Isolation, expression and characterization of a human apolipoprotein B 100-specific cDNA clone

The isolation and characterization of a human apolipoprotein B 100-specific cDNA clone (lambda gt-B1) containing a 1321 base pairs (bp) spanning insert is described. It encodes the 3'-nontranslated 281 bp long region up to the polyadenylation site and 1040 bp of the C-terminal coding region of 345 amino-acid residues of human apo B 100 and the stop codon. The lambda gt-B1 cDNA clone has been isolated from a human hepatoma cDNA expression library by immunoscreening using affinity-purified polyclonal anti apo B 100 antibodies. The nucleotide sequence of the apo B 100 insert has been determined. A part of the polypeptide sequence derived from this nucleotide sequence was identical with the amino-acid sequence obtained by protein sequencing of a purified cyanogen bromide fragment of apo B 100. The fusion protein consisting of beta-galactosidase and the 345 amino-acid residue long C-terminus of apo B 100 had an apparent molecular mass of 148 kDa in NaDodSO4 polyacrylamide gel electrophoresis. In Northern blot hybridization analysis the insert of the apo B 100-cDNA clone hybridized to a 20 to 22 kb mRNA from adult human liver.     

Indole alkaloids from cell suspension cultures of Rauwolfia serpentina benth

Twelve indole alkaloids belonging to the Ajmaline-, Sarpagine-, Yohimbine-, and Heteroyohimbine-type have been isolated and identified from cell suspension cultures of Rauwolfia serpentina. Ten of the alkaloids were found for the first time in cultured R. serpentina cells. The yield of the main alkaloid vomilenine was 51 times more than that of differentiated plants. Crude enzymes isolated from this cell suspension culture completely metabolize the biogenetic precursor strictosidine under formation of several alkaloidal compounds.     

Vibrational modes of tyrosines in cytochrome c oxidase from Paracoccus denitrificans: FTIR and electrochemical studies on Tyr-D4-labeled and on Tyr280His and Tyr35Phe mutant enzymes

A combined electrochemical and FTIR spectroscopic approach was used to identify the vibrational modes of tyrosines in cytochrome c oxidase from Paracoccus denitrificans which change upon electron transfer and coupled proton transfer. Electrochemically induced FTIR difference spectra of the Tyr-D4-labeled cytochrome c oxidase reveal that only small contributions arise from the tyrosines. Contributions between 1600 and 1560 cm(-1) are attributed to nu8a/8b(CC) ring modes. The nu19(CC) ring mode for the protonated form of tyrosines is proposed to absorb with an uncommonly small signal at 1525-1518 cm(-1) and for the deprotonated form at 1496-1486 cm(-1), accompanied by the increase of the nu19(CC) ring mode of the Tyr-D(4)-labeled oxidase at approximately 1434 cm(-1). A signal at 1270 cm(-1) can be tentatively attributed to the nu7'a(CO) and delta(COH) mode of a protonated tyrosine. Uncommon absorptions, like the mode at 1524 cm(-1), indicate the involvement of Tyr280 in the spectra. Tyr280 is a crucial residue close to the binuclear center and is covalently bonded to His276. The possible changes of the spectral properties are discussed together with the absorbance spectra of tyrosine bound to histidine. The vibrational modes of Tyr280 are further analyzed in combination with the mutation to histidine, which is assumed to abolish the covalent bonding. The electrochemically induced FTIR difference spectra of the Tyr280His mutant point to a change in protonation state in the environment of the binuclear center. Together with an observed decrease of a signal at 1736 cm(-1), previously assigned to Glu278, a possible functional coupling is reflected. In direct comparison to the FTIR difference spectra of the D4-labeled compound and comparing the spectra at pH 7 and 4.8, the protonation state of Tyr280 is discussed. Furthermore, a detailed analysis of the mutant is presented, the FTIR spectra of the CO adduct revealing a partial loss of Cu(B). Electrochemical redox titrations reflect a downshift of the heme a3 midpoint potential by 95 +/- 10 mV. Another tyrosine identified to show redox dependent changes upon electron transfer is Tyr35, a residue in the proposed D-pathway of the cytochrome c oxidase.     

Tracing the D-pathway in reconstituted site-directed mutants of cytochrome c oxidase from Paracoccus denitrificans

Heme-copper terminal oxidases use the free energy of oxygen reduction to establish a transmembrane proton gradient. While the molecular mechanism of coupling electron transfer to proton pumping is still under debate, recent structure determinations and mutagenesis studies have provided evidence for two pathways for protons within subunit I of this class of enzymes. Here, we probe the D-pathway by mutagenesis of the cytochrome c oxidase of the bacterium Paracoccus denitrificans; amino acid replacements were selected with the rationale of interfering with the hydrophilic lining of the pathway, in particular its assumed chain of water molecules. Proton pumping was assayed in the reconstituted vesicle system by a stopped-flow spectroscopic approach, allowing a reliable assessment of proton translocation efficiency even at low turnover rates. Several mutations at positions above the cytoplasmic pathway entrance (Asn 131, Asn 199) and at the periplasmic exit region (Asp 399) led to complete inhibition of proton pumping; one of these mutants, N131D, exhibited an ideal decoupled phenotype, with a turnover comparable to that of the wild-type enzyme. Since sets of mutations in other positions along the presumed course of the pathway showed normal proton translocation stoichiometries, we conclude that the D-pathway is too wide in most areas above positions 131/199 to be disturbed by single amino acid replacements.     

