Peroxisomal localization of sulfite oxidase separates it from chloroplast-based sulfur assimilation

Recently, we isolated the sulfite oxidase (SO) gene from Arabidopsis thaliana and characterized the purified SO protein. The purpose of the present study was to determine the subcellular localization of this novel plant enzyme. Immunogold electron-microscopic analysis showed the gold labels nearly exclusively in the peroxisomes. To verify this finding, green fluorescent protein was fused to full-length plant SO including the putative peroxisomal targeting signal 1 (PTS1) 'SNL' and expressed in tobacco leaves. Our results showed a punctate fluorescence pattern resembling that of peroxisomes. Co-labelling with MitoTracker-Red excluded that the observed fluorescence was due to mitochondrial sorting. By investigation of deleted or mutated PTS1, no functional peroxisomal targeting signal 2 (PTS2) could be detected in plant SO. This conclusion is supported by expression studies in Pichia pastoris mutants with defined defects either in PTS1- or PTS2-mediated peroxisomal import.     

Synthesis and amine transporter affinities of novel phenyltropane derivatives as potential positron emission tomography (PET) imaging agents

A series of novel fluoroalkyl-containing tropane derivatives (6-8, 10-14, 17, and 18) were synthesized from cocaine. Novel compounds were evaluated for affinity and selectivity in competitive radioligand binding assays selective for cerebral serotonin (5-HT), dopamine (DA), and norepinephrine (NE) transporters (SERT, DAT, and NET). The nortropane-fluoroalkyl esters (7, 10, 11) were most potent for SERT (K(i): 0.18, 0.24, and 0.30 nM, respectively). Tosylate esters 17 and 18, synthesized as precursors for [(18)F]-labeled, Positron Emission Tomography (PET) imaging agents, also showed high affinity for DAT.     

Synthesis and neuropharmacological evaluation of R(-)-N-alkyl-11-hydroxynoraporphines and their esters

We synthesized several N-substituted-11-hydroxynoraporphines and their esters of varying chain length, evaluated their binding affinity at dopamine (DA) receptor sites in rat caudate-putamen membranes, and quantified their effects on motor activity in normal adult male rats. The 11-hydroxyaporphines showed similar neuropharmacological properties to the corresponding 10,11-catecholaporphines. At moderate doses, their esters proved to have more prolonged behavioral actions and superior oral bioavailability.     

Binding of the anticonvulsant drug lamotrigine and the neurotoxin batrachotoxin to voltage-gated sodium channels induces conformational changes associated with block and steady-state activation

Voltage-gated sodium channels are dynamic membrane proteins characterized by rapid conformational changes that switch the molecule between closed resting, activated, and inactivated states. Sodium channels are specifically blocked by the anticonvulsant drug lamotrigine, which preferentially binds to the channel pore in the inactivated open state. Batrachotoxin is a lipid-soluble alkaloid that causes steady-state activation and binds in the inner pore of the sodium channel with overlapping but distinct molecular determinants from those of lamotrigine. Using circular dichroism spectroscopy on purified voltage-gated sodium channels from Electrophorus electricus, the secondary structures associated with the mixture of states present at equilibrium in the absence of these ligands were compared with specific stabilized states in their presence. As the channel shifts to open states, there appears to be a significant change in secondary structure to a more alpha-helical conformation. The observed changes are consistent with increased order involving the S6 segments that form the pore, the domain III-IV linker, and the P-loops that form the outer pore and selectivity filter. A molecular model has been constructed for the sodium channel based on its homology with the pore-forming regions of bacterial potassium channels, and automated docking of the crystal structure of lamotrigine with this model produces a structure in which the close contacts of the drug are with the residues previously identified by mutational studies as forming the binding site for this drug.     

The stromal cell-derived factor-1alpha/CXCR4 ligand-receptor axis is critical for progenitor survival and migration in the pancreas

The SDF-1alpha/CXCR4 ligand/chemokine receptor pair is required for appropriate patterning during ontogeny and stimulates the growth and differentiation of critical cell types. Here, we demonstrate SDF-1alpha and CXCR4 expression in fetal pancreas. We have found that SDF-1alpha and its receptor CXCR4 are expressed in islets, also CXCR4 is expressed in and around the proliferating duct epithelium of the regenerating pancreas of the interferon (IFN) gamma-nonobese diabetic mouse. We show that SDF-1alpha stimulates the phosphorylation of Akt, mitogen-activated protein kinase, and Src in pancreatic duct cells. Furthermore, migration assays indicate a stimulatory effect of SDF-1alpha on ductal cell migration. Importantly, blocking the SDF-1alpha/CXCR4 axis in IFNgamma-nonobese diabetic mice resulted in diminished proliferation and increased apoptosis in the pancreatic ductal cells. Together, these data indicate that the SDF-1alpha-CXCR4 ligand receptor axis is an obligatory component in the maintenance of duct cell survival, proliferation, and migration during pancreatic regeneration.     

