Modulation of higher-plant NAD(H)-dependent glutamate dehydrogenase activity in transgenic tobacco via alteration of beta subunit levels

Glutamate dehydrogenase (GDH; EC 1.4.1.2-1.4.1.4) catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate. In vascular plants the in vivo direction(s) of the GDH reaction and hence the physiological role(s) of this enzyme remain obscure. A phylogenetic analysis identified two clearly separated groups of higher-plant GDH genes encoding either the alpha- or beta-subunit of the GDH holoenzyme. To help clarify the physiological role(s) of GDH, tobacco (Nicotiana tabacum L.) was transformed with either an antisense or sense copy of a beta-subunit gene, and transgenic plants recovered with between 0.5- and 34-times normal leaf GDH activity. This large modulation of GDH activity (shown to be via alteration of beta-subunit levels) had little effect on leaf ammonium or the leaf free amino acid pool, except that a large increase in GDH activity was associated with a significant decrease in leaf Asp (similar to 51%, P=0.0045). Similarly, plant growth and development were not affected, suggesting that a large modulation of GDH beta-subunit titre does not affect plant viability under the ideal growing conditions employed. Reduction of GDH activity and protein levels in an antisense line was associated with a large increase in transcripts of a beta-subunit gene, suggesting that the reduction in beta-subunit levels might have been due to translational inhibition. In another experiment designed to detect post-translational up-regulation of GDH activity, GDH over-expressing plants were subjected to prolonged dark-stress. GDH activity increased, but this was found to be due more likely to resistance of the GDH protein to stress-induced proteolysis, rather than to post-translational up-regulation.

The ABC transporter gene in the sirodesmin biosynthetic gene cluster of Leptosphaeria maculans is not essential for sirodesmin production but facilitates self-protection

Epipolythiodioxopiperazine toxins are secreted by a range of fungi, including Leptosphaeria maculans, which produces sirodesmin, and Aspergillus fumigatus, which produces gliotoxin. The L. maculans biosynthetic gene cluster for sirodesmin includes an ABC transporter gene, sirA. Disruption of this gene led to increased secretion of sirodesmin into the medium and an altered ratio of sirodesmin to its immediate precursor. The transcription pattern of a peptide synthetase that catalyses an early step in sirodesmin biosynthesis was elevated in the sirA mutant by 47% over a 7-day period. This was consistent with the finding that the transporter mutant had elevated sirodesmin levels. Despite increased production of sirodesmin, the sit-A mutant was more sensitive to both sirodesmin and gliotoxin. The putative gliotoxin transporter gene, gliA, (a major facilitator superfamily transporter) from A.fumigatus complemented the tolerance of the L. maculans sirA mutant to gliotoxin, but not to sirodesmin. The results indicate that SirA contributes to self-protection against sirodesmin in L. maculans and suggest a transporter other than SirA is primarily responsible for efflux of endogenously produced sirodesmin. (C) 2004 Elsevier Inc. All rights reserved.

Unidirectional inversion of ibuprofen in Caco-2 cells: Developing a suitable model for presystemic chiral inversion study

Intestinal chiral inversion of ibuprofen is still lacking direct evidence. In a preliminary experiment, ibuprofen was found to undergo inversion in Caco-2 cells. This investigation was thus conducted to determine the characteristics and influence of some biochemical factors on the chiral inversion of ibuprofen in Caco-2 cells. The effects of substrate concentration (2.5-40 mu g/ml), cell density (0.5-2 x 10(6) cells/ well), content of serum (0-20%), coexistence of S ibuprofen (corresponding doses), sodium azide (10mm), exogenous Coenzyme A (CoA) (0.1 - 0.4 mm),. and palmitic acid (5-25 mu m) on inversion were examined. A stereoselective HPLC method based on the Chromasil-CHI-TBB column was developed for quantitative analysis of the drug in cell culture medium. The inversion ratio (F-i) and elimination rate constant were calculated as the indexes of inversion extent. Inversion of ibuprofen in Caeo-2 cells was found to be both dose and cell density dependent, indicating saturable characteristics. Addition of serum significantly inhibited the inversion, to an extent of 2.7 fold decrease at 20% content. Preexistence of S enantiomer exerted a significant inhibitory effect (p < 0.01 for all tests). Sodium azide decreased the inversion ratio from 0.43 to 0.32 (p < 0.01). Exogenous CoA and palmitic acid significantly promoted the inversion at all tested doses (p < 0.01 for all tests). This research provided strong evidence to the capacity and capability of intestinal chiral inversion. Although long incubation times up to 120 h were required, Caco-2 cells should be a suitable model for chiral inversion research of 2-APAs considering the human-resourced and well-defined characteristics from the present study.

