Structure of Cu2(indomethacin)4 and the reaction with superoxide in aprotic systems

The copper complex of indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyl-indole acetate), a common anti-inflammatory drug, was prepared and characterized. Crystal structure determination revealed the dimeric form of the 1:2 complex, namely Cu₂(indomethacin)₄ · L₂, in the unit cell. Suprisingly, the copper-copper distance (263 pm) was very close to metallic copper (256 pm). The two coordination sites in the copper-copper axis can be readily replaced by superoxide. An intriguing similarity to Cu₂(acetate)₄ was seen.Due to the lipophilic nature of the indomethacin ligand, this copper complex reacted with superoxide in aprotic solvents. The superoxide dismutating activity was successfully demonstrated in Me₂SO/water and acetonitrile/water mixtures using the nitro-blue tetrazolium assay and pulse radiolysis. The second-order rate constant of 6 · 10⁹ M^?1 · s^?1 in strictly aqueous systems dropped only slightly to 1.1 · 10⁹ M^?1 · s^?1 when aprotic solvents were used. This is the fastest rate constant ever observed for a copper-dependent dismutation of superoxide. The KO₂-induced lipid peroxidation in both erythrocytes and liver microsomes was suppressed by 70% in the presence of 1 · 10^?10 mol · ml^?1 of Cu₂(indomethacin)₄. The inhibitory action dropped to 25% when Cu₂Zn₂superoxide dismutase was employed. The formation of copper · indomethacin in rat serum after administration of indomethacin was shown in vitro and in vivo.     

Rabbit small intestine does not contain an annexin II/caveolin 1 complex as a target for 2-azetidinone cholesterol absorption inhibitors

Intestinal cholesterol absorption is specifically inhibited by the 2-azetidinone cholesterol absorption inhibitor ezetimibe. Photoreactive ezetimibe analogues specifically label a 145-kDa protein in the brush border membrane of enterocytes from rabbit small intestine identified as aminopeptidase N (CD13). In zebrafish and mouse small intestinal cytosol, a heterocomplex of M_r 52 kDa between annexin II and caveolin 1 was suggested as a target of ezetimibe. In contrast, in the cytosol and brush border membrane vesicles (BBMV) from rabbit small intestine of control animals or rabbits treated with the nonabsorbable cholesterol absorption inhibitor AVE 5530, both annexin II and caveolin 1 were exclusively present as monomers without any heterocomplex formation. Upon immunoprecipitation with annexin II a 52-kDa band was observed after immunostaining with annexin II antibodies, whereas no staining of a 52-kDa band occurred with anti-caveolin 1 antibodies. Vice versa, a 52-kDa band obtained by immunoprecipitation with caveolin 1 antibodies did not stain with annexin II-antibodies. The intensity of the 52-kDa band was dependent on the amount of antibody and was also observed with anti-actin or anti-APN antibodies suggesting that the 52-kDa band is a biochemical artefact. After incubation of cytosol or BBMV with radioactively labelled ezetimibe analogues, no significant amounts of the ezetimibe analogues could be detected in the immunoprecipitate with caveolin-1 or annexin II antibodies. Photoaffinity labelling of rabbit small intestinal BBMV with ezetimibe analogues did not result in labelling of proteins being immunoreactive with annexin II, caveolin 1 or a 52-kDa heterocomplex. These findings indicate that the rabbit small intestine does not contain an annexin II/caveolin 1 heterocomplex as a target for ezetimibe.     

Cell adhesion molecule in the hexactinellid Aphrocallistes vastus

Abstract. The Hexactinellida sponge Aphrocallistes vastus contains a soluble aggregation factor (AF) whose purification has been described in this communication. It is characterized by a S°_20.w value of 37 and a buoyant density of 1.45 g/cm³. The AF is a glycoporteinaceous particle composed of three major protein species; no core structure could be visualized. In the presence of Ca²⁺, the AF causes secondary aggregation of single cells. The aggregation process is temperature, pH, and ionic strength independent within a broad range. Evidence is presented indicating that two (or more) AF molecules are required for the establishment of a stable cell: cell interaction. In contrast to the AFs from demosponges, the hexactinellid AF functions species-unspecifically.     

