3-hydroxykynurenine and quinolinic acid, two neurotoxic metabolites that accumulate in the central nervous system of rats with experimental allergic encephalomyelitis. They also demonstrate that selective kynurenine 3-mono-oxygenase inhibitors reduce the neo-synthesis of these toxins.

Animals↗

PubMed2001

Poly(ADP-ribose) polymerase inhibitors attenuate necrotic but not apoptotic neuronal death in experimental models of cerebral ischemia.

An excessive activation of poly(ADP-ribose) polymerase (PARP) has been proposed to play a key role in post-ischemic neuronal death. We examined the neuroprotective effects of the PARP inhibitors benzamide, 6(5H)-phenanthridinone, and 3,4-dihydro-5-[4-1(1-piperidinyl)buthoxy]-1(2H)-isoquinolinone in three rodent models of cerebral ischemia. Increasing concentrations of the three PARP inhibitors attenuated neuronal injury induced by 60 min oxygen-glucose deprivation (OGD) in mixed cortical cell cultures, but were unable to reduce CA1 pyramidal cell loss in organotypic hippocampal slices exposed to 30 min OGD or in gerbils following 5 min bilateral carotid occlusion. We then examined the necrotic and apoptotic features of OGD-induced neurodegeneration in cortical cells and hippocampal slices using biochemical and morphological approaches. Cortical cells exposed to OGD released lactate dehydrogenase into the medium and displayed ultrastructural features of necrotic cell death, whereas no caspase-3 activation nor morphological characteristics of apoptosis were observed at any time point after OGD. In contrast, a marked increase in caspase-3 activity was observed in organotypic hippocampal slices after OGD, together with fluorescence and electron microscope evidence of apoptotic neuronal death in the CA1 subregion. Moreover, the caspase inhibitor Z-VAD-FMK reduced OGD-induced CA1 pyramidal cell loss. These findings suggest that PARP overactivation may be an important mechanism leading to post-ischemic neurodegeneration of the necrotic but not of the apoptotic type.

Animals↗

PubMed2001

The expression of glutamate transporter GLT-1 in the rat cerebral cortex is down-regulated by the antipsychotic drug clozapine.

We show here that clozapine, a beneficial antipsychotic, down-regulates the expression of the glutamate transporter GLT-1 in the rat cerebral cortex, thereby reducing glutamate transport and raising extracellular glutamate levels. Clozapine treatment (25--35 mg kg(-1) day(-1) orally) reduced GLT-1 immunoreactivity in several brain regions after 3 weeks; this effect was most prominent after 9 weeks and most evident in the frontal cortex. GLT-1 protein levels were reduced in the cerebral cortex of treated rats compared with controls and were more severely affected in the anterior (71.9 +/- 4.5%) than in the posterior (53.2 +/- 15.4%) cortex. L-[(3)H]-glutamate uptake in Xenopus laevis oocytes injected with mRNA extracted from the anterior cerebral cortex of rats treated for 9 weeks was remarkably reduced (to 30.6 +/- 8.6%) as compared to controls. In addition, electrophysiological recordings from oocytes following application of glutamate revealed a strong reduction in glutamate uptake currents (46.3 +/- 10.2%) as compared to controls. Finally, clozapine treatment led to increases in both the mean basal (8.1 +/- 0.7 microM) and the KCl-evoked (28.7 +/- 7.7 microM) output of glutamate that were 3.1 and 3.5, respectively, higher than in control rats. These findings indicate that clozapine may potentiate glutamatergic synaptic transmission by regulating glutamate transport.

ATP-Binding Cassette Transporters↗

PubMed2001

Presynaptic kynurenate-sensitive receptors inhibit glutamate release.