Carbamyl phosphate synthase deficiency: Diagnosed during pregnancy in a 41-year-old

Carbamyl phosphate synthase deficiency (CPS) is a rare urea cycle defect. We present a case of a 41-year-old woman diagnosed with CPS deficiency during pregnancy. She is the oldest CPS-deficient patient, at diagnosis, reported to date and the first to be diagnosed during pregnancy. This case highlights the need for consideration of inborn errors of metabolism in adults presenting with unusual neurological and psychiatric conditions. Crown Copyright (c) 2006 Published by Elsevier Ltd. All rights reserved.

Interpretation of the temperature dependence of equilibrium and rate constants

The objective of this review is to draw attention to potential pitfalls in attempts to glean mechanistic information from the magnitudes of standard enthalpies and entropies derived from the temperature dependence of equilibrium and rate constants for protein interactions. Problems arise because the minimalist model that suffices to describe the energy differences between initial and final states usually comprises a set of linked equilibria, each of which is characterized by its own energetics. For example, because the overall standard enthalpy is a composite of those individual values, a positive magnitude for AHO can still arise despite all reactions within the subset being characterized by negative enthalpy changes: designation of the reaction as being entropy driven is thus equivocal. An experimenter must always bear in mind the fact that any mechanistic interpretation of the magnitudes of thermodynamic parameters refers to the reaction model rather than the experimental system For the same reason there is little point in subjecting the temperature dependence of rate constants for protein interactions to transition-state analysis. If comparisons with reported values of standard enthalpy and entropy of activation are needed, they are readily calculated from the empirical Arrhenius parameters. Copyright (c) 2006 John Wiley & Sons, Ltd.

