Gender-dependent cellular and biochemical effects of maternal deprivation on the hippocampus of neonatal rats: a possible role for the endocannabinoid system

Adult animals submitted to a single prolonged episode of maternal deprivation (MD) [24 h, postnatal days (PND) 9-10] show behavioral alterations that resemble specific symptoms of schizophrenia. These behavioral impairments may be related to neuronal loss in the hippocampus triggered by elevated glucocorticoids. Furthermore, our previous data suggested functional relationships between MD stress and the endocannabinoid system. In this study, we addressed the effects of MD on hippocampal glial cells and the possible relationship with changes in plasma corticosterone (CORT) levels. In addition, we investigated the putative involvement of the endocannabinoid system by evaluating (a) the effects of MD on hippocampal levels of endocannabinoids (b) The modulation of MD effects by two inhibitors of endocannabinoids inactivation, the fatty acid amide hydrolase inhibitor N-arachidonoyl-serotonin (AA-5-HT), and the endocannabinoid reuptake inhibitor, OMDM-2. Drug treatments were administered once daily from PND 7 to PND 12 at a dose of 5 mg/kg, and the animals were sacrificed at PND 13. MD induced increased CORT levels in both genders. MD males also showed an increased number of astrocytes in CA1 and CA3 areas and a significant increase in hippocampal 2-arachidonoylglycerol. The cannabinoid compounds reversed the endocrine and cellular effects of maternal deprivation. We provide direct evidence for gender-dependent cellular and biochemical effects of MD on developmental hippocampus, including changes in the endocannabinoid system.     

Salicyl alcohol oxidase of the chemical defense secretion of two chrysomelid leaf beetles. Molecular and functional characterization of two new members of the glucose-methanol-choline oxidoreductase gene family

Salicyl alcohol oxidase is an extracellular enzyme that occurs in glandular reservoirs of chrysomelid leaf beetle larvae and catalyzes the formation of salicylaldehyde, a volatile deterrent used by the larvae against predators. Salicyl alcohol is the hydrolysis product of salicin, a plant-derived precursor taken up by the beetle larvae from the leaves of willow and poplar trees. The cDNA encoding salicyl alcohol oxidase from two related species Chrysomela tremulae and Chrysomela populi has been identified, cloned, and expressed in an active form in Escherichia coli. The open reading frame of 623 amino acids begins in both enzymes with an N-terminal signal peptide of 21 amino acids. Sequence comparison has revealed that salicyl alcohol oxidase belongs to the family of glucose-methanol-choline oxidoreductase-like sequences with mostly unknown function. Enzymes of this family share similar overall structure with an essentially identical FAD-binding site but possess different catalytic activities. The data suggest that salicyl alcohol oxidase, essential for the activation of the plant-derived precursor salicin, was originally recruited from an oxidase involved in the autogenous biosynthesis of iridoid monoterpenes and found in related chrysomelid leaf beetle species.     

E1a gene expression blocks the ERK1/2 signaling pathway by promoting nuclear localization and MKP up-regulation: implication in v-H-Ras-induced senescence

In response to oncogenic signals, cells have developed safe mechanisms to avoid transformation through activation of a senescence program. Upon v-H-Ras overexpression, normal cells undergo senescence through several cellular processes, including activation of the ERK1/2 pathway. Interestingly, the E1a gene from adenovirus 5 has been shown to rescue cells from senescence by a yet unknown mechanism. We investigated whether E1a was able to interfere with the ERK1/2 signaling pathway to rescue cells from v-H-Ras-mediated senescence. Our results show that, E1a overexpression blocks v-H-Ras-mediated ERK1/2 activation by two different and concomitant mechanisms. E1a through its ability to interfere with PKB/Akt activation induces the down-regulation of the PEA15 protein, an ERK1/2 nuclear export factor, leading to nuclear accumulation of ERK1/2. In addition to this, we show that E1a increases the expression of the inducible ERK1/2 nuclear phosphatases (MAPK phosphatases) MKP1/DUSP1 and DUSP5, which leads to ERK1/2 dephosphorylation. We confirmed our observations in the human normal diploid fibroblasts IMR90, in which we could also show that an E1a mutant, unable to bind retinoblastoma protein (pRb), cannot rescue cells from v-H-Ras-induced senescence. In conclusion, E1a is able to rescue from Ras-induced senescence by affecting ERK1/2 localization and phosphorylation.     

