Keyhole limpet hemocyanin type 2 (KLH2): detection and immunolocalization of a labile functional unit h

Keyhole limpet hemocyanin (KLH) is a mixture of two hemocyanin isoforms, termed KLH1 and KLH2. Within KLH1 eight oxygen-binding functional units (FUs), 1-a to 1-h, have been identified, in contrast to KLH2, which was previously thought to be organized in seven FUs (2-a to 2-g). By limited proteolysis of KLH2 subunits, isolation of the polypeptide fragments, and N-terminal sequencing, we have now identified an eighth FU of type h, with a molecular mass of 43 kDa. This is unusually small for a FU h from a gastropodan hemocyanin. It is also shown that KLH2 didecamers can be split into a stable and homogeneous population of decamers by dialysis against 50 mM Tris/HCl, pH 7.5, in the absence of divalent cations. Electron microscopic immunolocalization using a specific monoclonal antibody reveals that FU KLH2-h is located at the collar of the decamer.     

Reactive oxygen species derived from the mitochondrial respiratory chain are not responsible for the basal levels of oxidative base modifications observed in nuclear DNA of Mammalian cells

The mitochondrial electron transport chain (ETC) is the most important source of reactive oxygen species (ROS) in mammalian cells. To assess its relevance to the endogenous generation of oxidative DNA damage in the nucleus, we have compared the background (steady-state) levels of oxidative DNA base modifications sensitive to the repair glycosylase Fpg (mostly 7,8-dihydro-8-oxoguanine) in wild-type HeLa cells and HeLa rho0 cells. The latter are depleted of mitochondrial DNA and therefore are unable to produce ROS in the ETC. Although the levels of ROS measured by flow cytometry and redox-sensitive probes in rho0 cells were only 10-15% those of wild-type cells, steady-state levels of oxidative DNA base modifications were the same as in wild-type cells. Mitochondrial generation of ROS was then stimulated in HeLa wild-type cells using inhibitors interfering with the ETC. Although mitochondrial ROS production was raised up to 6-fold, none of the substances nor their combinations induced additional oxidative base modifications in the nuclear DNA. This was also true for glutathione-depleted cells. The results indicate that the contribution of mitochondria to the endogenously generated background levels of oxidative damage in the nuclear DNA is negligible.     

Acetyltransfer in natural product biosynthesis--functional cloning and molecular analysis of vinorine synthase

Vinorine synthase (EC 2.3.1.160) catalyses the acetyl-CoA- or CoA-dependent reversible formation of the alkaloids vinorine (or 11-methoxy-vinorine) and 16-epi-vellosimine (or gardneral). The forward reaction leads to vinorine, which is a direct biosynthetic precursor along the complex pathway to the monoterpenoid indole alkaloid ajmaline, an antiarrhythmic drug from the Indian medicinal plant Rauvolfia serpentina. Based on partial peptide sequences a cDNA clone was isolated and functionally expressed in Escherichia coli. The Km values of the native enzyme for gardneral and acetyl-CoA were determined to be 7.5 and 57 microM. The amino acid sequence of vinorine synthase has highest level of identity (28-31%) to that of Papaver salutaridinol acetyltransferase, Fragaria alcohol acyltransferase, and Catharanthus deacetylvindoline acetyltransferase involved in morphine, flavor, and vindoline biosynthesis, respectively. Vinorine synthase is a novel member of the BAHD superfamily of acyltransferases. Site-directed mutagenesis of 13 amino acid residues provided clear evidence that both, His160 and Asp164 of the consensus sequence HxxxD belong to the catalytic center. The mutations also showed that an amino acid triad is not characteristic of vinorine synthase. The experiments demonstrated the importance of the conserved motif SxL/I/VD near the N-terminus and the consensus sequence DFGWG near the C-terminal.     

Testing alternative vicariance scenarios in Western Mediterranean discoglossid frogs

Dated molecular phylogenies are often used to interpret evolutionary history with respect to paleogeographic events. Where more than one interpretation is possible, it is desirable but difficult to assess the alternatives in an objective manner. The present work demonstrates a formalized method for testing molecular clock calibrations and biogeographic scenarios based on them. We assessed the plausibility of several previously published biogeographic hypotheses, using the frog genera Alytes, Discoglossus, and Bombina as model groups. Our data set comprised ca. 900bp of partial mitochondrial 16S and 12S rRNA gene sequences (both genes evolved in a clock-like manner across genera) from nearly all the species and subspecies in the three genera. We tested several calibrations of a molecular clock, which resulted in competing temporal settings for the evolution of taxa. Although only one scenario was in complete accordance with paleogeographic data, statistical testing did not reject the alternatives. Limitations encountered with the present approach may be overcome by more comprehensive analyses in future.     