Studies on the mechanism of action on antitumour imidazotetrazinones

The imidazotetrazinones are clinically active antitumour agents, temozolomide currently proving successful in the treatment of melanomas and gliomas. The exact nature of the biological processes underlying response are as yet unclear.This thesis attempts to identify the cellular targets important to the cytotoxicity of imidazotetrazinones, to elucidate the pathways by which this damage leads to cell death, and to identify mechanisms by which tumour cells may circumvent this action. The levels of the DNA repair enzymes O6-alkylguanine-DNA-alkyltransferase (O6-AGAT) and 3-methyladenine-DNA-glycosylase (3MAG) have been examined in a range of murine and human cell lines with differential sensitivity to temozolomide. All the cell lines were proficient in 3MAG despite there being 40-fold difference in sensitivity to temozolomide. This suggests that while 3-methyladenine is a major product of temozolomide alkylation of DNA it is unlikely to be a cytotoxic lesion. In contrast, there was a 20-fold variation in O6-AGAT levels and the concentration of this repair enzyme correlated with variations in cytotoxicity. Furthermore, depletion of this enzyme in a resistant, O6-AGAT proficient cell line (Raji), by pre-treatment with the free base O6-methylguanine resulted in 54% sensitisation to the effects of temozolomide. These observations have been extended to 3 glioma cell lines; results that support the view that the cytotoxicity of temozolomide is related to alkylation at the O6-position of guanine and that resistance to this drug is determined by efficient repair of this lesion. It is clear, however, the other factors may influence tumour response since temozolomide showed little differential activity towards 3 established solid murine tumours in vivo, despite different tumour O6-AGAT levels. Unlike mitozolomide, temozolomide is incapable of cross-linking DNA and a mechanism by which O6-methylguanine may exert lethality is unclear. The cytotoxicity of the methyl group may be due to its disruption of DNA-protein interactions, or alternatively cell death may not be a direct result of the alkyl group itself, but manifested by DNA single-strand breaks. Enhanced alkaline elution rates were found for the DNA of Raji cells treated with temozolomide following alkyltransferase depletion, suggesting a relationship between O6-methylguanine and the induction single-strand breaks. Such breaks can activate poly(ADP-ribose) synthetase (ADPRT) an enzyme capable of rapid and lethal depletion of cellular NAD levels. However, at concentrations of temozolomlde relevant in vivo little change in adenine nucleotides was detected in cell lines, although this enzyme would appear important in modulating DNA repair since inhibition of ADPRT potentiated temozolomide cytotoxicity in Raji cells but not O6-AGAT deficient GM892A cells. Cell lines have been reported that are O6-AGAT deficient yet resistant to methylating agents. Thus, resistance to temozolomide may arise not only by removal of the methyl group from the O6-position of guanine, but also from another mechanism involving caffeine-sensitive post-replication repair or mismatch repair activity. A modification of the standard Maxam Gilbert sequencing technique was used to determine the sequence specificity of guanine-N7 alkylation. Temozolomide preferentially alkylated runs of guanines with the intensity of reaction increasing with the number of adjacent guanines in the DNA sequence. Comparable results were obtained with a polymerase-stop assay, although neither technique elucidates the sequence specificity of O6-guanine alkylation. The importance of such specificity to cytotoxicity is uncertain, although guanine-rich sequences are common to the promoter regions of oncogenes. Expression of a plasmid reporter gene under the control of the Ha-ras proto~oncogene promoter was inhibited by alkylation with temozolomide when transfected into cancer cell lines, However, this inhibition did not appear to be related to O6~guanine alkylation and therefore would seem unimportant to the chemotherapeutic activity of temozolomide.

Oxidative catabolism of tetrahydropterins

Mixed labelled folic acid was administerd to rats. Exposure to N2O was used to give an insight into the major route of scission within the monoglutamate pool, results suggest that THF formed during transport from the gut lumen to the plasma is the major route of scission within the gut. Peroxides in corn oil and arising as a result of lipid peroxidation and autoxidation increase catabolism of the monoglutamate pool and decrease incorporation of administered folates into the polyglutamate pool. It is suggested that peroxides may oxidise B12 resulting in inhibition of methionine synthetase, this results in diminished polyglutamation and increased urinary excretion of 5 CH3THF. Fats undergo peroxidation within tissues, the resulting peroxides increase catabolism of the polyglutamate pool. It is suggested that the NBT assay may reflect polyglutamate breakdown. Antioxidants such as vitamin E (and DES) decrease catabolism of the monoglutamate pool. Administration of DES resulted in changes similar to those observed during malignancy, it is suggested that these changes may precede the onset of tumour development. Vitamin E elevates brain DHPR activity. Since lowered DHPR levels and disturbed THB metabolism have been observed in aging and Down's syndrome it is proposed that vitamin E therapy may prove beneficial in situations where oxidative stress is increased. Brain DHPR activity was increased on administration of peroxides suggesting that in situations of oxidative stress (which may result in increased catabolism of THB) the salvage pathway may be stimulated and loss of THB minimised. N2O exposure had no effect on THB metabolism suggesting that the stimulatory role of 5 CH3THF is due to its role as a methyl donor.