Kynurenic acid is a tryptophan metabolite provided with antagonist activity on ionotropic glutamate and alpha7 nicotinic acetylcholine receptors. We noticed that in rats with a dialysis probe placed in the head of their caudate nuclei, local administration of kynurenic acid (30-100 nM) significantly reduced glutamate output. Qualitatively and quantitatively similar effects were observed after systemic administration of kynurenine hydroxylase inhibitors, a procedure able to increase brain kynurenate concentrations. Interestingly, in microdialysis studies, methyllycaconitine (0.3-10 nM), a selective alpha7 nicotinic receptor antagonist, also reduced glutamate output. In isolated superfused striatal synaptosomes, kynurenic acid (100 nM), but not methyllycaconitine, inhibited the depolarization (KCl 12.5 mM)-induced release of transmitter or previously taken-up [3H]-D-aspartate. This inhibition was not modified by glycine, N-methyl-D-aspartate or subtype-selective kainate receptor agents, while CNQX or DNQX (10 microM), two AMPA and kainate receptor antagonists, reduced kynurenic acid effects. Low concentrations of kynurenic acid, however, did not modify [3H]-kainate (high and low affinity) or [3H]-AMPA binding to rat brain membranes. Finally, because metabotropic glutamate (mGlu) receptors modulate transmitter release in striatal preparations, we evaluated, with negative results, kynurenic acid (1-100 nM) effects in cells transfected with mGlu1, mGlu2, mGlu4 or mGlu5 receptors. In conclusion, our data show that kynurenate-induced inhibition of glutamate release is not mediated by glutamate receptors. Nicotinic acetylcholine receptors, however, may contribute to the inhibitory effects of kynurenate found in microdialysis studies, but not in those found in isolated synaptosomes.

Animals↗

PubMed2000

Overexpression of wild type and mutated human ferritin H-chain in HeLa cells: in vivo role of ferritin ferroxidase activity.

Transfectant HeLa cells were generated that expressed human ferritin H-chain wild type and an H-chain mutant with inactivated ferroxidase activity under the control of the tetracycline-responsive promoter (Tet-off). The clones accumulated exogenous ferritins up to levels 14-16-fold over background, half of which were as H-chain homopolymers. This had no evident effect in the mutant ferritin clone, whereas it induced an iron-deficient phenotype in the H-ferritin wild type clone, manifested by approximately 5-fold increase of IRPs activity, approximately 2.5-fold increase of transferrin receptor, approximately 1.8-fold increase in iron-transferrin iron uptake, and approximately 50% reduction of labile iron pool. Overexpression of the H-ferritin, but not of the mutant ferritin, strongly reduced cell growth and increased resistance to H(2)O(2) toxicity, effects that were reverted by prolonged incubation in iron-supplemented medium. The results show that in HeLa cells H-ferritin regulates the metabolic iron pool with a mechanism dependent on the functionality of the ferroxidase centers, and this affects, in opposite directions, cellular growth and resistance to oxidative damage. This, and the finding that also in vivo H-chain homopolymers are much less efficient than the H/L heteropolymers in taking up iron, indicate that functional activity of H-ferritin in HeLa cells is that predicted from the in vitro data.

Anti-Bacterial Agents↗

PubMed2000

Antiferritin single-chain Fv fragment is a functional protein with properties of a partially structured state: comparison with the completely folded V(L) domain.

Differential scanning calorimetry and spectroscopic probes were applied to study folding and stability of the single-chain Fv fragment (scFv) of the anti-human ferritin antibody F11 and its isolated variable light-chain (V(L)) domain. The scFv fragment followed variable heavy-chain domain (V(H))-linker-V(L) orientation and contained (Gly(4)Ser)(3) linker peptide. The two proteins were produced in Escherichia coli and refolded from denaturant-solubilized inclusion bodies. The isolated V(L) domain demonstrated a typical immunoglobulin fold with well-defined secondary and tertiary structure and was capable of binding human ferritin with K(a) = 1.8 x 10(7) M(-)(1), approximately (1)/(30) of the affinity of the parent F11 antibody. Involvement of this V(L) domain into the two-domain scFv fragment yielded a distorted secondary and significantly destabilized tertiary structure in which neither of the two constituent domains attained complete folding. The thermal unfolding enthalpy of scFv F11 at pH 7.0 was as low as 5. 0 J.g(-)(1) versus 16.3 J.g(-)(1) obtained for the V(L) domain and 24.7 J.g(-)(1) for the parent F11 antibody (mouse IgG2a subclass). Intrinsic fluorescence and near-ultraviolet circular dichroic (CD) spectra, and binding of the hydrophobic probe 8-anilino-1-naphthalene sulfonate, confirmed partial loss of tertiary interactions in scFv. The spectroscopic and calorimetric properties of scFv F11 under physiological conditions are consistent with a model of a partially structured state with a distorted beta-sheet as a secondary structure and partial loss of tertiary interactions, which closely resembles the alternatively folded A-state adopted by an immunoglobulin at pH 2-3 [Buchner, J., Renner, M., Lilie, H., Hinz, H.-J., Jaenicke, R., Kiefhaber, T., and Rudolph, R. (1991) Biochemistry 30, 6922-6929]. However, scFv F11 demonstrated only an approximately 4-fold decrease in the antigen-binding affinity (K(a) = 1.3 x 10(8) M(-)(1)) versus the parent F11 antibody. The scFv fragment F11 provides the first description of a functional protein trapped under physiological conditions in a partially structured state. This state is either close to the native one in the antigen-binding affinity or, alternatively, initial weak binding of the antigenic epitope induces folding of scFv F11 into a more structured conformation that generates relatively high affinity.

