Neuronal and Glial Expression of the Multidrug Resistance Gene Product in an Experimental Epilepsy Model

1. Failure of anticonvulsive drugs to prevent seizures is a common complication of epilepsy treatment known as drug-refractory epilepsy but their causes are not well understood. It is hypothesized that the multidrug resistance P-glycoprotein (Pgp-170), the product of the MDR-1 gene that is normally expressed in several excretory tissues including the blood brain barrier, may be participating in the refractory epilepsy. 2. Using two monoclonal antibodies against Pgp-170, we investigated the expression and cellular distribution of this protein in the rat brain during experimentally induced epilepsy. Repeated seizures were induced in male Wistar rats by daily administration of 3-mercaptopropionic acid (MP) 45 mg/kg i.p. for either 4 days (MP-4) or 7 days (MP-7). Control rats received an equivalent volume of vehicle. One day after the last injection, rats were sacrificed and brains were processed for immunohistochemistry for Pgp-170. As it was previously described, Pgp-170 immunostaining was observed in some brain capillary endothelial cells of animals from control group. 3. Increased Pgp-170 immunoreactivity was detected in MP-treated animals. Besides the Pgp-170 expressed in blood vessels, neuronal, and glial immunostaining was detected in hippocampus, striatum, and cerebral cortex of MP-treated rats. Pgp-170 immunolabeled neurons and glial cells were observed in a nonhomogeneous distribution. MP-4 animals presented a very prominent Pgp-170 immunostaining in the capillary endothelium, surrounding astrocytes and some neighboring neurons while MP-7 group showed increased neuronal labeling. 4. Our results demonstrate a selective increase in Pgp-170 immunoreactivity in the brain capillary endothelial cells, astrocytes, and neurons during repetitive MP-induced seizures. 5. The role for this Pgp-170 overexpression in endothelium and astrocytes as a clearance mechanism in the refractory epilepsy, and the consequences of neuronal Pgp-170 expression remain to be disclosed.     

Differences among the cell wall galactomannans from Aspergillus wentii and Chaetosartorya chrysella and that of Aspergillus fumigatus

The alkali extractable and water-soluble cell wall polysaccharides F1SS from Aspergillus wentii and Chaetosartorya chrysella have been studied by methylation analysis, 1D- and 2D-NMR, and MALDI-TOF analysis. Their structures are almost identical, corresponding to the following repeating unit: [--> 3)-beta-D-Gal f -(1 --> 5)-beta-D-Gal f-(1 -->]n --> mannan core. The structure of this galactofuranose side chain differs from that found in the pathogenic fungus Aspergillus fumigatus, in other Aspergillii and members of Trichocomaceae: [--> 5)-beta-D-Gal f-(1 -->]n --> mannan core. The mannan cores have also been investigated, and are constituted by a (1 --> 6)-alpha-mannan backbone, substituted at positions 2 by chains from 1 to 7 residues of (1 --> 2) linked alpha-mannopyranoses.     

Bcl-2 family proteins regulate mitochondrial reactive oxygen production and protect against oxidative stress

Bcl-2 family proteins protect against a variety of forms of cell death, including acute oxidative stress. Previous studies have shown that overexpression of the antiapoptotic protein Bcl-2 increases cellular redox capacity. Here we report that cell lines transfected with Bcl-2 paradoxically exhibit increased rates of mitochondrial H(2)O(2) generation. Using isolated mitochondria, we determined that increased H(2)O(2) release results from the oxidation of reduced nicotinamide adenine dinucleotide-linked substrates. Antiapoptotic Bcl-2 family proteins Bcl-xL and Mcl-1 also increase mitochondrial H(2)O(2) release when overexpressed. Chronic exposure of cells to low levels of the mitochondrial uncoupler carbonyl cyanide 4-(triflouromethoxy)phenylhydrazone reduced the rate of H(2)O(2) production by Bcl-xL overexpressing cells, resulting in a decreased ability to remove exogenous H(2)O(2) and enhanced cell death under conditions of acute oxidative stress. Our results indicate that chronic and mild elevations in H(2)O(2) release from Bcl-2, Bcl-xL, and Mcl-1 overexpressing mitochondria lead to enhanced cellular antioxidant defense and protection against death caused by acute oxidative stress.     