The in vivo susceptibility of Leishmania donovani to sodium stibogluconate is drug specific and can be reversed by inhibiting glutathione biosynthesis

Resistance to pentavallent antimonial (Sb-v) agents such as sodium stibogluconate (SSG) is creating a major problem in the treatment of visceral leishmaniasis. In the present study the in vivo susceptibilities of Leishmania donovani strains, typed as SSG resistant (strain 200011) or SSG sensitive (strain 200016) on the basis of their responses to a single SSG dose of 300 mg of Sb-v/kg of body weight, to other antileishmanial drugs were determined. In addition, the role of glutathione in SSG resistance was investigated by determining the influence on SSG treatment of concomitant treatment with a nonionic surfactant vesicle formulation of buthionine sulfoximine (BSO), a specific inhibitor of the enzyme gamma-glutamylcysteine synthetase which is involved in glutathione biosynthesis, and SSG, on the efficacy of SSG treatment. L. donovani strains that were SSG resistant (strain 200011) and SSG sensitive (strain 200016) were equally susceptible to in vivo treatment with miltefosine, paromomycin and amphotericin B (Fungizone and AmBisome) formulations. Combined treatment with SSG and vesicular BSO significantly increased the in vivo efficacy of SSG against both the 200011 and the 200016 L. donovani strains. However, joint treatment that included high SSG doses was unexpectedly associated with toxicity. Measurement of glutathione levels in the spleens and livers of treated mice showed that the ability of the combined therapy to inhibit glutathione levels was also dependent on the SSG dose used and that the combined treatment exhibited organ-dependent effects. The SSG resistance exhibited by the L. donovani strains was not associated with cross-resistance to other classes of compounds and could be reversed by treatment with an inhibitor of glutathione biosynthesis, indicating that clinical resistance to antimonial drugs should not affect the antileishmanial efficacies of alternative drugs. In addition, it should be possible to identify a treatment regimen that could reverse antimony resistance.

Absolute configuration and biosynthesis of pahayokolide A from Lyngbya sp. Strain 15-2 of the Florida Everglades

Pahayokolides A-D are cytotoxic cyclic polypeptides produced by the freshwater cyanobacterium Lyngbya sp. strain 15-2 that possess an unusual β-amino acid, 3-amino-2,5,7,8-tetrahydroxy-10-methylundecanoic acid (Athmu). The absolute configuration of pahayokolides A-D was determined using advanced Marfey’s method. It was also confirmed that a pendant N-acetyl- N-methyl leucine moiety in pahayokolide A was absent in pahayokolides B and pahayokolides C-D were conformers of pahayokolide A. Feeding experiments indicated that the biosynthesis of the Athmu sidechain arises from leucine or α-ketoisovalerate, however could not be further extended by three rounds of condensation with malonate units. Putative four peptide and one unique polyketide synthetases in Lyngbya sp. strain 15-2 were identified by using a PCR method and degenerate primers derived from conserved core sequences of known NRPSs and PKSs. Identification of one unique KS domain conflicted with the logic rule that the long side chain of Athmu was assembled by three rounds of ketide extensions if PKSs were involved. A gene cluster (pah) encoding a peptide synthetase putatively producing pahayokolide was cloned, partially sequenced and characterized. Seven modules of the non-ribosomal peptide synthetase (NRPS) were identified. Ten additional opening reading frames (ORFs) were found, responsible for peptide resistance, transport and degradation. Although the predicted substrate specificities of NRPS agreed with the structure of pahayokolide A partially, the disagreement could be explained. However, no PKS gene was found in the pah gene cluster.