Amino Acid Sequence↗

PubMed2000

Functional and immunological analysis of recombinant mouse H- and L-ferritins from Escherichia coli.

The production and characterization of recombinant mouse H- and L-ferritin chains from Escherichia coli are described. The proteins were efficiently expressed and purified with yields of 7-40 mg per liter of cell culture. They had the expected molecular mass and showed a physical stability analogous to that of the corresponding human ferritins. Mouse H- and L-ferritins had a very similar mobility on denaturing SDS-PAGE, but could be readily separated on nondenaturing PAGE because of the distinct slow mobility of mouse L-ferritin. Direct comparative experiments showed that mouse and human H-ferritins had the same iron incorporation activity, whereas mouse L-ferritin incorporated iron less efficiently than human L-ferritin. The difference was attributed to the substitution of a residue exposed on the cavity surface (Glu140 --> Lys) in mouse L-ferritin, a hypothesis confirmed by the finding that the mouse L-ferritin mutant Lys140-Glu incorporated iron as efficiently as human L-ferritin. Rabbit antisera elicited by the recombinant mouse ferritins were specific for the H- and L-chains and did not cross-react with the human ferritins. The antibodies and the derived specific ELISA assays allow the determination of H- and L-ferritins in mouse tissues.

Amino Acid Sequence↗

PubMed2000

Expression, refolding, and ferritin-binding activity of the isolated VL-domain of monoclonal antibody F11.

Expression of the VL-domain of mouse monoclonal antibody F11 to human spleen ferritin in Escherichia coli cells is associated with the formation of insoluble protein aggregates (inclusion bodies). The aggregates were solubilized in the presence of guanidine hydrochloride and the recombinant VL-domain was purified by immobilized metal affinity chromatography (IMAC). Subsequent renaturation results in approximately 99% pure preparation with high yield. The VL-domain forms dimers at concentrations from 1 to 10 mg/ml. Monomeric form is detected only at protein concentrations below 0.5 mg/ml. Functional activity of the VL-domain was verified by two variants of ELISA. The affinity of the VL-domain ((0.2-1.2). 108 M(-1)) is similar to the affinity of the full-length parental antibody F11 because when the immobilized VL-domain was used, the binding constant of ferritin to the VL-domain was only 4-6-fold lower than that in the case of F11 antibody. In another ELISA system with immobilized ferritin, affinity was decreased 30-fold. The VL-domain of antibody F11 is the first example of the recombinant variable domain of the immunoglobulin light chain that preserves the antigen-binding activity in the absence of the partner VH-domain. The data indicate that the recombinant VL-domain can be used in construction of chimeric immunotoxins and other antigen-binding proteins in immunotherapy and in studies of correlations between folding, stability, and activity of immunoglobulins.

Amino Acid Sequence↗

PubMed1999

Overexpression of the hereditary hemochromatosis protein, HFE, in HeLa cells induces and iron-deficient phenotype.

