Conserved polynucleotide sequences among the genomes of papillomaviruses.

The DNAs of different members of the Papillomavirus genus of papovaviruses were analyzed for nucleotide sequence homology. Under standard hybridization conditions (Tm - 28 degrees C), no homology was detectable among the genomes of human papillomavirus type 1 (HPV-1), bovine papillomavirus type 2 (BPV-2), or cottontail rabbit (Shope) papillomavirus (CRPV). However, under less stringent conditions (i.e., Tm - 43 degrees C), stable hybrids were formed between radiolabeled DNAs of CRPV, BPV-1, or BPV-2 and the HindIII-HpaI A, B, and C fragments of HPV-1. Under these same conditions, radiolabeled CRPV and HPV-1 DNAs formed stable hybrids with HincII B and C fragments of BPV-2 DNA. These results indicate that there are regions of homology with as much as 70% base match among all these papillomavirus genomes. Furthermore, unlabeled HPV-1 DNA competitively inhibited the specific hybridization of radiolabeled CRPV DNA to bpv-2 DNA fragments, indicating that the homologous DNA segments are common among these remotely related papillomavirus genomes. These conserved sequences are specific for the Papillomavirus genus of papovaviruses as evidenced by the lack of hybridization between HPV-1 DNA and either simian virus 40 or human papovavirus BK DNA under identical conditions. These results indicate a close evolutionary relationship among the papillomaviruses and further establish the papillomaviruses and polyoma viruses as distinct genera.     

Polymorphism of carbon oxidation of drugs and clinical implications.

Eight volunteers previously phenotyped for their ability to hydroxylate debrisoquine (four extensive metabolisers (EM), four poor metabolisers (PM) were investigated for their metabolic handling of guanoxan and phenacetin. All three drugs are oxidised at carbon centres. Oxidative dealkylation of phenacetin was determined by measuring the rate of formation of paracetamol. The EM subjects excreted mostly metabolites of guanoxan (mean 29% of dose), whereas the PM group excreted large amounts of unchanged drug (48% of dose). The rate of formation of paracetamol was noticeably slower in the PM group, and, when analysed by minimum estimates of apparent first-order rate constants, the difference between the two phenotypes was significant. Thus the hydroxylation defect shown for debrisoquine metabolism carries over to the oxidative metabolism of phenacetin and guanoxan. Some 5% of the population are genetically defective hydroxylators of drugs. Thus methods for evaluating the metabolism of new drugs in respect of usage and side effects need to be revised.     

Involvement of 16S rRNA nucleotides G1338 and A1339 in discrimination of initiator tRNA

Three consecutive G-C pairs in the anticodon stem are a key discriminatory feature of initiator tRNA and are required for its selection by IF3. Here, we have mutated two 16S rRNA nucleotides, G1338 and A1339, which provide the sole contact to the G-C pairs of tRNA(fMet) bound to the ribosomal P site. We have tested their effects on translational activities in vivo and have affinity-purified mutant 30S subunits for functional analysis in vitro. Our results are consistent with the formation of Type II and I minor interactions, respectively, between G1338 and A1339 and the anticodon stem of tRNA and suggest that these interactions play a role in tRNA(fMet) discrimination by IF3. Moreover, our findings indicate that discrimination also involves recognition of at least one additional feature of the tRNA(fMet) anticodon stem loop.     

The Release of CO<Subscript>2</Subscript> from Riverwaters – the Contribution of Excess CO<Subscript>2</Subscript> from Groundwater

The dissolved CO ₂ concentration of stream waters is an important component of the terrestrial carbon cycle and an important pathway for release of CO₂ to the atmosphere. This study uses data from the UK's largest groundwater monitoring network to estimate the importance of groundwater in contributing excess dissolved CO₂ to the atmosphere. The study shows that:

FTIR difference spectra of Wolinella succinogenes quinol:fumarate reductase support a key role of Glu C180 within the "E-pathway hypothesis" of coupled transmembrane electron and proton transfer

