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.     

Modification of two peptides of bacteriorhodopsin with a pentaamminecobalt (III) complex

Bacteriorhodopsin (bR) was regenerated from the cation-depleted blue membrane with pentaammineaquocobalt(III) tetrafluoroborate [( Co(NH3)5H2O]3+[BF4-]3). Illumination of the sample with orange light decreased the extinction at 568 nm concomitantly with a hypsochromic shift of the absorption maximum. The photocycle of this sample was inhibited, and the rate of proton pumping was reduced. Chymotryptic cleavage of the corresponding apomembrane into the two fragments C1 and C2 and their subsequent separation revealed that cobalt label is only attached to C1. The maximal incorporation of Co into this peptide was 0.3 Co/C1. After cleavage of C1 with cyanogen bromide and subsequent proteolysis with trypsin and chymotrypsin, this modification could be associated with peptides from cyanogen bromide fragments 6 and 9. The sequences were determined to be 101Val-Asp-Ala-Asp-Gln and 228Ala-Ile-Phe-Gly-Glu-Ala-Glu-Ala. These peptides contain the sequences Asp-Ala-Asp and Glu-Ala-Glu, respectively, which might be constituents of the same cation binding site. The observation that the incorporation of Co into bacteriorhodopsin is enhanced under illumination with orange light indicates that this site might be involved in the proton uptake.     

Changes in the auditory system of locusts (Locusta migratoria and Schistocerca gregaria) after deafferentation

Deafferentation experiments during postembryonic development show morphological and/or physiological changes of receptor fibers and of identified auditory interneurons in the CNS of the locusts Locusta migratoria and Schistocerca gregaria after unilateral ablation of one tympanic organ either in the larva or the adult animal.

Assignment of the gene encoding the catalytic subunit Cβ of CAMP-dependent protein kinase to the p36 band on chromosome

Summary A cDNA for the human catalytic subunit (C) of cAMP-dependent protein kinase (PKA) has been cloned from a testis cDNA library. In the present study, we have determined the chromosomal localization of this gene using a cDNA for C as a probe. Southern blot analysis of genomic DNA from human/mouse cell hybrids revealed that the presence or absence of a 20-kbXbaI fragment, which hybridized with the C probe, was concordant with the presence of human chromosome 1.In situ hybridization to metaphase chromosome confirmed the somatic cell hybrid data and regionally mapped the C gene of PKA to the p36 band on chromosome 1.     

Isolation of third, fourth, and fifth instar larval midgut epithelia of the moth, Trichoplusia ni

A new tissue isolation technique was used to create intact midgut epithelial wholemounts from three Trichoplusia ni (Lepidoptera: Noctuidae) larval instars. The protease, dispase, removed the basal lamina and associated connective tissue and allowed for high resolution light microscopy of entire epithelia. Columnar, goblet, differentiating, and stem cells were characterized by double fluorescent labelling of f-actin and nuclei. A comparison of cell populations by digital image analysis revealed significant regional and temporal changes in the density and number of differentiating and stem cells. Growth of the midgut epithelium from third to fourth instar, and from fourth to fifth instar, was accomplished by both cell differentiation and cell division. Cell division however, was greatly reduced from fourth to fifth instar with a concomitant sharp decrease in the stem cell population.     

Isolation and regional localization of a large collection (2,000) of single-copy DNA fragments on human chromosome 3 for mapping and cloning tumor suppressor genes

Summary A collection of 2,000 lambda phage-carrying human single-copy inserts (> 700 bp) were isolated from two chromosome-3 flow-sorted libraries. The single-copy DNA fragments were first sorted into 3p and 3q locations and about 700 3p fragments were regionally mapped using a deletion mapping panel comprised of two humanhamster and two-human-mouse cell hybrids, each containing a chromosome 3 with different deletions in the short arm. The hybrids were extensively mapped with a set of standard 3p markers physically localized or ordered by linkage. The deletion mapping panel divided the short arm into five distinct subregions (A-E). The 3p fragments were distributed on 3p regions as follows: region A, 26%; B, 31%; C, 4%; D, 4% and E, 35%. We screened 300 single-copy DNA fragments from the distal part of 3p (regions A and B) with ten restriction endonucleases for their ability to detect restriction fragment length polymorphisms (RFLPs). Of these fragments 110 (36%) were found to detect useful RFLPs: 35% detected polymorphisms with frequency of heterozygosity of 40% or higher, and 25% with frequency of 30% or higher. All polymorphisms originated from single loci and most of them were of the base pair substitution type. These RFLP markers make it possible to construct a fine linkage map that will span the distal part of chromosome 3p and encompasses the von Hippel-Lindau disease locus. The large number of single-copy fragments (2,000) spaced every 100–150 kb on chromosome 3 will make a significant contribution to mapping and sequencing the entire chromosome 3. The 300 conserved chromosome 3 probes will increase the existing knowledge of man-mouse homologies.     

