Omega-3 fatty acids deprivation affects ontogeny of glutamatergic synapses in rats: Relevance for behavior alterations

Essential omega-3 polyunsaturated fatty acids (ω3) are crucial to brain development and function, being relevant for behavioral performance. In the present study we examined the influence of dietary ω3 in the development of the glutamatergic system and on behavior parameters in rats. Female rats received isocaloric diets, either with ω3 (ω3 group) or a ω3 deficient diet (D group). In ontogeny experiments of their litters, hippocampal immunocontent of ionotropic NMDA and AMPA glutamatergic receptors subunits (NR2 A\B and GluR1, respectively) and the alpha isoform of the calcium-calmodulin protein kinase type II (αCaMKII) were evaluated. Additionally, hippocampal [³H]glutamate binding and uptake were assessed. Behavioral performance was evaluated when the litters were adult (60 days old), through the open-field, plus-maze, inhibitory avoidance and flinch-jump tasks. The D group showed decreased immunocontent of all proteins analyzed at 02 days of life (P2) in comparison with the ω3 group, although the difference disappeared at 21 days of life (except for αCaMKII, which content normalized at 60 days old). The same pattern was found for [³H]glutamate binding, whereas [³H]glutamate uptake was not affected. The D group also showed memory deficits in the inhibitory avoidance, increased in the exploratory pattern in open-field, and anxiety-like behavior in plus-maze. Taken together, our results suggest that dietary ω3 content is relevant for glutamatergic system development and for behavioral performance in adulthood. The putative correlation among the neurochemical and behavioral alterations caused by dietary ω3 deficiency is discussed.     

Interactions Between Chronic Stress and Chronic Consumption of Caffeine on the Enzymatic Antioxidant System

We studied the effect of chronic caffeine on parameters related to oxidative stress in different brain regions of stressed and non-stressed rats. Wistar rats were divided into three groups: control (receiving water), caffeine 0.3 g/L and caffeine 1.0 g/L (in the drinking water). These groups were subdivided into non-stressed and stressed (repeated restraint stress during 40 days). Lipid peroxide levels and the total radical-trapping potential were assessed, as well as antioxidant enzyme activities superoxide dismutase, gluthatione peroxidase, and catalase in hippocampus, striatum and cerebral cortex. Results showed interactions between stress and caffeine, especially in the cerebral cortex, since caffeine increased the activity of some antioxidant enzymes, but not in stressed animals. We concluded that chronic administration of caffeine led, in some cases, to increased activity of antioxidant enzymes. However, these effects were not observed in the stressed animals.     

Therapeutic Efficacy of Cintredekin Besudotox (IL13-PE38QQR) in Murine Lung Fibrosis Is Unaffected by Immunity to Pseudomonas aeruginosa Exotoxin A

Background

We have previously explored a therapeutic strategy for specifically targeting the profibrotic activity of IL-13 during experimental pulmonary fibrosis using a fusion protein comprised of human IL-13 and a mutated form of Pseudomonas aeruginosa exotoxin A (IL13-PE) and observed that the intranasal delivery of IL13-PE reduced bleomycin-induced pulmonary fibrosis through its elimination of IL-13-responsive cells in the lung. The aim of the present study was to determine whether the presence of an immune response to P. aeruginosa and/or its exotoxin A (PE) would diminish the anti-fibrotic properties of IL13-PE.

Methodology/Principal Findings

Fourteen days after P. aeruginosa infection, C57BL/6 mice were injected with bleomycin via the intratracheal route. Other groups of mice received 4 doses of saline or IL13-PE by either intranasal or intraperitoneal application, and were challenged i.t. with bleomycin 28 days later. At day 21 after bleomycin, all mice received either saline vehicle or IL13-PE by the intranasal route and histopatological analyses of whole lung samples were performed at day 28 after bleomycin. Intrapulmonary P. aeruginosa infection promoted a neutralizing IgG2A and IgA antibody response in BALF and serum. Surprisingly, histological analysis showed that a prior P. aeruginosa infection attenuated the development of bleomycin-induced pulmonary fibrosis, which was modestly further attenuated by the intranasal administration of IL13-PE. Although prior intranasal administration of IL13-PE failed to elicit an antibody response, the systemic administration of IL13-PE induced a strong neutralizing antibody response. However, the prior systemic sensitization of mice with IL13-PE did not inhibit the anti-fibrotic effect of IL13-PE in fibrotic mice.