Dexamethasone treatment causes resistance to insulin-stimulated cellular potassium uptake in the rat

Patients treated with glucocorticoids have elevated skeletal muscle ouabain binding sites. The major Na⁺-K⁺-ATPase (NKA) isoform proteins found in muscle, ₂ and ₁, are increased by 50% in rats treated for 14 days with the synthetic glucocorticoid dexamethasone (DEX). This study addressed whether the DEX-induced increase in the muscle NKA pool leads to increased insulin-stimulated cellular K⁺ uptake that could precipitate hypokalemia. Rats were treated with DEX or vehicle via osmotic minipumps at one of two doses: 0.02 mg·kg^–1·day^–1 for 14 days (low DEX; n = 5 pairs) or 0.1 mg·kg^–1·day^–1 for 7 days (high DEX; n = 6 pairs). Insulin was infused at a rate of 5 mU·kg^–1·min^–1 over 2.5 h in conscious rats. Insulin-stimulated cellular K⁺ and glucose uptake rates were assessed in vivo by measuring the exogenous K⁺ infusion () and glucose infusion (G_inf) rates needed to maintain constant plasma K⁺ and glucose concentrations during insulin infusion. DEX at both doses decreased insulin-stimulated glucose uptake as previously reported. G_inf (in mmol·kg^–1·h^–1) was 10.2 ± 0.6 in vehicle-treated rats, 5.8 ± 0.8 in low-DEX-treated rats, and 5.2 ± 0.6 in high-DEX-treated rats. High DEX treatment also reduced insulin-stimulated K⁺ uptake. (in mmol·kg^–1·h^–1) was 0.53 ± 0.08 in vehicle-treated rats, 0.49 ± 0.14 in low-DEX-treated rats, and 0.27 ± 0.08 in high-DEX-treated rats. DEX treatment did not alter urinary K⁺ excretion. NKA ₂-isoform levels in the low-DEX-treated group, measured by immunoblotting, were unchanged, but they increased by 38 ± 15% (soleus) and by 67 ± 3% (gastrocnemius) in the high-DEX treatment group. The NKA ₁-isoform level was unchanged. These results provide novel evidence for the insulin resistance of K⁺ clearance during chronic DEX treatment. Insulin-stimulated cellular K⁺ uptake was significantly depressed despite increased muscle sodium pump pool size. skeletal muscle; sodium pump; Na⁺-K⁺-ATPase     

Influence of physical stress, distress cues, and predator kairomones on the foraging behavior of Ozark zigzag salamanders, Plethodon angusticlavius

An animal's response to environmentally induced stress can have important fitness consequences. For example, an animal may reduce activity in response to stress related to increased predation risk, but this response carries the potential cost of missed foraging or mating opportunities. We used laboratory bioassays to determine whether Ozark zigzag salamanders, Plethodon angusticlavius, decreased their feeding behavior following increased levels of threat-related stress. Stress level was manipulated in three experiments where salamanders were exposed to direct or indirect indicators of stress. The direct cue was a physical attack. The indirect cues were chemical cues from distressed conspecifics and chemical cues ("kairomones") from predatory ringneck snakes, Diadophis punctatus. For all three cues, stressed salamanders showed lower foraging success than unstressed salamanders. The physical attack did not result in a more intense response than the other treatments, possibly because the attack occurred away from the foraging area. The intensity of the response to stress may depend more on the nature of the threat (identity and imminence) than the level of physical disturbance.     

Face transplantation: where do we stand?

The recent clinical cases of hand and composite tissue allotransplantation opened a new era in the practice of reconstructive surgery. Some have suggested that face (allo)transplantation could be the next step to benefit patients whose conditions cannot be addressed by conventional techniques of reconstructive surgery using autologous tissues. This article reviews the current status of science regarding the prospect of human face transplantation. The main issues fall into three categories: (1) the surgical challenge of the procedure, specifically regarding vascular viability and functional recovery of the graft; (2) the risks of side effects from life-long immunosuppression necessary to prevent graft rejection; and (3) the ethical debate and the effects of the procedure on the population. Although face transplantation could one day be performed and extend the boundaries of reconstructive surgery, there are currently many obstacles that need to be overcome first.     

