Heteroaryl urea inhibitors of fatty acid amide hydrolase: Structure–mutagenicity relationships for arylamine metabolites

The structure–activity relationships for a series of heteroaryl urea inhibitors of fatty acid amide hydrolase (FAAH) are described. Members of this class of inhibitors have been shown to inactivate FAAH by covalent modification of an active site serine with subsequent release of an aromatic amine from the urea electrophile. Systematic Ames II testing guided the optimization of urea substituents by defining the structure–mutagenicity relationships for the released aromatic amine metabolites. Potent FAAH inhibitors were identified having heteroaryl amine leaving groups that were non-mutagenic in the Ames II assay.     

3-(7-Azaindolyl)-4-arylmaleimides as potent, selective inhibitors of glycogen synthase kinase-3

A novel series of acyclic 3-(7-azaindolyl)-4-(aryl/heteroaryl)maleimides was synthesized and evaluated for activity against GSK-3beta and selectivity versus PKC-betaII, as well as a broad panel of protein kinases. Compounds 14 and 17c potently inhibited GSK-3beta (IC(50)=7 and 26 nM, respectively) and exhibited excellent selectivity over PKC-betaII (325 and >385-fold, respectively). Compound 17c was also highly selective against 68 other protein kinases. In a cell-based functional assay, both 14 and 17c effectively increased glycogen synthase activity by inhibiting GSK-3beta.     

Succession and soil development in man-made coastal ecosystems at the Baltic Sea

By the creation of Køge Bay Seaside Park in 1978 the opportunity was given to study the establishment of vegetation and primary succession in a man-made coastal area. As the soil consists of marine material from the Baltic Sea, no organic matter or seedbank was present. The first steps of primary succession were followed in 1980, when the initial inventories of flora immigration and soil development were carried out in coastal grasslands and plantings. The surveys were repeated in 1992 and 1993. Except for the planted woody species and a few sown grasses, all other species of plants have reached the area through natural dispersal of diaspores. The total number of species in the permanent plots has increased from 26 in 1980 to 91 in 1993. The results indicate that this number will continue to increase in the coming years until a certain level. Then it will probably decrease as a result of competition from woody species, unless the vegetation is kept in a steady state by disturbances or management. Today the area is very far from the initial situation, and the off-shore barrier has changed towards a landscape dominated by small groves and grasslands of an urban common type.     

N-terminal region of gelsolin induces apoptosis of activated hepatic stellate cells by a caspase-dependent mechanism

Activated hepatic stellate cells (HSCs) are the major source for alteration of extracellular matrix in fibrosis and cirrhosis. Conditioned medium (CM) collected from immortalized human hepatocytes (IHH) have earlier been shown to be responsible for apoptosis of HSCs. In this study, we have shown that antibodies raised against a peptide derived from a linear B-cell epitope in the N-terminal region of gelsolin identified a gelsolin fragment in IHH CM. Analysis of activated stellate cell death by CM collected from Huh7 cells transfected with plasmids encoding gelsolin deletion mutants suggested that the N-terminal half of gelsolin contained sequences which were responsible for stellate cell death. Further analysis determined that this activity was restricted to a region encompassing amino acids 1-70 in the gelsolin sequence; antibody directed to an epitope within this region was able to neutralize stellate cell death. Gelsolin modulation of cell death using this fragment involved upregulation of TRAIL-R1 and TRAIL-R2, and involved caspase 3 activation by extrinsic pathway. The apoptotic activity of N-terminal gelsolin fragments was restricted to activated but not quiescent stellate cells indicating its potential application in therapeutic use as an anti-fibrotic agent. Gelsolin fragments encompassing N-terminal regions in polypeptides of different molecular sizes were detected by N-terminal peptide specific antiserum in IHH CM immunoprecipitated with chronically HCV infected patient sera, suggesting the presence of autoantibodies generated against N-terminal gelsolin fragments in patients with chronic liver disease.     

