Electrophysiological evidence for glial-subtype glutamate transporter functional expression in rat cerebellar granule neurons

A glutamate-sensitive inward current (Iglu) is described in rat cerebellar granule neurons and related to a glutamate transport mechanism. We examined the features of Iglu using the patch-clamp technique. In steady-state conditions the Iglu measured 8.14 ± 1.9 pA. Iglu was identified as a voltage-dependent inward current showing a strong rectification at positive potentials. L-Glutamate activated the inward current in a dose-dependent manner, with a half-maximal effect at about 18 µM and a maximum increase of 51.2 ± 4.4%. The inward current was blocked by the presence of dihydrokainate (0.5 mM), shown by others to readily block the GLT1 isoform. We thus speculate that Iglu could be attributed to the presence of a native glutamate transporter in cerebellar granule neurons.

Silibinin improves palmitate-induced insulin resistance in C2C12 myotubes by attenuating IRS-1/PI3K/Akt pathway inhibition

The present study investigated the effect of silibinin, the principal potential anti-inflammatory flavonoid contained in silymarin, a mixture of flavonolignans extracted from Silybum marianum seeds, on palmitate-induced insulin resistance in C2C12 myotubes and its potential molecular mechanisms. Silibinin prevented the decrease of insulin-stimulated 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose) uptake and the downregulation of glutamate transporter type 4 (GLUT4) translocation in C2C12 myotubes induced by palmitate. Meanwhile, silibinin suppressed the palmitate-induced decrease of insulin-stimulated Akt Ser473 phosphorylation, which was reversed by wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase (PI3K). We also found that palmitate downregulated insulin-stimulated Tyr632 phosphorylation of insulin receptor substrate 1 (IRS-1) and up-regulated IRS-1 Ser307 phosphorylation. These effects were rebalanced by silibinin. Considering several serine/threonine kinases reported to phosphorylate IRS-1 at Ser307, treatment with silibinin downregulated the phosphorylation of both c-Jun N-terminal kinase (JNK) and nuclear factor-κB kinase β (IKKβ), which was increased by palmitate in C2C12 myotubes mediating inflammatory status, whereas the phosphorylation of PKC-θ was not significantly modulated by silibinin. Collectively, the results indicated that silibinin prevented inhibition of the IRS-1/PI3K/Akt pathway, thus ameliorating palmitate-induced insulin resistance in C2C12 myotubes.

The 245 kb amplified chromosome of Leishmania (V.) braziliensis contains a biopterin transporter gene

Leishmania (V.) braziliensis M2903 presents a small linear and stable 245 kb chromosome originating from a genomic amplification. Similar amplifications present in other species of Leishmania contain a gene coding for a biopterin transporter. Since Leishmania is auxotrophic for this metabolite, this amplification could result from the need to better capture biotpterin from growth media under specific circumstances. In this paper we show that this gene is also present in L. (V.) braziliensis small chromosome, which shares sequences with other genomic amplifications already described.

Antibodies against the Plasmodium falciparum glutamate-rich protein from naturally exposed individuals living in a Brazilian malaria-endemic area can inhibit in vitro parasite growth

The glutamate-rich protein (GLURP) is an exoantigen expressed in all stages of the Plasmodium falciparum life cycle in humans. Anti-GLURP antibodies can inhibit parasite growth in the presence of monocytes via antibody-dependent cellular inhibition (ADCI), and a major parasite-inhibitory region has been found in the N-terminal R0 region of the protein. Herein, we describe the antiplasmodial activity of anti-GLURP antibodies present in the sera from individuals naturally exposed to malaria in a Brazilian malaria-endemic area. The anti-R0 antibodies showed a potent inhibitory effect on the growth of P. falciparum in vitro, both in the presence (ADCI) and absence (GI) of monocytes. The inhibitory effect on parasite growth was comparable to the effect of IgGs purified from pooled sera from hyperimmune African individuals. Interestingly, in the ADCI test, higher levels of tumour necrosis factor alpha (TNF-α) were observed in the supernatant from cultures with higher parasitemias. Our data suggest that the antibody response induced by GLURP-R0 in naturally exposed individuals may have an important role in controlling parasitemia because these antibodies are able to inhibit the in vitro growth of P. falciparum with or without the cooperation from monocytes. Our results also indicate that TNF-α may not be relevant for the inhibitory effect on P. falciparum in vitro growth.