Differential cycles of range contraction and expansion in European high mountain plants during the Late Quaternary: insights from Pritzelago alpina (L.) O. Kuntze (Brassicaceae)

Nuclear DNA sequence variation of the internal transcribed spacer (ITS) and amplified fragment length polymorphisms (AFLPs) were used to illuminate the evolutionary history of Pritzelago alpina, a herbaceous perennial of (sub)alpine to nival habitats of the European high mountains. Maximum likelihood analysis of ITS sequences of P. alpina, Hornungia petraea and Hymenolobus procumbens (the 'Pritzelago alliance') resolved P. alpina and H. petraea as sister taxa. ITS divergence estimates support an origin for P. alpina in the Late Tertiary, while intraspecific diversification started in the Late Quaternary (0.4-0.9 million years ago). AFLP analysis of 76 individuals of P. alpina, representing 24 localities across its entire west-east distribution, identified four mountain lineages in Cantabria, the Pyrenees, (south-) western Alps, and northeastern Alps/Tatras/Carpathians. In an analysis of molecular variance (amova), 14.3% of the total variation derived from this separation. However, relationships among these lineages remained unresolved in neighbour-joining and principal co-ordinates analyses, suggesting a population history of near simultaneous vicariance. Comparison with our previous ITS/AFLP study of Anthyllis montana (Fabaceae) indicates that the two co-distributed but altitudinally differentiated plant species exhibit temporally concordant but spatially discordant patterns of genetic variation. Moreover, levels of AFLP divergence were significantly lower in P. alpina than in the submediterranean, lower-elevation A. montana. Together, these data are consistent with a 'displacement refugia model', which predicts that European mountain plant species associated with lower- and upper-elevation habitats had a different cycle of range contraction into (long-term) glacial and (short-term) interglacial refugia, respectively.     

How does the eye breathe? Evidence for neuroglobin-mediated oxygen supply in the mammalian retina

Visual performance of the vertebrate eye requires large amounts of oxygen, and thus the retina is one of the highest oxygen-consuming tissues of the body. Here we show that neuroglobin, a neuron-specific respiratory protein distantly related to hemoglobin and myoglobin, is present at high amounts in the mouse retina (approximately 100 microm). The estimated concentration of neuroglobin in the retina is thus about 100-fold higher than in the brain and is in the same range as that of myoglobin in the muscle. Neuroglobin is expressed in all neurons of the retina but not in the retinal pigment epithelium. Neuroglobin mRNA was detected in the perikarya of the nuclear and ganglion layers of the neuronal retina, whereas the protein was present mainly in the plexiform layers and in the ellipsoid region of photoreceptor inner segment. The distribution of neuroglobin correlates with the subcellular localization of mitochondria and with the relative oxygen demands, as the plexiform layers and the inner segment consume most of the retinal oxygen. These findings suggest that neuroglobin supplies oxygen to the retina, similar to myoglobin in the myocardium and the skeletal muscle.     

Molecular evolution of the arthropod hemocyanin superfamily

Arthropod hemocyanins are members of a protein superfamily that also comprises the arthropod phenoloxidases (tyrosinases), crustacean pseudohemocyanins (cryptocyanins), and insect storage hexamerins. The evolution of these proteins was inferred by neighbor-joining, maximum-parsimony, and maximum-likelihood methods. Monte Carlo shuffling approaches provided evidence against a discernible relationship of the arthropod hemocyanin superfamily and molluscan hemocyanins or nonarthropodan tyrosinases. Within the arthropod hemocyanin superfamily, the phenoloxidase probably emerged early in the (eu-)arthropod stemline and thus form the most likely outgroup. The respiratory hemocyanins evolved from these enzymes before the radiation of the extant euarthropodan subphyla. Due to different functional constraints, replacement rates greatly vary between the clades. Divergence times were thus estimated assuming local molecular clocks using several substitution models. The results were consistent and indicated the separation of the cheliceratan and crustacean hemocyanins close to 600 MYA. The different subunit types of the multihexameric cheliceratan hemocyanin have a rather conservative structure and diversified in the arachnidan stemline between 550 and 450 MYA. By contrast, the separation of the crustacean (malacostracan) hemocyanin subunits probably occurred only about 200 MYA. The nonrespiratory pseudohemocyanins evolved within the Decapoda about 215 MYA. The insect hemocyanins and storage hexamerins emerged independently from the crustacean hemocyanins. The time of divergence of the insect proteins from the malacostracan hemocyanins was estimated to be about 430-440 MYA, providing support for the notion that the Hexapoda evolved from the same crustacean lineage as the Malacostraca.     

Vomilenine reductase--a novel enzyme catalyzing a crucial step in the biosynthesis of the therapeutically applied antiarrhythmic alkaloid ajmaline

Delineation of the biochemical pathway leading to the antiarrhythmic Rauvolfia alkaloid ajmaline has been an important target in biosynthetic research for many years. The biosynthetic sequence starting with tryptamine and the monoterpene secologanin consists of about 10 different steps. Most of the participating enzymes have been detected and characterized previously, except those catalyzing the reduction of the intermediate vomilenine. A novel NADPH-dependent enzyme that reduces the intermediate has been isolated from Rauvolfia serpentina cell suspension cultures. Vomilenine reductase (M(r )43 kDa, temp opt 30 degrees C, pH opt 5.7-6.2), saturates the indolenine double bond of vomilenine with stereospecific formation of 2beta(R)-1,2-dihydrovomilenine. The described detection, enrichment and properties of the reductase not only closes a gap in ajmaline biosynthesis but is also a prerequisite for overexpressing the protein heterologously for final clarification of its molecular properties.