Absolute Configuration and Biosynthesis of Pahayokolide A from Lyngbya sp. Strain 15-2 of the Florida Everglades

Pahayokolides A-D are cytotoxic cyclic polypeptides produced by the freshwater cyanobacterium Lyngbya sp. strain 15-2 that possess an unusual β-amino acid, 3-amino-2,5,7,8-tetrahydroxy-10-methylundecanoic acid (Athmu). The absolute configuration of pahayokolides A-D was determined using advanced Marfey’s method. It was also confirmed that a pendant N-acetyl-N-methyl leucine moiety in pahayokolide A was absent in pahayokolides B and pahayokolides C-D were conformers of pahayokolide A. Feeding experiments indicated that the biosynthesis of the Athmu sidechain arises from leucine or α-ketoisovalerate, however could not be further extended by three rounds of condensation with malonate units. Putative four peptide and one unique polyketide synthetases in Lyngbya sp. strain 15-2 were identified by using a PCR method and degenerate primers derived from conserved core sequences of known NRPSs and PKSs. Identification of one unique KS domain conflicted with the logic rule that the long side chain of Athmu was assembled by three rounds of ketide extensions if PKSs were involved. A gene cluster (pah) encoding a peptide synthetase putatively producing pahayokolide was cloned, partially sequenced and characterized. Seven modules of the non-ribosomal peptide synthetase (NRPS) were identified. Ten additional opening reading frames (ORFs) were found, responsible for peptide resistance, transport and degradation. Although the predicted substrate specificities of NRPS agreed with the structure of pahayokolide A partially, the disagreement could be explained. However, no PKS gene was found in the pah gene cluster.

Potencial fisiológico de sementes de soja enriquecidas com molibdênio no armazenamento e sua influência na atividade de enzimas do metabolismo do nitrogênio

Molybdenum is one of the essential micronutrients for soybeans, acting directly on nitrogen metabolism as enzyme cofactor of nitrogenase. Usually, this nutrient is supplied to the plants through seed treatment or foliar application. The aim of this study was to evaluate the molybdenum effects by foliar in the physiological potential of soybean seeds and verify its interference in the enzyme activities involved in nitrogen metabolism. Soybean seeds of BMX Turbo cultivar were used, produced in Erechim, RS, harvest 2013, from plants treated with the following Mo concentrations: 0; 25; 50 and 75 g ha-1, supplied through two commercial products (Biomol and Molybdate) and stored during 0 and 6 months in uncontrolled conditions. The first experiment was conducted in Seedtes Seed Analysis Laboratory in Pato Branco, PR. The used design was completely randomized in a factorial analysis 4 x 2 x 2 with four replications each. The physiological potential of the seeds was evaluated by the germination test, seedling growth, accelerated aging and emergence on the soil. The second experiment was conducted in a greenhouse, where the seeds derived from treatments with different concentrations of Mo: 0; 25; 50 and 75 g ha-1 supplied through two commercial products (Biomol and Molybdate) were grown in vases. The used design was completely randomized in a factorial analysis 4 x 2 with four replications. Evaluations were performed when the plants reached the R1 phenological stage concerning the nodulation, dry matter of root and shoot of the plants and the determination of the activity of the enzymes glutamine synthetase and glutamate synthetase and the content of total soluble proteins. The data were submitted to variance analysis and when significant they were assessed by Tukey’s test for comparison of products and seed storage and with regression study to the concentrations at 5% probability. Analyses were performed using SISVAR statistical software. The soybean seed storage under uncontrolled conditions affected the seed vigour produced with Mo, regardless of the commercial product used during production. The application of Mo through foliar positively influences the production of soya beans which presented increasing responses in the germination and vigour with the application of Mo above 25 g ha-1 . The enrichment of Mo through foliar did not affect the nodulation of plants of the next generation, however, the use of Mo above 25 g ha-1 provided an increase in the activity of enzymes involved in nitrogen metabolism as well as on the total protein content.

Proteomic and phosphoproteomic analysis of polyethylene glycol-induced osmotic stress in root tips of common bean (Phaseolus vulgaris L.)