Complement factor H binds to denatured rather than to native pentameric C-reactive protein

Binding of the complement regulatory protein, factor H, to C-reactive protein has been reported and implicated as the biological basis for association of the H402 polymorphic variant of factor H with macular degeneration. Published studies utilize solid-phase or fluid-phase binding assays to show that the factor H Y402 variant binds C-reactive protein more strongly than H402. Diminished binding of H402 variant to C-reactive protein in retinal drusen is posited to permit increased complement activation, driving inflammation and pathology. We used well validated native human C-reactive protein and pure factor H Y402H variants to test interactions. When factor H variants were incubated with C-reactive protein in the fluid phase at physiological concentrations, no association occurred. When C-reactive protein was immobilized on plastic, either non-specifically by adsorption in the presence of Ca(2+) to maintain its native fold and pentameric subunit assembly or by specific Ca(2+)-dependent binding to immobilized natural ligands, no specific binding of either factor H variant from the fluid phase was observed. In contrast, both factor H variants reproducibly bound to C-reactive protein immobilized in the absence of Ca(2+), conditions that destabilize the native fold and pentameric assembly. Both factor H variants strongly bound C-reactive protein that was denatured by heat treatment before immobilization, confirming interaction with denatured but not native C-reactive protein. We conclude that the reported binding of factor H to C-reactive protein results from denaturation of the C-reactive protein during immobilization. Differential binding to C-reactive protein, thus, does not explain association of the Y402H polymorphism with macular degeneration.     

The anti-fibrotic effect of liver growth factor is associated with decreased intrahepatic levels of matrix metalloproteinases 2 and 9 and transforming growth factor beta 1 in bile duct-ligated rats

Liver growth factor (LGF), a mitogen for liver cells, behaves as an anti-fibrotic agent even in extrahepatic sites, but its mechanistic basis is unknown. We aimed to determine the intrahepatic expression pattern of key modulators of liver fibrosis in bile duct-ligated rats (BDL) after injection of LGF. BDL rats received either LGF (4.5 microg/ratXdose, two doses/week, at time 0 or 2 or 5w after operation, depending on the group (BDL+LGF groups, n=20) or saline (BDL+S groups, n=20). Groups were compared in terms of fibrosis (histomorphometry), liver function (aminopyrine breath test), matrix metalloproteinases MMP-2 and MMP-9, transforming growth factor beta 1 (TGF-beta1) and liver endoglin content (Western blotting), and serum tissue inhibitor of metalloproteinases 1 (TIMP-1) levels (ELISA). In BDL+LGF rats, the fibrotic index was significantly lower at 5w, p=0.006, and at 8w, p=0.04, than in BDL+S rats. Liver function values in BDL+LGF rats were higher than those obtained in BDL+S rats (80% at 5w and 79% at 8w, versus 38% and 29%, p<0.01, taking healthy controls as 100%). Notably, in BDL+LGF rats the intrahepatic expression levels of both MMPs were lower at 2w (MMP-2, p=0.03; MMP-9, p=0.05) and 5w (MMP-2, p=0.05, MMP-9, p=0.04). In addition, the hepatic TGF-beta1 level in BDL+LGF rats was lower at 2w (36%, p=0.008), 5w (50%) and 8wk (37%), whereas intrahepatic endoglin expression remained constant in all BDL rats studied. LGF ameliorates liver fibrosis and improves liver function in BDL rats. The LGF-induced anti-fibrotic effect is associated with a decreased hepatic level of MMP-2, MMP-9 and TGF-beta1 in fibrotic rats.     

Isolation of a novel chromium(III) binding protein from bovine liver tissue after chromium(VI) exposure

In the ongoing investigation into the biological importance and toxicity issues surrounding the bioinorganic chemistry of chromium, the accepted literature procedure for the isolation of the biological form of chromium, low molecular weight chromium binding protein (LMWCr) or chromodulin, was investigated for its specificity. When chromium(VI) is added to bovine liver homogenate, results presented here indicate at least four chromium(III) binding peptides and proteins are produced and that the process is non-specific for the isolation of LMWCr. A novel trivalent chromium containing protein (1) has been isolated to purity and initial characterization is reported here. Chromium(III) identification was determined by optical spectroscopy and diphenylcarbazide testing. This chromium binding protein has a molecular weight of 15.6kDa, which was determined from both gel-electrophoresis and mass spectrometry. The protein is comprised primarily of Asx, Glx, His, Gly/Thr, Ala, and Lys in a 1.00:2.51:0.37:2.09:0.39:1.17 ratio and is anionic at pH 7.4. In addition, the protein binds approximately 2.5 chromium(III) ions per molecule.     

Hematologic Values of Free-ranging Cebus cay and Cebus nigritus in Southern Brazil

We evaluated blood samples obtained from 80 free-ranging healthy capuchins (Cebus cay and C. nigritus) to establish hematological reference values and to assess the influence of sex and age on them. We caught the monkeys in the Paraná River region of Southern Brazil via manual or automatic traps. We anesthetized them intramuscularly with 3.6 mg/kg tiletamine/zolazepam hydrochlorides. After physical examinations, we divided the sample according to sex and age: 26 females (13 adults and 13 juveniles) and 54 males (27 adults and 27 juveniles). We collected blood and determined hematological values via traditional published methods. We analyzed data via 2-way ANOVA to test the effect of sex, age, and interactions between the 2 factors. The packed-cell volume was higher in adult males and the numbers of white blood cells and lymphocytes were higher in juveniles. There is no other significant difference.