A transfectant HeLa cell clone expressing HFE under the control of a tetracycline-repressible promoter was generated. HFE expression was fully repressed by the presence of doxycycline, while it was strongly induced by growth in the absence of doxycycline. HFE accumulation was accompanied by a large (approximately 10-fold) decrease in H- and L-ferritin levels, by a approximately 3-4-fold increase in transferrin receptor, and a approximately 2-fold increase in iron regulatory protein activity. These indices of cellular iron deficiency were reversed by iron supplementation complexes. The overexpressed HFE immunoprecipitated together with transferrin receptor, indicating a physical association which is the likely cause for the observed approximately 30% decrease in 55Fe-transferrin incorporation after 18 h incubation. In the HFE-expressing cells the reduction in transferrin-mediated iron incorporation was partially compensated by a approximately 30% increase in non-transferrin iron incorporation from 55Fe-NTA, evident after prolonged, 18 h, incubations. The findings indicate that HFE binding to transferrin receptor reduces cellular iron availability and regulates the balance between transferrin-mediated and non-transferrin-mediated cellular iron incorporation.

Apoferritins↗

PubMed1999

A parallel noise-robust algorithm to recover depth information from radial flow fields.

A parallel algorithm operating on the units ('neurons') of an artificial retina is proposed to recover depth information in a visual scene from radial flow fields induced by ego motion along a given axis. The system consists of up to 600 radii with fewer than 65 radially arranged neurons on each radius. Neurons are connected only to their nearest neighbors, and they are excited as soon as a sufficiently strong gray-level change occurs. The time difference of two subsequently activated neurons is then used by the last-excited neuron to compute the depth information. All algorithmic calculations remain strictly local, and information is exchanged only between adjacent active neurons (except for the final read-out). This, in principle, permits parallel implementation. Furthermore, it is demonstrated that the calculation of the object coordinates requires only a single multiplication with a constant, which is dependent on only the retinal position of the active neuron. The initial restriction to local operations makes the algorithm very noise sensitive. In order to solve this problem, a predication mechanism is introduced. After an object coordinate has been determined, the active neuron computes the time when the next neuronal excitation should take place. This estimated time is transferred to the respective next neuron, which will wait for this excitation only within a certain time window. If the excitation fails to arrive within this window, the previously computed object coordinate is regarded as noisy and discarded. We will show that this predictive mechanism relies also on only a (second) single multiplication with another neuron-dependent constant. Thus, computational complexity remains low, and noisy depth coordinates are efficiently eliminated. Thus, the algorithm is very fast and operates in real time on 128 x 128 images even in a serial implementation on a relatively slow computer. The algorithm is tested on scenes of growing complexity, and a detailed error analysis is provided showing that the depth error remains very low in most cases. A comparison to standard flow-field analysis shows that our algorithm outperforms the older method by far. The analysis of the algorithm also shows that it is generally applicable despite its restrictions, because it is fast and accurate enough such that a complete depth percept can be composed from radial flow field segments. Finally, we suggest how to generalize the algorithm, waiving the restriction of radial flow.

Algorithms↗

PubMed1999

Neuroprotective effects of kynurenine-3-hydroxylase inhibitors in models of brain ischemia.

The neuroprotective effects of two kynurenine hydroxylase inhibitors, (m-nitrobenzoyl)-alanine (mNBA) and 3,4-dimethoxy-[-N-4-(nitrophenyl)thiazol-2yl]-benzenesulfona mide (Ro 61-8048), were studied in vitro and in vivo. In organotypic hippocampal slice cultures deprived of oxygen and glucose, these inhibitors significantly reduced neuronal damage. In gerbils subjected to bilateral carotid occlusion for 5 min, the administration of mNBA (400 mg/kg i.p., 3 times) or Ro 61-8048 (40 mg/kg i.p., 3 times) dramatically decreased the percentage of damaged pyramidal neurones in the hippocampal CA1 region. Finally, in rats with permanent occlusion of the middle cerebral artery, mNBA (200-400 mg/kg i.p.) and Ro 61-8048 (40 mg/kg i.p.) administration reduced the infarct volume. Our results demonstrate that ischemic neuronal damage may be significantly decreased by inhibiting kynurenine hydroxylase.

Alanine↗

PubMed1999

Protection with metabotropic glutamate 1 receptor antagonists in models of ischemic neuronal death: time-course and mechanisms.