Genetic polymorphisms in the glutamate-rich protein of Plasmodium falciparum field isolates from a malaria-endemic area of Brazil

The genetic diversity displayed by Plasmodium falciparum, the most deadly Plasmodium species, is a significant obstacle for effective malaria vaccine development. In this study, we identified genetic polymorphisms in P. falciparum glutamate-rich protein (GLURP), which is currently being tested in clinical trials as a malaria vaccine candidate, from isolates found circulating in the Brazilian Amazon at variable transmission levels. The study was performed using samples collected in 1993 and 2008 from rural villages situated near Porto Velho, in the state of Rondônia. DNA was extracted from 126 P. falciparum-positive thick blood smears using the phenol-chloroform method and subjected to a nested polymerase chain reaction protocol with specific primers against two immunodominant regions of GLURP, R0 and R2. Only one R0 fragment and four variants of the R2 fragment were detected. No differences were observed between the two time points with regard to the frequencies of the fragment variants. Mixed infections were uncommon. Our results demonstrate conservation of GLURP-R0 and limited polymorphic variation of GLURP-R2 in P. falciparum isolates from individuals living in Porto Velho. This is an important finding, as genetic polymorphisms in B and T-cell epitopes could have implications for the immunological properties of the antigen.

A novel ABCG-like transporter of Trypanosoma cruziis involved in natural resistance to benznidazole

Benznidazole (BZ) is one of the two drugs used for Chagas disease treatment. Nevertheless therapeutic failures of BZ have been reported, which were mostly attributed to variable drug susceptibility among Trypanosoma cruzistrains. ATP-binding cassette (ABC) transporters are involved in a variety of translocation processes and some members have been implicated in drug resistance. Here we report the characterisation of the first T. cruzi ABCG transporter gene, named TcABCG1, which is over-expressed in parasite strains naturally resistant to BZ. Comparison ofTcABCG1 gene sequence of two TcI BZ-resistant strains with CL Brener BZ-susceptible strain showed several single nucleotide polymorphisms, which determined 11 amino acid changes. CL Brener transfected with TcI transporter genes showed 40-47% increased resistance to BZ, whereas no statistical significant increment in drug resistance was observed when CL Brener was transfected with the homologous gene. Only in the parasites transfected with TcI genes there was 2-2.6-fold increased abundance of TcABCG1transporter protein. The analysis in wild type strains also suggests that the level of TcABCG1transporter is related to BZ natural resistance. The characteristics of untranslated regions of TcABCG1genes of BZ-susceptible and resistant strains were investigated by computational tools.

Distribution of AMPA-type glutamate receptor subunits in the chick visual system

Several glutamate receptor (GluR) subunits have been characterized during the past few years. In the present study, subunit-specific antisera were used to determine the distribution of the AMPA-type glutamate receptor subunits GluR1-4 in retinorecipient areas of the chick brain. Six white leghorn chicks (Gallus gallus, 7-15 days old, unknown sex) were deeply anesthetized and perfused with 4% buffered paraformaldehyde and brain sections were stained using immunoperoxidase techniques. The AMPA-type glutamate receptor subunits GluR1, GluR2/3 and GluR4 were present in several retinorecipient areas, with varying degrees of colocalization. For example, perikarya in layers 2, 3, and 5 of the optic tectum contained GluR1, whereas GluR2/3 subunits appeared mainly in neurons of layer 13. The GluR4 subunit was only detected in a few cells of the tectal layer 13. GluR1 and GluR2/3 were observed in neurons of the nucleus geniculatus lateralis ventralis, whereas GluR4 was only present in its neuropil. Somata in the accessory optic nucleus appeared to contain GluR2/3 and GluR4, whereas GluR1 was the dominant subunit in the neuropil of this nucleus. These results suggest that different subpopulations of visual neurons might express different combinations of AMPA-type GluR subunits, which in turn might generate different synaptic responses to glutamate derived from retinal ganglion cell axons

Control of the phosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in the immature rat hippocampus by glutamate and calcium ions: possible key factor in astrocytic plasticity