Striking convergence in the mouthpart evolution of stream-living algae grazers

This scanning-electron microscopic study demonstrates the convergent evolution of the mouthparts of various herbivorous stream animals (insects from different orders, an isopod, snails, fish, and a tadpole) feeding on epilithic algal pastures. This food source is rich but is often difficult to harvest. Nevertheless, a large number of species can live on it because they have evolved highly specialized mouthparts. There are four functional problems that an algae grazer has to solve: the algae must be removed from the stone, they have to be collected and crushed, and a current shield is needed to prevent the water flow sweeping away the food. Among the 30 algae grazers examined in this study, a limited number of morphological solutions have been found for each of these adaptational problems. There are multiple evolutionary pathways for mouthpart adaptation and even closely related species have often evolved different types of tools for the same function. This refects the existence of a certain amount of evolutionary scope. Such freedom of evolution is present, however, only at the beginning of the adaptiogenesis of an algae grazer. Once one of the evolutionary pathways is taken, further improvement of the mouthparts is possible only by the refinement of the ‘chosen’ type of tools. The consequence of this is that a large number of astonishing convergences have occurred in algae grazers that have independently trodden the same evolutionary pathway.     

Influence of interferon-gamma and extracellular tryptophan on indoleamine 2,3-dioxygenase activity in T24 cells as determined by a non-radiometric assay.

The indoleamine 2,3-dioxygenase (EC 1.13.11.17) activity in human T24 cells has been investigated in cell extracts by using a non-radioactive assay. It is enhanced in a dose-dependent manner up to 25-fold by interferon-gamma. The maximum reaction velocity is increased rather than the Km, which remains at 4 mumol/l. Induction of activity starts 3 h after stimulation and reaches a plateau at 21-48 h. Decreased stimulation was observed in the presence of high L-tryptophan concentrations.     

Localization of immunoreactive epidermal growth factor receptors in human nervous system

Epidermal growth factor is a well-defined peptide which stimulates cell growth and elicits cell responses in a variety of tissues by binding to specific receptors, EGF-R. A specific antiserum against the EGF receptor, which has previously been used to characterize EGF-R in human skin, fibroblasts, and smooth muscle, was used to survey the distribution of EGF-R in human nervous system. Portions of formalin-fixed, paraffin-embedded autopsy specimens were examined by use of immunohistochemical staining (PAP technique) with EGF-R antiserum. Many types of nerve cells, e.g., cerebral cortical pyramidal cells, hippocampal pyramidal cells, Purkinje cells, anterior horn cells, and dorsal root ganglion neurons, contained immunoreactive EGF-R. However, immunoreactive EGF-R were not detected in astrocytes, oligodendrogliocytes, and other small neurons such as granule cells. Intense immunostaining for EGF-R was also detected in ependymal cells from choroidal and extrachoroidal locations. Although immunoreactive EGF-R is widely distributed in human nervous system, the functional role of EGF and its receptor in the nervous system remains unknown.     

Mapping sequenced E.coli genes by computer: software, strategies and examples.

Methods are presented for organizing and integrating DNA sequence data, restriction maps, and genetic maps for the same organism but from a variety of sources (databases, publications, personal communications). Proper software tools are essential for successful organization of such diverse data into an ordered, cohesive body of information, and a suite of novel software to support this endeavor is described. Though these tools automate much of the task, a variety of strategies is needed to cope with recalcitrant cases. We describe such strategies and illustrate their application with numerous examples. These strategies have allowed us to order, analyze, and display over one megabase of E. coli DNA sequence information. The integration task often exposes inconsistencies in the available data, perhaps caused by strain polymorphisms or human oversight, necessitating the application of sound biological judgment. The examples illustrate both the level of expertise required of the database curator and the knowledge gained as apparent inconsistencies are resolved. The software and mapping methods are applicable to the study of any genome for which a high resolution restriction map is available. They were developed to support a weakly coordinated sequencing effort involving many laboratories, but would also be useful for highly orchestrated sequencing projects.     

Plathelminth abundance in North Sea salt marshes: environmental instability causes high diversity

Although supralittoral salt marshes are habitats of high environmental instability, the meiofauna is rich in species and abundance is high. The community structure of free-living Plathelminthes (Turbellaria) in these salt marshes is described. On an average, 104 individuals are found below an area of 10 cm². The average species density in ungrazed salt marshes is 11.3 below 10 cm² and 45.2 below 100 cm², indicating strong small-scale heterogenity. The faunal similarity between sediment and the corresponding above-ground vegetation is higher than between adjacent sample sites. Species prefer distinct ranges of salinity. In the lower part of the supralittoral salt marshes, the annual fluctuations of salinity are strongest and highly unpredictable. This region is richest in plathelminth species and abundance; diversity is highest, and the faunal composition of parallel samples is quite similar. In the upper part of the supralittoral salt marshes, the annual variability of salinity is lower, plathelminths are poor in species diversity and abundance. Parallel samples often have no species in common. Thus, those salt marsh regions with the most unstable environment are inhabited by the most diverse species assemblage. Compared to other littoral zones of the North Sea, however, plathelminth diversity in salt marshes is low. The observed plathelminth diversity pattern can apparently be explained by the dynamic equilibrium model (Huston, 1979).