Electrochemically induced static FTIR difference spectroscopy has been employed to investigate redox-driven protonation changes of individual amino acid residues in the quinol:fumarate reductase (QFR) from Wolinella succinogenes. The difference spectra presented were taken in the mid-infrared region from 1800 to 1000 cm(-1), and the signals obtained represent transitions between the reduced and oxidized states of the enzyme. Analysis of the difference spectra shows evidence for structural reorganizations of the polypeptide backbone upon the induced redox reaction. Furthermore, spectral contributions were found above 1710 cm(-1) where stretching vibrations of protonated carboxyl groups from aspartic or glutamic acid side chains absorb. With the help of site-directed mutagenesis and hydrogen/deuterium isotope exchange, it was possible to identify amino acid residue Glu C180, which is located in the membrane-spanning, diheme-containing subunit C of QFR, as being partially responsible for the derivative-shaped spectral feature with a peak/trough at 1741/1733 cm(-1) in the reduced-minus-oxidized difference spectrum. This signal pattern is interpreted as a superposition of a protonation/deprotonation and a change of the hydrogen-bonding environment of Glu C180. This residue is the principal constituent of the recently proposed "E-pathway hypothesis" of coupled transmembrane proton and electron transfer in QFR [Lancaster, C. R. D. (2002) Biochim. Biophys. Acta 1565, 215-231]. Thus, the study presented yields experimental evidence which supports a key role of Glu C180 within the framework of the E-pathway hypothesis.     

Characterization of the menaquinone reduction site in the diheme cytochrome b membrane anchor of Wolinella succinogenes NiFe-hydrogenase

The majority of bacterial membrane-bound NiFe-hydrogenases and formate dehydrogenases have homologous membrane-integral cytochrome b subunits. The prototypic NiFe-hydrogenase of Wolinella succinogenes (HydABC complex) catalyzes H2 oxidation by menaquinone during anaerobic respiration and contains a membrane-integral cytochrome b subunit (HydC) that carries the menaquinone reduction site. Using the crystal structure of the homologous FdnI subunit of Escherichia coli formate dehydrogenase-N as a model, the HydC protein was modified to examine residues thought to be involved in menaquinone binding. Variant HydABC complexes were produced in W. succinogenes, and several conserved HydC residues were identified that are essential for growth with H2 as electron donor and for quinone reduction by H2. Modification of HydC with a C-terminal Strep-tag II enabled one-step purification of the HydABC complex by Strep-Tactin affinity chromatography. The tagged HydC, separated from HydAB by isoelectric focusing, was shown to contain 1.9 mol of heme b/mol of HydC demonstrating that HydC ligates both heme b groups. The four histidine residues predicted as axial heme b ligands were individually replaced by alanine in Strep-tagged HydC. Replacement of either histidine ligand of the heme b group proximal to HydAB led to HydABC preparations that contained only one heme b group. This remaining heme b could be completely reduced by quinone supporting the view that the menaquinone reduction site is located near the distal heme b group. The results indicate that both heme b groups are involved in electron transport and that the architecture of the menaquinone reduction site near the cytoplasmic side of the membrane is similar to that proposed for E. coli FdnI.     

Discovery and characterization of a Coenzyme A disulfide reductase from Pyrococcus horikoshii. Implications for this disulfide metabolism of anaerobic hyperthermophiles

We have cloned NADH oxidase homologues from Pyrococcus horikoshii and P. furiosus, and purified the recombinant form of the P. horikoshii enzyme to homogeneity from Escherichia coli. Both enzymes (previously referred to as NOX2) have been shown to act as a coenzyme A disulfide reductases (CoADR: CoA-S-S-CoA + NAD(P)H + H+-->2CoA-SH + NAD(P)+). The P. horikoshii enzyme shows a kcat app of 7.2 s(-1) with NADPH at 75 degrees C. While the enzyme shows a preference for NADPH, it is able to use both NADPH and NADH efficiently, with both giving roughly equal kcats, while the Km for NADPH is roughly eightfold lower than that for NADH. The enzyme is specific for the CoA disulfide, and does not show significant reductase activity with other disulfides, including dephospho-CoA. Anaerobic reductive titration of the enzyme with NAD(P)H proceeds in two stages, with an apparent initial reduction of a nonflavin redox center with the first reduction resulting in what appears to be an EH2 form of the enzyme. Addition of a second of NADPH results in the formation of an apparent FAD-NAD(P)H complex. The behavior of this enzyme is quite different from the mesophilic staphylococcal version of the enzyme. This is only the second enzyme with this activity discovered, and the first from a strict anaerobe, an Archaea, or hyperthermophilic source. P. furiosus cells were assayed for small molecular mass thiols and found to contain 0.64 micromol CoA.g dry weight(-1) (corresponding to 210 microM CoA in the cell) consistent with CoA acting as a pool of disulfide reducing equivalents.