The present review describes recent research on the regulation by glutamate and Ca2+ of the phosphorylation state of the intermediate filament protein of the astrocytic cytoskeleton, glial fibrillary acidic protein (GFAP), in immature hippocampal slices. The results of this research are discussed against a background of modern knowledge of the functional importance of astrocytes in the brain and of the structure and dynamic properties of intermediate filament proteins. Astrocytes are now recognized as partners with neurons in many aspects of brain function with important roles in neural plasticity. Site-specific phosphorylation of intermediate filament proteins, including GFAP, has been shown to regulate the dynamic equilibrium between the polymerized and depolymerized state of the filaments and to play a fundamental role in mitosis. Glutamate was found to increase the phosphorylation state of GFAP in hippocampal slices from rats in the post-natal age range of 12-16 days in a reaction that was dependent on external Ca2+. The lack of external Ca2+ in the absence of glutamate also increased GFAP phosphorylation to the same extent. These effects of glutamate and Ca2+ were absent in adult hippocampal slices, where the phosphorylation of GFAP was completely Ca2+-dependent. Studies using specific agonists of glutamate receptors showed that the glutamate response was mediated by a G protein-linked group II metabotropic glutamate receptor (mGluR). Since group II mGluRs do not act by liberating Ca2+ from internal stores, it is proposed that activation of the receptor by glutamate inhibits Ca2+ entry into the astrocytes and consequently down-regulates a Ca2+-dependent dephosphorylation cascade regulating the phosphorylation state of GFAP. The functional significance of these results may be related to the narrow developmental window when the glutamate response is present. In the rat brain this window corresponds to the period of massive synaptogenesis during which astrocytes are known to proliferate. Possibly, glutamate liberated from developing synapses during this period may signal an increase in the phosphorylation

Monosodium glutamate (MSG)-obese rats develop glucose intolerance and insulin resistance to peripheral glucose uptake

Different levels of insulin sensitivity have been described in several animal models of obesity as well as in humans. Monosodium glutamate (MSG)-obese mice were considered not to be insulin resistant from data obtained in oral glucose tolerance tests. To reevaluate insulin resistance by the intravenous glucose tolerance test (IVGTT) and by the clamp technique, newborn male Wistar rats (N = 20) were injected 5 times, every other day, with 4 g/kg MSG (N = 10) or saline (control; N = 10) during the first 10 days of age. At 3 months, the IVGTT was performed by injecting glucose (0.75 g/kg) through the jugular vein into freely moving rats. During euglycemic clamping plasma insulin levels were increased by infusing 3 mU . kg-1 . min-1 of regular insulin until a steady-state plateau was achieved. The basal blood glucose concentration did not differ between the two experimental groups. After the glucose load, increased values of glycemia (P<0.001) in MSG-obese rats occurred at minute 4 and from minute 16 to minute 32. These results indicate impaired glucose tolerance. Basal plasma insulin levels were 39.9 ± 4 µU/ml in control and 66.4 ± 5.3 µU/ml in MSG-obese rats. The mean post-glucose area increase of insulin was 111% higher in MSG-obese than in control rats. When insulinemia was clamped at 102 or 133 µU/ml in control and MSG rats, respectively, the corresponding glucose infusion rate necessary to maintain euglycemia was 17.3 ± 0.8 mg . kg-1 . min-1 for control rats while 2.1 ± 0.3 mg . kg-1 . min-1 was sufficient for MSG-obese rats. The 2-h integrated area for total glucose metabolized, in mg . min . dl-1, was 13.7 ± 2.3 vs 3.3 ± 0.5 for control and MSG rats, respectively. These data demonstrate that MSG-obese rats develop insulin resistance to peripheral glucose uptake

Involvement of hippocampal AMPA glutamate receptor changes and the cAMP/protein kinase A/CREB-P signalling pathway in memory consolidation of an avoidance task in rats

Training in step-down inhibitory avoidance (0.3-mA footshock) is followed by biochemical changes in rat hippocampus that strongly suggest an involvement of quantitative changes in glutamate AMPA receptors, followed by changes in the dopamine D1 receptor/cAMP/protein kinase A (PKA)/CREB-P signalling pathway in memory consolidation. AMPA binding to its receptor and levels of the AMPA receptor-specific subunit GluR1 increase in the hippocampus within the first 3 h after training (20-70%). Binding of the specific D1 receptor ligand, SCH23390, and cAMP levels increase within 3 or 6 h after training (30-100%). PKA activity and CREB-P levels show two peaks: a 35-40% increase 0 h after training, and a second increase 3-6 h later (35-60%). The results correlate with pharmacological findings showing an early post-training involvement of AMPA receptors, and a late involvement of the D1/cAMP/PKA/CREB-P pathway in memory consolidation of this task

Characterization of Plasmodium falciparum glutamate dehydrogenase-soluble antigen