Conclusions

Thus, IL13-PE therapy in pulmonary fibrosis works regardless of the presence of a humoral immune response to Pseudomonas exotoxin A. Interestingly, a prior infection with P. aeruginosa markedly attenuated the pulmonary fibrotic response suggesting that the immune elicitation by this pathogen exerts anti-fibrotic effects.     

Light- and electron microscopic observations on parathyroid glands in different age groups of rats

The parathyroid hormone (PTH) acts on bones, intestines, and kidneys to maintain the calcium homeostasis which, in turn, is a main factor in controling the parathyroid (PT) gland activity. In all mammals studied, the chief cells of PT glands changed their size, shape, and cytoplasmic structure due to different functional states which vary the serum calcium levels. The chief cells of the rat PT glands were classified as dark and light. The dark cells may constitute an active form, characterized mainly by the abundant free ribosomes, conspicuous rough endoplasmic reticulum, and GOLGI complexes, greater number of secretory granules (SG) and increased tortuosity of the plasma membranes as compared to the light ones which were considered as a less active type of cells. Due to different calcium requirements in newborn and young rats for the ossification of growing skeleton and in adult and senile rats with consolidate mature bones, the PT glands studied with electron microscope showed various cytological features. The parenchyma of newborn and young PT glands was composed by dark chief cells. The light chief cells were more frequent in adult and senile animals as a less active type of cell. Mature SG were only occasionally observed in dark cells of newborn, young and adult PT glands. They may constitute a reserve supply of PTH but probably not the main way of secretion, according to their little number. Another pool of PTH probably answers the needs for the small basal variations in the steady-state secretion and may be represented by the vesicles observed in the chief cells cytoplasm.     

Hydroxyl Radicals and a Thylakoid-Bound Endopeptidase are Involved in Light- and Oxygen-Induced Proteolysis in Oat Chloroplasts

The hypothesis that light- and oxygen-induced proteolysis inchloroplasts is mediated by active oxygen species was examined.In order to determine whether or not H₂O₂ and/or ^{dot}OH radicalsare involved in these degradative processes we compared thedegradation of proteins in isolated oat chloroplasts exposedto white light at 80 W m^-2 with that in chloroplasts incubatedin darkness in the absence or presence of H₂O₂ or a ^{dot}OH-generatingsystem composed by ascorbic acid, FeCl₃ and H₂O₂ (Asc-Fe-H₂O₂).Light enhanced the rate of degradation of at least 18 polypeptides,while proteolysis was almost negligible in darkness in the abscenceof additives. H₂O₂ had a very small effect. However, Asc-Fe-H₂O₂-treatedchloroplasts in darkness showed a pattern of protein degradationalmost identical to that observed in the light. A thylakoid-boundendopeptidase (EP), the activity of which increased under photooxidativeenvironmental conditions and treatment with an ^{dot}OH-generatingsystem, was partially purified and characterized as a serinetypeprotease. Treatments with inhibitors of serine-type proteaseprevented both light- and Asc- Fe-H₂O₂-induced proteolysis.EP was more active against both soluble and membranous proteinsthat had been pretreated with Asc-Fe-H₂O₂ than against untreatedproteins. It is proposed that a high dose of light irradiationpromotes proteolysis by increasing the formation of ^{dot}OH,which may modify proteins such that they become more susceptibleto EP-catalyzed hydrolysis. ¹Fisiología Vegetal, Dept. de Biología Vegetal,Universidad de Alcalá de Henares, Present address: 28871Alcalá de Henares (Madrid), España.     

A chitin-like component in Aedes aegypti eggshells, eggs and ovaries

An insoluble white substance was prepared from extracts of eggshells of Aedes aegypti, the yellow fever mosquito and dengue vector. Its infrared and proton NMR spectra were similar to that of standard commercial chitin. This putative chitin-like material, also obtained from ovaries, newly laid and dark eggs, was hydrolyzed in acid and a major product was identified by HPLC to be glucosamine. The eggshell acid hydrolysate was also analyzed by ESI-MS and an ion identical to a glucosamine monoprotonated species was detected. The presence of chitin was also analyzed during different developmental stages of the ovary using a fluorescent microscopy technique and probes specific for chitin. The results showed that a chitin-like material accumulates in oocytes during oogenesis. Streptomyces griseus chitinase pre-treatment of oocytes greatly reduced the chitin-derived fluorescence. Chitinase activity was detected in newborn larvae and eggs prior to hatching. Feeding experiments indicated that the chitin synthesis inhibitor lufenuron inhibited chitin synthesis, either when mosquitoes were allowed to feed directly on lufenuron-treated chickens or when an artificial feeding system was used. Lufenuron inhibited egg hatch, larval development and reduced mosquito viability. These data demonstrate for the first time that (1) a chitin-like material is present in A. aegypti eggs, ovaries and eggshells; (2) a chitin synthesis inhibitor can be used to inhibit mosquito oogenesis; and (3) chitin synthesis inhibitors have potential for controlling mosquito populations.     