Carotenoid:methyl-beta-cyclodextrin formulations: an improved method for supplementation of cultured cells

BC. Two days after supplementation with 5 microM BC in MbetaCD, cellular BC levels reached a maximum of 140+/-11 pmol/microg DNA, leveling off to 100+/-15 pmol/microg DNA until day 8. Incubation with BC dissolved in THF/DMSO resulted in a lower BC uptake of 105+/-14 pmol/microg DNA and 64+/-20 pmol/microg DNA respectively. No cytotoxic effects of these formulations were detected. The results show that the MbetaCD formulation is an improved method for investigations of carotenoids and other lipophilic compounds in in vitro test systems compared to methods using organic solvents.     

An as-1-like motif controls the level of expression of the gene for the pathogenesis-related protein 1a from tobacco

Pathogenesis-related proteins of group 1 (PR-1) are strongly induced in plants by pathogen attack, exposure of the plants to (acetyl)salicylic acid (ASA, SA), and by developmental cues. Functional analysis of the PR-1a promoter identified a region of 139 bp (from -691 to -553) mediating expression of the GUS reporter gene in response to ASA. Inspection of this region revealed two TGACG elements reminiscent of activation sequence-1 (as-1). Recently, as-1 has been reported to be responsive to SA in the context of the CaMV 35S RNA promoter. To address the question of whether the as-1-like sequence may be of functional significance for the expression of the PR-1a gene, gel shift assays were performed with TGA1a, a protein been shown to interact with as-1 in vitro. TGA1a was found to bind to the PR-1a as-1-like sequence with similar specificity and affinity as to as-1. Furthermore, mutations were introduced in the as-1-like sequence in the context of the inducible 906 bp PR-1a promoter which are impaired in binding TGA1a in vitro. Significantly reduced levels of GUS reporter gene activity were obtained with the mutant promoter regions as compared to the wild-type PR-1a promoter in response to all stimuli in transgenic tobacco plants. Yet, mutation of the as-1-like sequence did not abolish induction of reporter gene expression. Taken together, these results suggest that the level of expression of the tobacco PR-1a gene is controlled by an as-1-like sequence motif in the PR-1a upstream region, possibly interacting with a factor related to TGA1a.     

Deposition and cycling of carbon and nitrogen in carbonate mud of the lagoons of Arlington and Sudbury Reefs, Great Barrier Reef

The dynamics of carbon and nitrogen in carbonate mud were examined in the lagoons of Arlington and Sudbury Reefs, Great Barrier Reef. Most (89–93%) of the organic carbon and total nitrogen depositing to the carbonate mud zones was mineralized over a sediment depth of 1 m, with ∼50% of CO₂ produced during microbial decomposition involved in carbonate precipitation/dissolution reactions. There was proportionally little burial of organic carbon (10–11%) or nitrogen (7–10%). Nitrogen budgets suggest rapid turnover of porewater inorganic N pools on the order of hours to a few days. Incubation experiments indicate carbonate dissolution in surface deposits (≤20 cm depth) and carbonate precipitation in deeper sediments. Depth-integrated reaction rates indicate net carbonate precipitation of 7–10 mol CaCO₃ m² year⁻¹ over a depth of 1 m. Budget calculations at the whole-reef scale imply that deposition of CaCO₃ in the mud zones of both lagoons may equate to 50–90% of total reef carbonate production, with organic carbon fluxes equating to nearly all net primary production on each reef. These biogeochemical estimates point to the functional importance of carbonate mud zones in the lagoons of the shelf reefs of the Great Barrier Reef.     

Spectroscopic study on the communication between a heme a3 propionate, Asp399 and the binuclear center of cytochrome c oxidase from Paracoccus denitrificans

The proton pumping mechanism of cytochrome c oxidase on a molecular level is highly disputed. Recently theoretical calculations and real time electron transfer measurements indicated the involvement of residues in the vicinity of the ring A propionate of heme a3, including Asp399 and the CuB ligands His 325, 326. In this study we probed the interaction of Asp399 with the binuclear center and characterize the protonation state of its side chain. Redox induced FTIR difference spectra of mutations at the site in direct comparison to wild type, indicate that below pH 5 Asp 399 displays signals typical for the deprotonation of the acidic residue with reduction of the enzyme. Interestingly at a pH higher than 5, no contributions from Asp 399 are evident. In order to probe the interaction of the site with the binuclear center we followed the rebinding of CO by infrared spectroscopy for mutations on residue Asp399 to Glu, Asn and Leu. Previously different CO conformers have been identified for bacterial cytochrome c oxidases, and its pH dependent behaviour discussed to be relevant for catalysis. Interestingly we observe the lack of this pH dependency and a strong influence on the observable conformers for all mutants studied here, clearly suggesting a communication of the site with the heme-copper center and the nearby histidine residues.