Pig-tailed macaques (<Emphasis Type="Italic">Macaca nemestrina</Emphasis>) possess six MHC-E families that are conserved among macaque species: implication for their binding to natural killer receptor variants

MHC loci encode highly polymorphic molecules involved in the presentation of self and non-self peptides to cells of the adaptive and innate immune systems. Although variable, MHC-E genes are well conserved among primates and provide signals to natural killer cells. In this study, we sequenced and analyzed MHC-E alleles of pig-tailed macaque (Macaca nemestrina), a nonhuman primate used for HIV pathogenesis and vaccine studies. Among a group of seven macaques, the characterization of eight Mane-E alleles revealed an increased number of polymorphic sites compared with human HLA-E alleles. Phylogenetic analyses of MHC-E alleles from pig-tailed macaque, rhesus monkey (Macaca mulatta) and cynomolgus macaque (Macaca fascicularis) demonstrated that the three macaque species shared six families of macaque MHC-E alleles and indicated that these families existed in the common ancestor 5.5 million years ago. Polymorphic Mane-E sites were not concentrated within the peptide-binding pockets, but were distributed throughout the entire ORF. The peptide-binding domain of Mane-E is similar to its human analogue, and peptide substrates theoretically capable of binding to Mane-E molecules were found in the leader sequence of classical Mane-A and -B molecules. Additionally, the polymorphic amino acids located in the ₁ and ₂ domains of Mane-E molecules have side chains expected to be oriented toward solvent and away from the peptide-binding groove, suggesting that some of them (positions 19, 73, 79 and 145) might be available for interaction with polymorphic receptors of natural killer cells.     

Analysis of the gyrA gene of clinical Yersinia ruckeri isolates with reduced susceptibility to quinolones

Antimicrobial susceptibility of seven clinical strains of Yersinia ruckeri representative of those isolated between 1994 and 2002 from a fish farm with endemic enteric redmouth disease was studied. All isolates displayed indistinguishable pulsed-field gel electrophoresis restriction patterns, indicating that they represented a single strain. However, considering both inhibition zone diameters (IZD) and MICs, the isolates recovered in 2001-2002 formed a separate cluster with lower levels of susceptibility to all the quinolones tested, especially nalidixic acid (NA) and oxolinic acid (OA), compared with the isolates recovered between 1994 and 1998. Analysis of the PCR product of the quinolone resistance-determining region of the gyrA gene from clinical isolates of Y. ruckeri with reduced susceptibility to OA and NA revealed a single amino acid substitution, Ser-83 to Arg-83 (Escherichia coli numbering). Identical substitution was observed in induced OA-resistant mutant strains, which displayed IZD and MICs of quinolones similar to those of the clinical isolates of Y. ruckeri with reduced susceptibility to these antimicrobial agents. These data indicate in that for Y. ruckeri, the substitution of Ser by Arg at position 83 of the gyrA gene is associated with reduced susceptibility to quinolones.     

Development of an optical immunosensor based on the fluorescence of Cyanine-5 for veterinarian diagnostics

A model optical immunosensor was developed to quantify an antibody present in a sample by measuring the fluorescence of Cyanine-5 conjugated with the antibody, using a competitive and a sandwich immunoreaction configuration, with the antigen immobilised in controlled pore glass beads. At pH 2, 94% of the antigen-antibody complex was dissociated, allowing reutilisation. Photobleaching had no effect on the fluorescence. This model system was used to detect Brucella sp. infection and could quantify anti-Brucella sp. antibodies in ovine serum samples in the range from 0.005 to 0.11 mg ml(-1).     

A Highly Active ATP-Insensitive K+ Import Pathway in Plant Mitochondria

We describe here a regulated and highly active K+ uptake pathway in potato (Solanum tuberosum), tomato (Lycopersicon esculentum), and maize (Zea mays) mitochondria. K+ transport was not inhibited by ATP, NADH, or thiol reagents, which regulate ATP-sensitive K+ channels previously described in plant and mammalian mitochondria. However, K+ uptake was completely prevented by quinine, a broad spectrum K+ channel inhibitor. Increased K+ uptake in plants leads to mitochondrial swelling, respiratory stimulation, heat release, and the prevention of reactive oxygen species formation. This newly described ATP-insensitive K+ import pathway is potentially involved in metabolism regulation and prevention of oxidative stress.