Heat shock protein expression and change of cytochrome c oxidase activity: presence of two phylogenic old systems to protect tissues in ischemia and reperfusion

Induction of heat shock proteins (hsp) has been shown to protect cells from ischemia by providing transient tolerance against myocardial injury and improving postischemic functional recovery. Attenuation of ATP depletion and earlier restoration of ATP content on reperfusion are thought to play a role in this scenario. Hsp induction is accompanied by altered enzyme activity of the respiratory chain, the major generator of ATP under physiological conditions. This report addresses the question whether processing and final assembly of the active holoenzyme cytochrome c oxidase (CcO, complex IV), member of the respiratory chain, is compromised under hypoxic conditions unless protected by stress proteins. Special focus is laid on function of the enzyme’s subunits and importance of cellular energy availability and maintenance.     

An Evaluation of Habitat Rehabilitation on Coastal Dune Forests in Northern KwaZulu-Natal, South Africa

The rehabilitation program conducted by Richards Bay Minerals (RBM) of areas exposed to opencast surface mining of sand dunes north of Richards Bay (28°43'S, 32°12'E) on the coast of northern KwaZulu-Natal Province commenced 16 years before this study and has resulted in the development of a series of known-aged stands of vegetation. By assuming that these spatially separated stands develop along a similar pathway over time, instantaneous sampling should reveal successional or other changes usually associated with aging and should provide an opportunity to evaluate the success of rehabilitation. We compare relative densities of pioneer and secondary species, species richness, and a similarity index of the herbaceous layer, tree, beetle, millipede, bird, and small-mammal communities of rehabilitating areas of known age with those of 30-year-old unmined forests and unmined forests of unknown age adjacent to the rehabilitating area. Species richness for all but the mammalian taxa increased with increasing age of rehabilitating stands. For all taxa but the mammals and herbaceous layer, the unmined stands harbored more species than the mined rehabilitating stands. The relative densities of pioneer species of all the taxa decreased with an increase in the age of rehabilitating stands, whereas those of the secondary species increased with an increase in habitat age. Similarity between unmined stands and rehabilitating stands of different ages increased with increasing regeneration age of rehabilitating stands, suggesting that rehabilitating communities, in terms of species composition and relative densities, are developing towards the status of unmined communities. Rehabilitation based on RBM's management program of limited interference is occurring and may result in the reestablishment of a coastal dune forest ecosystem. But rehabilitation resulting from succession depends on the availability of species sources from which colonization can take place. In the Richards Bay mining operation the present mining path is laid out so that such refuges are present.     

Multiple Propofol-binding Sites in a γ-Aminobutyric Acid Type A Receptor (GABAAR) Identified Using a Photoreactive Propofol Analog

Propofol acts as a positive allosteric modulator of γ-aminobutyric acid type A receptors (GABA_ARs), an interaction necessary for its anesthetic potency in vivo as a general anesthetic. Identifying the location of propofol-binding sites is necessary to understand its mechanism of GABA_AR modulation. [³H]2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (azietomidate) and R-[³H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), photoreactive analogs of 2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (etomidate) and mephobarbital, respectively, have identified two homologous but pharmacologically distinct classes of intersubunit-binding sites for general anesthetics in the GABA_AR transmembrane domain. Here, we use a photoreactive analog of propofol (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol ([³H]AziPm)) to identify propofol-binding sites in heterologously expressed human α1β3 GABA_ARs. Propofol, AziPm, etomidate, and R-mTFD-MPAB each inhibited [³H]AziPm photoincorporation into GABA_AR subunits maximally by ∼50%. When the amino acids photolabeled by [³H]AziPm were identified by protein microsequencing, we found propofol-inhibitable photolabeling of amino acids in the β3-α1 subunit interface (β3Met-286 in β3M3 and α1Met-236 in α1M1), previously photolabeled by [³H]azietomidate, and α1Ile-239, located one helical turn below α1Met-236. There was also propofol-inhibitable [³H]AziPm photolabeling of β3Met-227 in βM1, the amino acid in the α1-β3 subunit interface photolabeled by R-[³H]mTFD-MPAB. The propofol-inhibitable [³H]AziPm photolabeling in the GABA_AR β3 subunit in conjunction with the concentration dependence of inhibition of that photolabeling by etomidate or R-mTFD-MPAB also establish that each anesthetic binds to the homologous site at the β3-β3 subunit interface. These results establish that AziPm as well as propofol bind to the homologous intersubunit sites in the GABA_AR transmembrane domain that binds etomidate or R-mTFD-MPAB with high affinity.