Derivation of cytotoxic T cell lines that express T cell receptor but exhibit reversible loss of stable antigen binding

Stable cell lines lacking cytotoxic activity against specific target cells were derived from highly active murine CTL clones by the omission of antigen from the culture for several weeks. Several independent CTL clones cultured in the absence of antigen showed a gradual decline in cytotoxic activity, resulting in complete loss by 5 to 10 wk. Such noncytotoxic (NC) cells lacked the ability to form stable conjugates with specific target cells, but were able to kill all target cells tested in the presence of Con A. It was shown by subcloning at limiting dilution that all cells in the starting population were cytolytically active, and that all cells in the NC population derived from such a clone were cytolytically inactive against target cells bearing an appropriate antigen under normal assay conditions. By using the monoclonal antibody F23.1, which reacted with the antigen receptors of two of the CTL clones, it was shown that the NC cells derived from these clones continued to express the receptor at normal levels. Levels of expression of Thy-1.2, Lyt-2.2, and LFA-1 were also similar in all cytotoxic cell lines and their noncytotoxic derivatives. The F23.1 antibody induced an increase in cytoplasmic free Ca2+ in both CTL and NC cells, and NC cells lysed F23.1 hybridoma cells in the absence of Con A. When cells expressing appropriate target cell antigen were added back to cultures of NC cells, cytotoxic activity of appropriate specificity was fully recovered in 2 wk. These results indicate that expression of an apparently functional antigen receptor alone is insufficient for stable binding of CTL to specific target cells, and that other factors dependent upon antigen stimulation may be involved in the recognition process. A difference in affinity for antigen between CTL and NC cells is suggested as a possible explanation for these observations.     

Construction of novel class I histocompatibility antigens by interspecies exon shuffling

Human and mouse class I histocompatibility antigens share considerable structural homology at both the protein and DNA sequence level. This homology has allowed the production of hybrid class I molecules by the reciprocal exchange of DNA sequences corresponding to equivalent domains of HLA-B7 and either H-2Ld or H-2Dd. It is shown that these genes give rise to protein products that are stably expressed on the surface of murine L cells after DNA-mediated gene transfer. These proteins express only those monoclonal antibody-defined H-2 determinants that are expected based on their genetic construction. The molecules have allowed the localization of a number of polymorphic and monomorphic HLA-specific epitopes. In all but one case, expression of an epitope on a domain does not appear to be influenced by the replacement of adjacent human domains with their murine equivalents, suggesting a considerable degree of structural independence of the domains. Cells expressing the hybrid molecules have also been tested as targets for a panel of HLA-B7-specific cytotoxic T cell clones. The results show that the polymorphic determinants recognized by these clones map to the alpha 1 and alpha 2 domains of the HLA-B7 molecule. No evidence for an influence of species-related amino acid sequence differences in the third extracellular domain on T cell recognition was seen. The results are discussed in light of the proposed domain structure of the class I proteins and the potential use of such molecules for further functional studies.     

Seasonal variation in catabolic enzyme activities in breast muscle of some migratory birds

Summary Five bird species were examined in order to ascertain if any changes in flight muscle catabolism take place between breeding season and migration. Two different patterns were discovered. The first consists of a high oxidative capacity and a low glycolytic and anaerobic capacity during migration. The converse occurs during the breeding season, i.e. low oxidative, high glycolytic and anaerobic capacity. The pattern was found in those species that deposit large amounts of fat prior to migration. The second pattern was similar to the first, but there was no change in fatty acid oxidation capacity between breeding season and migration. The pattern was found in those species that do not deposit much fat towards migration. These changes are believed to reflect differences in migration strategy and differences in locomotory activity during different seasons. Deviations from these patterns are discussed.