Abnormal brain activation to visual stimulation in cocaine abusers

Chronic cocaine abuse has been associated with cerebrovascular pathology. This is likely to reflect its vasoactive effects; cocaine produces vasoconstriction and reduces cerebral blood flow. We propose that cerebrovascular pathology in chronic cocaine abusers would result in abnormal BOLD [blood oxygenation level dependent] responses to activation stimuli. Here, we used fMRI to compared the BOLD response to photic visual stimulation in neurologically intact active cocaine abusers to that in non-drug-using healthy controls. Cocaine abusers showed a significantly enhanced positive BOLD response to photic stimulation when compared to control subjects. The enhanced activation in the cocaine abusers could result from low resting cerebral blood flow secondary to increased vasoconstriction and/or from low oxidative metabolism during activation. Alternatively, the larger signal intensity in the cocaine abusers could result from inefficient neuronal processing as has been shown to occur in other conditions of cerebral pathology. These findings provide evidence of cerebral dysfunction with chronic cocaine abuse, which could reflect cerebral blood flow or neuronal changes. Further studies are required to determine if the cerebrovascular changes we observed in the cocaine abusers recover with detoxification and to assess their functional consequences.     

Association of the low-density lipoprotein receptor with caveolae in hamster and rat liver

The association of the low-density lipoprotein (LDL) receptor with detergent resistant hepatic membranes was investigated using discontinuous sucrose gradients. In liver homogenates from both hamsters and rats, the fractions with the highest concentrations of LDL receptor coincided with the location of caveolin-1, a marker of the cholesterol-rich caveolae. Feeding the animals diets enriched in cholesterol slightly shifted both LDL receptor and caveolin-1 to positions of lower density. The cholesterol content of the caveolae fractions was increased 2-fold in animals fed cholesterol-supplemented diets. In homogenates of CHO cells, fractionated in the same manner, the LDL receptor was absent from the caveolae fractions but was present in denser fractions near the bottom of the gradient. Addition of caveolin-1 antibody to solubilized caveolae from liver coimmunoprecipitated the LDL receptor. These observations suggest that in liver, the LDL receptor is mainly located in caveolae. This location contrasts with the clathrin-coated pit location observed in fibroblasts and CHO cells.     

In vivo imaging of the brain cannabinoid receptor

The CB1 cannabinoid receptor is expressed in the brain at levels sufficient to serve as potential target for in vivo imaging using positron emission tomography (PET) or single photon emission computed tomography methodology. To date, the most promising radioligands for the in vivo imaging of this receptor have structures based on that of the cannabinoid antagonist, SR141716A. Rodent data obtained using these in vivo radiotracers has demonstrated that both the behavioral and neurochemical effects of cannabinoids occur at very low levels of receptor occupancy. More recently, an agonist radiotracer based on the structure of aminoalkylindole cannabinoids has also been examined for in vivo labeling of CB1 receptors. Although rodent studies have indicated that in vivo imaging of CB1 receptors is feasible, at the present time this receptor has still to be successful imaged in a human PET study.     

A new species of Thelastomathidae (Nematoda) a parasite of Neocurtilla claraziana Saussure (Orthoptera,Gryllotalpidae) in Argentina

Gryllophila cephalobulata n. sp. (Nematoda, Thelastomatidae) a parasite of the mole cricket Neocurtilla claraziana (Orthoptera, Gryllotalpidae) isolated in Buenos Aires Province, is described and illustrated. It is characterized by cuticle annulated all along the length of the body; the first ring has 4 lobules, the second one has 14 lobules, the others rings are simple, the stoma is short and has 4 small teeth, the genital papillae are arranged in 5 pairs, of which 3 pairs are preanal and 2 pairs are postanal. The tail appendage of the male is long and filiform.