Immunologic memory response induced by a meningococcal serogroup C conjugate vaccine using the P64k recombinant protein as carrier

In this study, we used an adoptive lymphocyte transfer experiment to evaluate the ability of the P64k recombinant protein to recruit T-helper activity and induce immunologic memory response to the polysaccharide moiety in a meningococcal serogroup C conjugate vaccine. Adoptive transfer of splenocytes from mice immunized with the glycoconjugate conferred antipolysaccharide immunologic memory to naive recipient mice. The observed anamnestic immune response was characterized by more rapid kinetics, isotype switching from IgM to IgG and higher antipolysaccharide antibody titers compared with those reached in groups transferred with splenocytes from plain polysaccharide or phosphate-immunized mice. The memory response generated was also long lasting. Sera from mice transferred with cells from conjugate-immunized mice were the only protective in the infant rat passive protection assay, and also showed higher bactericidal titers. We demonstrated that priming the mice immune system with the glycoconjugate using the P64k protein as carrier induced a memory response to the polysaccharide, promoting a switch of the T-cell-independent response to a T-cell dependent one.     

Nalidixic acid-resistant V79 cells with reduced DNA topoisomerase II activity and amplification prone phenotype.

Spontaneously nalidixic acid-resistant lines (NAr lines) were selected from a V79 Chinese hamster cell line and phenotypically characterized. NAr lines showed an increased doubling time, a higher number of spontaneous SCE, and more interestingly, decreased DNA topoisomerase II activity. These lines were also cross-resistant to the eukaryotic topoisomerase II inhibitors etoposide and adriamycin, but showed the same level of sensitivity as the parental line to the DNA topoisomerase I inhibitor camptothecin. NAr lines were cross-resistant to other drugs, such as PALA, MTX and MPA, resistance to which has been shown to arise by amplification of the target genes. This last feature, together with enhanced cross-resistance to PALA and MTX when employed simultaneously, suggests that NAr lines have an 'amplification prone' phenotype. From these results the decreased activity of topoisomerase II seems to be involved in the generation of amplified sequences possibly by affecting recombinational events underlying gene amplification.     

A carbamate-based approach to primaquine prodrugs: antimalarial activity, chemical stability and enzymatic activation

O-Alkyl and O-aryl carbamate derivatives of the antimalarial drug primaquine were synthesised as potential prodrugs that prevent oxidative deamination to the inactive metabolite carboxyprimaquine. Both O-alkyl and O-aryl carbamates undergo hydrolysis in alkaline and pH 7.4 phosphate buffers to the parent drug, with O-aryl carbamates being ca. 10(6)-10(10) more reactive than their O-alkyl counterparts. In human plasma O-alkyl carbamates were stable, whereas in contrast their O-aryl counterparts rapidly released the corresponding phenol product, with primaquine being released only slowly over longer incubation periods. Activation of the O-aryl carbamates in human plasma appears to be catalysed by butyrylcholinesterase (BuChE), which leads to carbamoylation of the catalytic serine of the enzyme followed by subsequent slow enzyme reactivation and release of parent drug. Most of the O-aryl and O-alkyl carbamates are activated in rat liver homogenates with half-lives ranging from 9 to 15 h, while the 4-nitrophenyl carbamate was hydrolysed too rapidly to determine an accurate rate constant. Antimalarial activity was studied using a model consisting of Plasmodium berghei, Balb C mice and Anopheles stephensi mosquitoes. When compared to controls, ethyl and n-hexyl carbamates were able to significantly reduce the percentage of infected mosquitos as well as the mean number of oocysts per infected mosquito, thus indicating that O-alkyl carbamates of primaquine have the potential to be developed as transmission-blocking antimalarial agents.