In order to study the role of metabotropic glutamate 1 (mGlu1) receptors in ischemic neuronal death, we examined the effects of the recently characterized and relatively selective mGlu1 receptor antagonists 1-aminoindan-1,5-dicarboxylic acid (AIDA) and (S)-(+)-2-(3'-carboxybicyclo[1.1.1]pentyl)-glycine (CBPG) in murine cortical cell cultures and rat organotypic hippocampal slices exposed to oxygen glucose deprivation (OGD) and in vivo, following transient global ischemia in gerbils. AIDA and CBPG significantly reduced neuronal death when added to the incubation medium during the OGD insult and the subsequent recovery period. Neuroprotection was observed even when these compounds were added up to 60 min (in cortical neurons) or 30 min (in hippocampal slices) after OGD. In vivo, i.c.v. administration of AIDA and CBPG reduced hippocampal CA1 pyramidal cell injury following transient global ischemia. Neuroprotection was also observed when AIDA was added to the hippocampal perfusion fluid in microdialysis experiments, and this effect was associated with an increase in the basal output of GABA. These findings demonstrate that AIDA and CBPG are neuroprotective when administered during the maturation of ischemic damage and that different mechanisms are likely to be involved in mediating their effects following blockade of mGlu1 receptors in cortical and hippocampal neurons.

Animals↗

PubMed1999

1-Aminoindan-1,5-dicarboxylic acid and (S)-(+)-2-(3'-carboxybicyclo[1.1.1] pentyl)-glycine, two mGlu1 receptor-preferring antagonists, reduce neuronal death in in vitro and in vivo models of cerebral ischaemia.

Metabotropic glutamate (mGlu) receptors have been implicated in a number of physiological and pathological responses to glutamate, but the exact role of group I mGlu receptors in causing postischaemic injury is not yet clear. In this study, we examined whether the recently-characterized and relatively selective mGlu1 receptor antagonists 1-aminoindan-1,5-dicarboxylic acid (AIDA) and (S)-(+)-2-(3'-carboxybicyclo[1.1.1]pentyl)-glycine (CBPG) could reduce neuronal death in vitro, following oxygen-glucose deprivation (OGD) in murine cortical cell and rat organotypic hippocampal cultures, and in vivo, after global ischaemia in gerbils. When present in the incubation medium during the OGD insult and the subsequent 24 h recovery period, AIDA and CBPG significantly reduced neuronal death in vitro. The extent of protection was similar to that observed with the nonselective mGlu receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine [(+)MCPG] and with typical ionotropic glutamate (iGlu) receptor antagonists. Neuroprotection was also observed when AIDA or CBPG were added only after the OGD insult was terminated. Neuronal injury was not attenuated by the inactive isomer (-)MCPG, but was significantly enhanced by the nonselective mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid [(1S,3R)-ACPD] and the group I mGlu receptor agonist 3,5-dihydroxyphenylglycine (3,5-DHPG). The antagonists (+)MCPG, AIDA and CBPG were also neuroprotective in vivo, because i. c.v. administration reduced CA1 pyramidal cell degeneration examined 7 days following transient carotid occlusion in gerbils. Our results point to a role of mGlu1 receptors in the pathological mechanisms responsible for postischaemic neuronal death and propose a new target for neuroprotection.

Animals↗

PubMed1999

Kynurenine hydroxylase inhibitors reduce ischemic brain damage: studies with (m-nitrobenzoyl)-alanine (mNBA) and 3,4-dimethoxy-[-N-4-(nitrophenyl)thiazol-2yl]-benzenesulfonamide (Ro 61-8048) in models of focal or global brain ischemia.

