Mechanisms of the antinociceptive action of (?) Epicatechin obtained from the hydroalcoholic fraction of Combretum leprosum Mart & Eic in rodents

ABSTRACT: BACKGROUND: The mechanisms of the antinociceptive activity of () epicatechin (EPI), a compound isolated from the hydroalcoholic fraction of Combreum leprosum Mart & Eicher. METHODS: were assessed in the model of chemical nociception induced by glutamate (20 mumol/paw). To evaluate the mechanisms involved, the animals , male Swiss mice (25-30 g), received EPI (50 mg/kg p.o.) after pretreatment with naloxone (2 mg/kg s.c. opioid antagonist), glibenclamide (2 mg/kg s.c. antagonist K + channels sensitive to ATP), ketanserin (0.3 mg/kg s.c. antagonist of receptor 5-HT2A), yoimbine (0.15 mg/kg s.c. alpha2 adrenergic receptor antagonist), pindolol (1 mg/kg s.c. 5-HT1a/1b receptor antagonist), atropine (0.1 mg/kg s.c. muscarinic antagonist) and caffeine (3 mg/kg s.c. adenosine receptor antagonist), ondansetron (0.5 mg/kg s.c. for 5-HT3 receptor) and L-arginine (600 mg/kg i.p.). RESULTS: The antinociceptive effect of EPI was reversed by pretreatment with naloxone and glibenclamide, ketanserin, yoimbine, atropine and pindolol, which demonstrates the involvement of opioid receptors and potassium channels sensitive to ATP, the serotoninergic (receptor 5HT1A and 5HT2A), adrenergic (receptor alpha 2) and cholinergic (muscarinic receptor) systems in the activities that were observed. The effects of EPI, however, were not reversed by pretreatment with caffeine, L-arginine or ondansetron, which shows that there is no involvement of 5HT3 receptors or the purinergic and nitrergic systems in the antinociceptive effect of EPI. In the Open Field and Rotarod test, EPI had no significant effect, which shows that there was no central nervous system depressant or muscle relaxant effect on the results. CONCLUSIONS: This study demonstrates that the antinociceptive activity of EPI in the glutamate model involves the participation of the opioid system, serotonin, adrenergic and cholinergic.     

Role of interactions involving C-terminal nonpolar residues of hirudin in the formation of the thrombin-hirudin complex

The role of interactions involving C-terminal nonpolar residues of hirudin in the formation of the thrombin-hirudin complex has been investigated by site-directed mutagenesis. The residues Phe56, Pro60, and Tyr63 of hirudin were replaced by a number of different amino acids, and the kinetics of the inhibition of thrombin by the mutant proteins were determined. Phe56 could be replaced by aromatic amino acids without significant loss in binding energy. While substitution of Phe56 by alanine decreased the binding energy (delta G degrees b by only 1.9 kJ mol-1, replacement of this residue by amino acids with branched side chains caused larger decreases in delta G degrees b. For example, the mutant Phe56----Val displayed a decrease in delta G degrees b of 10.5 kJ mol-1. Substitution of Pro60 by alanine or glycine resulted in a decrease in delta G degrees b of about 6 kJ mol-1. Tyr63 could be replaced by phenylalanine without any loss in binding energy, and replacement of this residue by alanine caused a decrease of 2.2 kJ mol-1 in delta G degrees b. Substitution of Tyr63 by residues with branched side chains resulted in smaller decreases in delta G degrees b than those seen with the corresponding substitutions of Phe56; for example, the mutant Tyr63----Val showed a decrease in binding energy of 5.1 kJ mol-1. The effects of the mutations are discussed in terms of the crystal structure of the thrombin-hirudin complex.     

A gene for hypotrichosis simplex of the scalp maps to chromosome 6p21.3

Hypotrichosis simplex of the scalp (HSS) is an autosomal dominant form of isolated alopecia causing almost complete loss of scalp hair, with onset in childhood. After exclusion of candidate regions previously associated with hair-loss disorders, we performed a genomewide linkage analysis in two Danish families and localized the gene to chromosome 6p21.3. This was confirmed in a Spanish family, with a total LOD score of 11.97 for marker D6S1701 in all families. The combined haplotype data identify a critical interval of 14.9 cM between markers D6S276 and D6S1607. Localization of the locus for HSS to 6p21.3 is a first step toward identification of the gene. The gene will give important insights into the molecular and cellular basis of hair growth on the scalp.     

Regional neural activation defines a gateway for autoreactive T cells to cross the blood-brain barrier

Although it is believed that neural activation can affect immune responses, very little is known about the neuroimmune interactions involved, especially the regulators of immune traffic across the blood-brain barrier which occurs in neuroimmune diseases such as multiple sclerosis (MS). Using a mouse model of MS, experimental autoimmune encephalomyelitis, we show that autoreactive T?cells access the central nervous system via the fifth lumbar spinal cord. This location is defined by IL-6 amplifier-dependent upregulation of the chemokine CCL20 in associated dorsal blood vessels, which in turn depends on gravity-induced activation of sensory neurons by the soleus muscle in the leg. Impairing soleus muscle contraction by tail suspension is sufficient to reduce localized chemokine expression and block entry of pathogenic T?cells at the fifth lumbar cord, suggesting that regional neuroimmune interactions may offer therapeutic targets for a variety of neurological diseases.     