The major aim of this study was to characterize a soluble Plasmodium falciparum antigen from the plasma of malaria-infected humans and Plasmodium falciparum culture supernatants, using immunoabsorbent techniques and Western blotting. An Mr 60-kDa protein was isolated from the plasma of patients with Plasmodium falciparum malaria by affinity chromatography using rabbit anti-Proteus spp GDH(NADP+) serum as ligand. This protein, present in plasma of patients with acute Plasmodium falciparum infection, in Plasmodium falciparum culture supernatants, and in immune complexes, was tested with Plasmodium falciparum malaria hyperimmune serum from patients living in hyperendemic areas and rabbit anti-Proteus spp GDH(NADP+) serum prepared in the laboratory. In this report, we describe the results of a study showing that parasite GDH(NADP+) can be used to detect the presence of Plasmodium falciparum. It appears that this technique permits the chromatographic detection of a Plasmodium falciparum excretion antigen that may be used in the production of monoclonal antibodies to improve immunodiagnostic assays for the detection of antigenemia, and opens the possibility of its use as a non-microscopic screening method.

Uptake of NO-releasing drugs by the P2 nucleoside transporter in trypanosomes

Nitric oxide (NO·) has been identified as a principal regulatory molecule of the immune system and the major cytotoxic mediator of activated immune cells. NO· can also react rapidly with a variety of biological species, particularly with the superoxide radical anion O2·- at almost diffusion-limited rates to form peroxynitrite anion (ONOO-). ONOO- and its proton-catalyzed decomposition products are capable of oxidizing a great diversity of biomolecules and can act as a source of toxic hydroxyl radicals. As a consequence, a strategy for the development of molecules with potential trypanocidal activities could be developed to increase the concentration of nitric oxide in the parasites through NO·-releasing compounds. In this way, the rate of formation of peroxynitrite from NO· and O2·- would be faster than the rate of dismutation of superoxide radicals by superoxide dismutases which constitute the primary antioxidant enzymatic defense system in trypanosomes. The adenosine transport systems of parasitic protozoa, which are also in certain cases implicated in the selective uptake of active drugs such as melarsoprol or pentamidine, could be exploited to specifically target these NO·-releasing compounds inside the parasites. In this work, we present the synthesis, characterization and biological evaluation of a series of molecules that contain both a group which would specifically target these drugs inside the parasites via the purine transporter, and an NO·-donor group that would exert a specific pharmacological effect by increasing NO level, and thus the peroxynitrite concentration inside the parasite.

Defense reaction induced by a metabotropic glutamate receptor agonist microinjected into the dorsal periaqueductal gray of rats

The behavioral effects of trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD), a metabotropic glutamate receptor (mGluR) agonist, or 0.9% (w/v) saline, injected into the dorsal periaqueductal gray (DPAG), was investigated. Male Wistar rats showed defense reactions characterized by jumps toward the top edges of the cages (saline = 0 vs t-ACPD = 6.0, medians P<0.05) and gallops (saline = 0 vs t-ACPD = 10.0, medians P<0.05) during the 60-s period after the beginning of the injection. In another experiment animals were placed inside an open arena for 5 min immediately after injection. Their behavior was recorded by a video camera and a computer program analyzed the videotapes. Eleven of fifteen rats injected with t-ACPD showed a short-lasting (about 1 min) flight reaction. No saline-treated animal showed this reaction (P<0.0005, chi-square test). The drug induced an increase in turning behavior (P = 0.002, MANOVA) and a decrease in the number of rearings (P<0.001, MANOVA) and grooming episodes (P<0.001, MANOVA). These results suggest that mGluRs play a role in the control of defense reactions in the DPAG.

Effect of inhibitory avoidance training on [3H]-glutamate binding in the hippocampus and parietal cortex of rats

Glutamate receptors have been implicated in memory formation. The aim of the present study was to determine the effect of inhibitory avoidance training on specific [3H]-glutamate binding to membranes obtained from the hippocampus or parietal cortex of rats. Adult male Wistar rats were trained (0.5-mA footshock) in a step-down inhibitory avoidance task and were sacrificed 0, 5, 15 or 60 min after training. Hippocampus and parietal cortex were dissected and membranes were prepared and incubated with 350 nM [3H]-glutamate (N = 4-6 per group). Inhibitory avoidance training induced a 29% increase in glutamate binding in hippocampal membranes obtained from rats sacrificed at 5 min (P<0.01), but not at 0, 15, or 60 min after training, and did not affect glutamate binding in membranes obtained from the parietal cortex. These results are consistent with previous evidence for the involvement of glutamatergic synaptic modification in the hippocampus in the early steps of memory formation.