Two kynurenine hydroxylase inhibitors, (m-nitrobenzoyl)-alanine (mNBA) and 3,4-dimethoxy-[-N-4-(nitrophenyl)thiazol-2yl]-benzenesulfona mide (Ro 61-8048), have been tested as neuroprotective agents on brain lesions induced by bilateral carotid occlusion in gerbils or by middle cerebral artery occlusion in rats. The percentage of lesioned pyramidal neurones found in the hippocampal CA1 region of gerbils subjected to bilateral carotid occlusion for 5 minutes decreased from 92+/-10% in vehicle-treated animals to 7+/-6% after mNBA (400 mg/kg intraperitoneally, three times at 1, 30, and 180 minutes after occlusion) or to 10+/-11% after Ro 61-8048 (40 mg/kg intraperitoneally, three times). A significant reduction in infarct volumes also was found when the kynurenine hydroxylase inhibitors were given to rats after permanent middle cerebral artery occlusion (from 207+/-111 mm3 in vehicle-treated rats to 82+/-18 and to 62+/-57 mm3 in rats treated with mNBA, 400 mg/kg intraperitoneally, or with Ro 61-8048, 40 mg/kg intraperitoneally, respectively). The administration of mNBA (400 mg/kg intraperitoneally) or Ro 61-8048 (40 mg/kg intraperitoneally) to gerbils with a dialysis probe in their dorsal hippocampus or to rats with a dialysis probe in their parietal cortex significantly increased kynurenic acid concentration in the dialysates. The data suggest that inhibition of kynurenine hydroxylase could be a new avenue to reduce neuronal loss in brain ischemia.

Alanine↗

PubMed1998

Presynaptic mGlu1 type receptors potentiate transmitter output in the rat cortex.

In the present study we used freely moving rats with a microdialysis probe placed in their parietal cortex to study the effects of local application of agonists and antagonists of metabotropic glutamate (mGlu) receptors on glutamate release. (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD; 0.1-1 mM), a non-selective agonist of metabotropic glutamate (mGlu) receptors, increased glutamate concentration in the dialysate up to 3-fold. A significant increase in glutamate output in cortical dialysates was also obtained with (RS)-3,5-dihydroxyphenylglycine (DHPG; 0.5-1 mM), a group 1-selective mGlu receptor agonist, suggesting the involvement of group 1 mGlu receptors in 1S,3R-ACPD effects. S-4-carboxyphenylglycine (S-4CPG; 0.3 microM), a mGlu1 receptor antagonist with a mild agonist action on mGlu2 receptors, antagonised, in a surmountable manner, the effects of 1S,3 R-ACPD. Similarly, 1-aminoindan-1,5-dicarboxylic acid (AIDA; 0.03-1 mM) a selective group 1 antagonist with a preferential action on mGlu1 type receptors, antagonised the effects of 1S,3R-ACPD. Finally, (S)-(+)-2-(3'-Carboxybicyclo[1.1.1]pentyl)-glycine (UPF596; 30-300 microM), a potent mGlu1 antagonist with modest agonist activity on mGlu5, antagonised 1S,3R-ACPD-induced glutamate release. In conclusion, our data showed that 1S,3R-ACPD-induced glutamate release in the parietal cortex is mediated by mGlu1 receptors and that, under basal conditions, these receptors are not tonically activated.

Animals↗

PubMed1998

Transient overexpression of human H- and L-ferritin chains in COS cells.

The understanding of the in vitro mechanisms of ferritin iron incorporation has greatly increased in recent years with the studies of recombinant and mutant ferritins. However, little is known about how this protein functions in vivo, mainly because of the lack of cellular models in which ferritin expression can be modulated independently from iron. To this aim, primate fibroblastoid COS-7 cells were transiently transfected with cDNAs for human ferritin H- and L-chains under simian virus 40 promoter and analysed within 66 h. Ferritin accumulation reached levels 300-500-fold higher than background, with about 40% of the cells being transfected. Thus ferritin concentration in individual cells was increased up to 1000-fold over controls with no evident signs of toxicity. The exogenous ferritin subunits were correctly assembled into homopolymers, but did not affect either the size or the subunit composition of the endogenous heteropolymeric fraction of ferritin, which remained essentially unchanged in the transfected and non-transfected cells. After 18 h of incubation with [59Fe]ferric-nitrilotriacetate, cellular iron incorporation was similar in the transfected and non-transfected cells and most of the protein-bound radioactivity was associated with ferritin heteropolymers, while H- and L-homopolymers remained iron-free. Cell co-transfection with cDNAs for H- and L-chains produced ferritin heteropolymers that also did not increase cellular iron incorporation. It is concluded that transient transfection of COS cells induces a high level of expression of ferritin subunits that do not co-assemble with the endogenous ferritins and have no evident activity in iron incorporation/metabolism.

Animals↗