Ozone-sensitive arabidopsis rcd1 mutant reveals opposite roles for ethylene and jasmonate signaling pathways in regulating superoxide-dependent cell death

We have isolated a codominant Arabidopsis mutant, radical-induced cell death1 (rcd1), in which ozone (O(3)) and extracellular superoxide (O(2)(*)-), but not hydrogen peroxide, induce cellular O(2)(*)- accumulation and transient spreading lesions. The cellular O(2)(*)- accumulation is ethylene dependent, occurs ahead of the expanding lesions before visible symptoms appear, and is required for lesion propagation. Exogenous ethylene increased O(2)(*)--dependent cell death, whereas impairment of ethylene perception by norbornadiene in rcd1 or ethylene insensitivity in the ethylene-insensitive mutant ein2 and in the rcd1 ein2 double mutant blocked O(2)(*)- accumulation and lesion propagation. Exogenous methyl jasmonate inhibited propagation of cell death in rcd1. Accordingly, the O(3)-exposed jasmonate-insensitive mutant jar1 displayed spreading cell death and a prolonged O(2)(*)- accumulation pattern. These results suggest that ethylene acts as a promoting factor during the propagation phase of developing oxyradical-dependent lesions, whereas jasmonates have a role in lesion containment. Interaction and balance between these pathways may serve to fine-tune propagation and containment processes, resulting in alternate lesion size and formation kinetics.     

Imaging synaptic vesicle exocytosis and endocytosis with FM dyes

FM dyes have been used to label and then monitor synaptic vesicles, secretory granules and other endocytic structures in a variety of preparations. Here, we describe the general procedure for using FM dyes to study endosomal trafficking in general, and synaptic vesicle recycling in particular. The dye, dissolved in normal saline solution, is added to a chamber containing the preparation to be labeled. Stimulation evokes exocytosis, and compensatory endocytosis that follows traps FM dye inside the retrieved vesicles. The extracellular dye is then washed from the chamber, and labeled endocytic structures are examined with a fluorescence microscope. Fluorescence intensity provides a direct measure of the labeled vesicle number, a good measure of the amount of exocytosis. If the preparation is stimulated again, without dye in the chamber, dimming of the preparation provides a measure of exocytosis of labeled vesicles. With a synaptic preparation on hand, this protocol requires 1 day.     

Inhibition of neurite extension by overexpression of individual domains of LIM kinase 1

Lin-11, Isl-1 and Mec-3 (LIM) kinases are serine/threonine kinases that phosphorylate cofilin, an actin depolymerizing protein. LIM kinases have a highly modular structure composed of two N-terminal LIM domains (LIM 1/2), a PSD-95, Dlg and ZO-1 (PDZ) domain and a C-terminal protein kinase domain. Here, we overexpressed individual domains of mouse LIM kinase 1 (LIMK1) in PC12 cells and investigated their effects on neurite outgrowth. Although none of the LIMK1 domains had an effect on spontaneous neurite outgrowth, the N-terminal LIM 1/2 domains strongly inhibited differentiation of PC12 cells after stimulation with both nerve growth factor (NGF) and the Rho-kinase inhibitor Y-27632. In contrast, the overexpressed PDZ domain reduced neurite outgrowth only when differentiation had been induced by Y-27632, but not by NGF. Our data suggest that the different non-catalytic N-terminal domains of LIMK1 contribute to the regulation of neurite extension by using distinct signal transduction pathways.     

Mapping of antigenic epitopes on the alpha 1 subunit of the inhibitory glycine receptor.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel protein that occurs in developmentally regulated isoforms in the vertebrate central nervous system. Monoclonal antibodies (mAbs) against the GlyR distinguish neonatal and adult GlyR proteins by identifying distinct alpha subunit variants within these receptor isoforms. Here, bacterially expressed fusion proteins of the rat GlyR alpha 1 subunit were used to localize the major antigenic epitopes of this protein within its N-terminal 105 amino acids. Synthetic peptides allowed further fine mapping of two mAb binding domains. MAb 2b, specific for the adult alpha 1 subunit, bound to a peptide corresponding to amino acids 1-10, whereas mAb 4a, which recognizes both neonatal and adult GlyR isoforms, reacted with a peptide representing residues 96-105 of the alpha 1 polypeptide. These data define unique and common antigenic epitopes on GlyR alpha subunit variants.     

Transport activities and expression patterns of glycine transporters 1 and 2 in the developing murine brain stem and spinal cord

Glycine serves as a neurotransmitter in spinal cord and brain stem, where it activates inhibitory glycine receptors. In addition, it serves as an essential co-agonist of excitatory N-methyl-d-aspartate receptors. In the central nervous system, extracellular glycine concentrations are regulated by two specific glycine transporters (GlyTs), GlyT1 and GlyT2. Here, we determined the relative transport activities and protein levels of GlyT1 and GlyT2 in membrane preparations from mouse brain stem and spinal cord at different developmental stages. We report that early postnatally (up to postnatal day P5) GlyT1 is the predominant transporter isoform responsible for a major fraction of the GlyT-mediated [(3)H]glycine uptake. At later stages (≥ P10), however, the transport activity and expression of GlyT2 increases, and in membrane fractions from adult mice both GlyTs contribute about equally to glycine uptake. These alterations in the activities and expression profiles of the GlyTs suggest that the contributions of GlyT1 and GlyT2 to the regulation of extracellular glycine concentrations at glycinergic synapses changes during development.     

Metabolism of the obligatory aerobic yeast Rhodotorula gracilis

Summary D-Glucose and D-xylose addition to not-growing Rhodotorula gracilis cells brings about alterations in pyruvate kinase and phosphoenolpyruvate carboxykinase activities characteristic for glycolysis and gluconeogenesis, respectively.Abbreviations used PK Pyruvate kinase (EC 2.7.1.40) - PEPCK Phosphoenolpyruvate carboxykinase (EC 4.1.1.32) - PFK Phosphofructokinase (EC 2.7.1.11)