3-Hydroxyglutaric acid induces oxidative stress and decreases the antioxidant defenses in cerebral cortex of young rats

Glutaryl-CoA dehydrogenase deficiency (GDD) is an inherited neurometabolic disorder biochemically characterized by tissue accumulation of glutaric, 3-hydroxyglutaric (3-OHGA) and glutaconic acids and clinically by severe neurological symptoms and cerebral atrophy whose pathophysiology is poorly known. In the present study we investigated the effect of 3-OHGA, considered the main neurotoxin in GDD, on the lipoperoxidation parameters chemiluminescence and thiobarbituric acid-reactive species (TBA-RS), and on the amount of nitric oxide metabolites in cerebral cortex of young rats. Total radical-trapping antioxidant potential (TRAP), which reflects the tissue antioxidant defenses, was also examined. We observed that 3-OHGA significantly increased chemiluminescence, TBA-RS and nitric oxide metabolites, in contrast to TRAP, which was decreased by the metabolite. The data indicate a stimulation of lipid peroxidation and free radical production, and a reduction of the tissue antioxidant defenses caused by the metabolite. In case these findings also occur in the human condition, it may be presumed that oxidative stress is involved in the brain damage observed in GDD.     

Correlation between CGRP immunoreactivity and firing activity in cat abducens motoneurons

Intercellular communication through gap junction channels is a prominent feature of the developing cerebral cortex. In the first 2 weeks after birth, a time critical in the development of the rat neocortex, extensive cell coupling has been documented that diminishes as the cortex matures. Among the family of gap junction proteins, connexins 26, 36, and 43 are differentially expressed during cortical development. We used intracellular dye injections and connexin immunohistochemistry to investigate the coupling patterns and connexin expression between the different neuronal and glial cell types of the developing cortex of the rat. We found that neurons and glia couple homotypically and heterotypically at postnatal days 7 and 14. Although the prevalence of coupling was homotypic, there was considerable heterotypic coupling that involved pyramidal and nonpyramidal neurons, the principal neuronal cell types of the cortex, or neurons and astrocytes. Coupling between different cell types appeared to be mediated by differential expression of connexins 26, 36, and 43. It may be that coupling between cells in the developing neocortex is a function of the spatial and temporal expression of these and other connexin proteins.     

The hemostatic agent ethamsylate enhances P-selectin membrane expression in human platelets and cultured endothelial cells

Ethamsylate possesses antihemorrhagic properties, but whether or not it directly activates blood platelets is unclear. Here we investigated the platelet activation potential of ethamsylate, by measuring membrane P-selectin expression with flow cytometry in human whole blood and also by immunofluorescence imaging of isolated human platelets. Moreover, we measured membrane P-selectin expression in the SV40-transformed aortic rat endothelial cell line (SVAREC) and ¹⁴C-ethamsylate membrane binding and/or uptake in platelets and endothelial cells. Whole blood flow cytometry showed a modest, but statistically significant increase by ethamsylate in the percentage of platelets expressing P-selectin (from 2% to 4–5%, p<0.05). Immunofluorescence showed a sizable (39%) and significant (p<0.01) enhancement of P-selectin expression at the lowest concentration of ethamsylate tested (1 μM), with maximal enhancement of P-selectin expression (75–90%) at 10 μM ethamsylate. Similar results were obtained in SVAREC endothelial cells. ¹⁴C-ethamsylate specifically bound to platelets and endothelial cell membranes, without significant uptake into the cell interior. In conclusion, ethamsylate enhances membrane P-selectin expression in human platelets and in cultured endothelial cells. Ethamsylate specifically binds to some protein receptor in platelet and endothelial cell membranes, receptor which can signal for membrane P-selectin expression. These results support the view that ethamsylate acts on the first step of hemostasis, by improving platelet adhesiveness and restoring capillary resistance.     

Hypokalemic periodic paralysis as first manifestation of hyperthyroidism: case report

We report a case of thyrotoxicosis in a 37 year old male, in whom hypokalemic periodic paralysis was the first manifestation of the disease. We comment about the differential diagnosis with other types of periodic paralysis, and the importance of a correct diagnosis to avoid permanent muscle damage.     

Microcin E492, a channel-forming bacteriocin from Klebsiella pneumoniae, induces apoptosis in some human cell lines

The cytotoxic effect of microcin E492, a low-molecular-mass channel-forming bacteriocin (7,887 Da) produced by a strain of Klebsiella pneumoniae, was characterized in HeLa cells. At low (5 microg/ml) and intermediate (10 microg/ml) concentrations, microcin E492 induced biochemical and morphological changes typical of apoptosis, such as cell shrinkage, DNA fragmentation, extracellular exposure of phosphatidylserine, caspase activation, and loss of mitochondrial membrane potential. Treatment with zVAD-fmk, a general caspase inhibitor, completely blocked the cytotoxic effect of this bacteriocin. At higher microcin concentrations (>20 microg/ml) a necrotic phenotype was observed. Induction of apoptosis by microcin E492 was associated with the release of calcium from intracellular stores, probably after microcin-triggered ion channel formation. Microcin E492 also presented a cytotoxic effect on Jurkat and RJ2.25 cells, but had no effect on KG-1 cells nor on a primary culture of human tonsil endothelial cells, suggesting that there is a specific interaction of the bacteriocin with components of the target cell surface. This report describes a bacteriocin that has the capacity to induce apoptosis in human cell lines.     

A methodological bias toward overestimation of molecular evolutionary time scales

There is presently a conflict between fossil- and molecular-based evolutionary time scales. Molecular approaches for dating the branches of the tree of life frequently lead to substantially deeper times of divergence than those inferred by paleontologists. The discrepancy between molecular and fossil estimates persists despite the booming growth of sequence data sets, which increasingly feeds the interpretation that molecular estimates are older than stratigraphic dates because of deficiencies in the fossil record. Here we show that molecular time estimates suffer from a methodological handicap, namely that they are asymmetrically bounded random variables, constrained by a nonelastic boundary at the lower end, but not at the higher end of the distribution. This introduces a bias toward an overestimation of time since divergence, which becomes greater as the length of the molecular sequence and the rate of evolution decrease.     

Cell cycle deregulation in liver lesions of rats with and without genetic predisposition to hepatocarcinogenesis

Preneoplastic and neoplastic hepatocytes undergo c-Myc up-regulation and overgrowth in rats genetically susceptible to hepatocarcinogenesis, but not in resistant rats. Because c-Myc regulates the pRb-E2F pathway, we evaluated cell cycle gene expression in neoplastic nodules and hepatocellular carcinomas (HCCs), induced by initiation/selection (IS) protocols 40 and 70 weeks after diethylnitrosamine treatment, in susceptible Fisher 344 (F344) rats, and resistant Wistar and Brown Norway (BN) rats. No interstrain differences in gene expression occurred in normal liver. Overexpression of c-myc, Cyclins D1, E, and A, and E2F1 genes, at messenger RNA (mRNA) and protein levels, rise in Cyclin D1-CDK4, Cyclin E-CDK2, and E2F1-DP1 complexes, and pRb hyperphosphorylation occurred in nodules and HCCs of F344 rats. Expression of Cdk4, Cdk2, p16(INK4A), and p27(KIP1) did not change. In nodules and/or HCCs of Wistar and BN rats, low or no increases in c-myc, Cyclins D1, E, and A, and E2F1 expression, and Cyclin-CDKs complex formation were associated with p16(INK4A) overexpression and pRb hypophosphorylation. In conclusion, these results suggest deregulation of G1 and S phases in liver lesions of susceptible rats and block of G1-S transition in lesions of resistant strains, which explains their low progression capacity.