Previous studies have shown that polyethylene glycol (PEG)-induced osmotic stress (OS) reduces cell-wall (CW) porosity and limits aluminium (Al) uptake by root tips of common bean (Phaseolus vulgaris L.). A subsequent transcriptomic study suggested that genes related to CW processes are involved in adjustment to OS. In this study, a proteomic and phosphoproteomic approach was applied to identify OS-induced protein regulation to further improve our understanding of how OS affects Al accumulation. Analysis of total soluble proteins in root tips indicated that, in total, 22 proteins were differentially regulated by OS; these proteins were functionally categorized. Seventy-seven per- cent of the total expressed proteins were involved in metabolic pathways, particularly of carbohydrate and amino acid metabolism. An analysis of the apoplastic proteome revealed that OS reduced the level of five proteins and increased that of seven proteins. Investigation of the total soluble phosphoproteome suggested that dehydrin responded to OS with an enhanced phosphorylation state without a change in abundance. A cellular immunolocalization analysis indicated that dehydrin was localized mainly in the CW. This suggests that dehydrin may play a major protective role in the OS-induced physical breakdown of the CW structure and thus maintenance of the reversibility of CW extensibility during recovery from OS. The proteomic and phosphoproteomic analyses provided novel insights into the complex mechanisms of OS-induced reduction of Al accumulation in the root tips of common bean and highlight a key role for modification of CW structure.

Biosynthesis and mode of action of ribosomally synthesized and post-translationally modified antimicrobial peptides

One of the greatest sources of biologically active compounds is natural products. Often these compounds serve as platforms for the design and development of novel drugs and therapeutics. The overwhelming amount of genomic information acquired in recent years has revealed that ribosomally synthesized and post-translationally modified natural products are much more widespread than originally anticipated. Identified in nearly all forms of life, these natural products display incredible structural diversity and possess a wide range of biological functions that include antimicrobial, antiviral, anti-inflammatory, antitumor, and antiallodynic activities. The unique pathways taken to biosynthesize these compounds offer exciting opportunities for the bioengineering of these complex molecules. The studies described herein focus on both the mode of action and biosynthesis of antimicrobial peptides. In Chapter 2, it is demonstrated that haloduracin, a recently discovered two-peptide lantibiotic, possesses nanomolar antimicrobial activity against a panel of bacteria strains. The potency of haloduracin rivals that of nisin, an economically and therapeutically relevant lantibiotic, which can be attributed to a similar dual mode of action. Moreover, it was demonstrated that this lantibiotic of alkaliphile origin has better stability at physiological pH than nisin. The molecular target of haloduracin was identified as the cell wall peptidoglycan precursor lipid II. Through the in vitro biosynthesis of haloduracin, several analogues of Halα were prepared and evaluated for their ability to inhibit peptidoglycan biosynthesis as well as bacterial cell growth. In an effort to overcome the limitations of in vitro biosynthesis strategies, a novel strategy was developed resulting in a constitutively active lantibiotic synthetase enzyme. This methodology, described in Chapter 3, enabled the production of fully-modified lacticin 481 products with proteinogenic and non-proteinogenic amino acid substitutions. A number of lacticin 481 analogues were prepared and their antimicrobial activity and ability to bind lipid II was assessed. Moreover, site-directed mutagenesis of the constitutively active synthetase resulted in a kinase-like enzyme with the ability to phosphorylate a number of peptide substrates. The hunt for a lantibiotic synthetase enzyme responsible for installing the presumed dehydro amino acids and a thioether ring in the natural product sublancin, led to the identification and characterization of a unique post-translational modification. The studies described in Chapter 4, demonstrate that sublancin is not a lantibiotic, but rather an unusual S-linked glycopeptide. Its structure was revised based on extensive chemical, biochemical, and spectroscopic characterization. In addition to structural investigation, bioinformatic analysis of the sublancin gene cluster led to the identification of an S-glycosyltransferase predicted to be responsible for the post-translational modification of the sublancin precursor peptide. The unprecedented glycosyltransferase was reconstituted in vitro and demonstrated remarkable substrate promiscuity for both the NDP-sugar co-substrate as well as the precursor peptide itself. An in vitro method was developed for the production of sublancin and analogues which were subsequently evaluated in bioactivity assays. Finally, a number of putative biosynthetic gene clusters were identified that appear to harbor the necessary genes for production of an S-glycopeptide. An additional S-glycosyltransferase with more favorable intrinsic properties including better expression, stability, and solubility was reconstituted in vitro and demonstrated robust catalytic abilities.

MECHANISM OF UPREGULATION OF PHOSPHATIDYLCHOLINE SYNTHESIS DURING PICORNAVIRUS INFECTION AND ITS ROLE IN THE DEVELOPMENT OF VIRAL REPLICATION ORGANELLES

Picornaviruses are a group of human and animal pathogens capable of inflicting serious public health diseases and economic burdens. Treatments options through vaccines for prevention or antivirals to cure infection are not available for the vast majority of these viruses. These shortcomings, in the development of vaccines or antivirals therapeutic, are linked to the genetic diversity and to an incomplete understanding of the biology of these viruses. Despite the diverse host range, this group of positive-strand RNA viruses shares the same replication mechanisms, including the development of membranous structures (replication organelles) in the cytoplasm of infected cells. The development of these membranous structures, which serve as sites for the replication of the viral RNA genome, has been linked to the hijacking of elements of the cellular membrane metabolism pathways. Here we show that upon picornavirus infection, there is a specific activation of acyl-CoA synthetase enzymes resulting in strong import and accumulation of long chain fatty acids in the cytoplasm of infected cells. We show that the newly imported fatty acids serve as a substrate for the upregulation of phosphatidylcholine synthesis required for the structural development of replication organelles. In this work, we identified that acyl-CoA synthetase long chain 3 (ACSL3) is required for the upregulation of lipids syntheses and the replication of poliovirus. We have shown that the poliovirus protein 2A was required but not sufficient for the activation of import of long chain fatty acids in infected cells. We demonstrated that the fatty acid import is upregulated upon infection by diverse picornaviruses and that such upregulation is not dependent on activation of ER stress response or the autophagy pathways. In this work, we have demonstrated that phosphatidylcholine was required for the structural development of replication organelles. Phosphatidylcholine synthesis was dispensable for the production of infectious particles at high MOI but required at a low MOI for the protection of the replication complexes from the cellular innate immunity mechanisms.

Potential protective role of reactive astrocytes in the periventricular parenchyma in congenital hydrocephalus

Background Cerebrospinal fluid accumulation in hydrocephalus produces an elevation of intraventricular pressure with pathological consequences on the periventricular brain parenchyma including ischemia, oedema, oxidative stress, and accumulation of metabolic waste products. Here we studied in the hyh mouse, an animal model of congenital hydrocephalus, the role of reactive astrocytes in this clinical degenerative condition. Materials and Methods Wild type and hydrocephalic hyh mice at 30 days of postnatal age were used. Three metabolites related to the oxidative and neurotoxic conditions were analysed in ex vivo samples (glutathione, glutamine and taurine) using High Resolution Magic Angle Spinning (HR-MAS). Glutathione synthetase and peroxidase, glutamine synthetase, kidney-type glutaminase (KGA), and taurine/taurine transporter were immunolocated in brain sections. Results Levels of the metabolites were remarkably higher in hydrocephalic conditions. Glutathione peroxidase and synthetase were both detected in the periventricular reactive astrocytes and neurons. Taurine was mostly found free in the periventricular parenchyma and in the reactive astrocytes, and the taurine transporter was mainly present in the neurons located in such regions. Glutamine synthetase was found in reactive astrocytes. Glutaminase was also detected in the reactive astrocytes and in periventricular neurons. These results suggest a possible protective response of reactive astrocytes against oxidative stress and neurotoxic conditions. Conclusions Astrocyte reaction seems to trigger an anti-oxidative and anti-neurotoxic response in order to ameliorate pathological damage in periventricular areas of the hydrocephalic mice.