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Biomedical subjects

A J Davison

Publications and source records attributed to A J Davison.

At least 109 records · Page 6Linked to original sources
PubMed1984

Redox state of cytochrome c in the presence of the 6-hydroxydopamine/oxygen couple: oscillations dependent on the presence of hydrogen peroxide or superoxide.

The reduction of ferricytochrome c in the presence of 6-hydroxydopamine/O2 mixtures was examined under various reaction conditions. As the autoxidation of 6-hydroxydopamine progressed to completion, there were fluctuations in the net redox reactivity between reducing and oxidizing steady states. This was reflected in a sequence of damped oscillations in the redox state of cytochrome c. Corresponding to the time when 6-hydroxydopamine was 75-100% exhausted, reoxidation of the ferrocytochrome c occurred (prevented by catalase or catalase plus superoxide dismutase). After the H2O2, in turn, was mostly consumed, the next phase commenced in which the cytochrome c became reduced for a second time. This reductive phase was 52% inhibited by superoxide dismutase. In the subsequent and final phase of the process, a progressive oxidation of cytochrome c lasting at least 24 h was observed. Of the initial reduction of ferricytochrome c, at most 37% can be attributed to direct reduction by 6-hydroxydopamine or its semiquinone. This initial net reduction of cytochrome c was inhibited 51% by superoxide dismutase and 41% by catalase. However, since either catalase or superoxide dismutase inhibited the autoxidation of 6-hydroxydopamine by at least as much as it slowed the reduction of cytochrome c, their effects in slowing the reduction of cytochrome c resulted largely from the decreased production of those free radicals which reduce ferricytochrome c, and only in part from accelerated removal. Elimination of the actions of transition metal ions (whether by passage of the buffer solutions through Chelex 100 resins or by addition of desferrioxamine to the reaction medium) slowed both the reoxidation and rereduction by up to 96%. Addition of mannitol decreased the rate of the first reoxidation by 25% and increased the rate of the rereduction by 7%. In general, the oscillations are explicable in terms of changes in the steady state levels of O-2 and H2O2, with metal ions playing a major role and hydroxyl radicals a minor role in both the reoxidation and rereduction.

Chemical Phenomena↗
PubMed1984

Structure of the genome termini of varicella-zoster virus.

The DNA molecule of varicella-zoster virus (VZV) is represented structurally as L-S, where L is a unique sequence and S is a unique sequence flanked by an inverted repeat. S may be present in either orientation in virion DNA molecules, but, to date, L has been found in only one orientation. DNA sequences were determined at the L-S joint and genome termini, which were cloned using methods designed to conserve either the 3' or 5' terminal nucleotide. Molecular hybridization experiments and analysis of the sequences showed that: the genome is not terminally redundant; the unique sequence in L is flanked by an inverted repeat of 88.5 base pairs; a single unpaired nucleotide is located at each 3' terminus of the genome, such that fusion of the termini would produce a sequence identical to that at the L-S joint; approx. 5% of virions contain genomes with L inverted. The implications of these results in possible mechanisms for VZV DNA replication are discussed.

Base Sequence↗
PubMed1983

Fragments from both termini of the herpes simplex virus type 1 genome contain signals required for the encapsidation of viral DNA.

A 535 base pair DNA fragment which maps entirely within the IRS/TRS regions of the herpes simplex virus type 1 (HSV-1) genome and contains all the cis-acting signals necessary for it to function as an origin of viral DNA replication has previously been identified (N.D. Stow and E.C. McMonagle, Virology, in press). When BHK cells were transfected with circular plasmid molecules containing cloned copies of this DNA fragment, and superinfected with wt HSV-1 as helper, amplification of the input plasmid was detected. Two observations indicated that the amplified DNA was not packaged into virus particles. Firstly, when the transfected cells were disrupted the amplified DNA was susceptible to digestion by added DNase, and secondly, it was not possible to further propagate the DNA when virus from the cells was passaged. Fragments from the joint region and from both termini of the viral genome were inserted into origin-containing plasmids and the resulting constructs analysed. In all cases the inserted fragment allowed the amplified DNA to be further passaged, and a proportion to become resistant to digestion with DNase. These observations suggest that signals required for the encapsidation of HSV-1 DNA are located within DNA sequences shared by the inserted fragments and therefore lie within the reiterated 'a' sequence of the viral genome.

Animals↗
PubMed1983

Aminooxyacetic acid provides transient protection against seizures induced by hyperbaric oxygen.

In an attempt to demonstrate a protective effect against convulsions induced by hyperbaric oxygen (OHP), aminooxyacetic acid (AOAA at 2.5 mg/kg) was administered subcutaneously to 60 white leghorn cockerels (aged 21-28 days) at periods of 1-72 h before exposure to OHP. A transient protection was noted 1-8 h after administration of the AOAA. This took the form of a 51-67% increase in the latent period before OHP induced convulsions began. By 12 h after injection, the protective effect had disappeared. Thereafter a phase of increased susceptibility to hyperbaric oxygen commenced. This phase lasted for at least a further 60 h.

Acetates↗
PubMed1983

DNA sequence of the US component of the varicella-zoster virus genome.

The linear duplex DNA molecule of varicella-zoster virus is 120 000 bp in size and has the sequence arrangement UL-IRS-US-TRS, where UL and US are unique sequences and IRS and TRS are inverted repeats flanking US. The primary structure of the cloned SstI g DNA fragment containing US (5232 bp) and adjacent portions of IRS and TRS (426 bp of each) was determined, and the following model for genetic expression was derived from an analysis of the sequence. The region specifies four mRNAs encoding primary translation products with mol. wts. of 11, 44, 39 and either 74 or 70 kd. The 39-and 70-kd proteins have primary structures characteristic of membrane proteins. The mRNAs encoding the 11- and 74/70-kd proteins extend from opposite sides of US into IRS/TRS, thus sharing a common 3' terminus. These proteins do not share a common carboxy terminus because the coding region for the 11-kd protein terminates at the junction between US and IRS, whereas that for the 74/70-kd protein extends into TRS. The analysis affirms the hypothesis that the extent of inverted repeats in herpesvirus genomes is primarily a result of constraints imposed by adjacent protein coding sequences.

Amino Acid Sequence↗
PubMed1983

Inversion of the two segments of the herpes simplex virus genome in intertypic recombinants.

We have analysed by restriction site mapping the structures of the termini and L-S joint in several HSV-1/HSV-2 intertypic recombinants, including Bx1(28-1), the virion DNA of which has a marked overabundance of one orientation of the L segment, and subclones of Bx1(28-1). All recombinants with both orientations of L present in equal amounts contain TRL and IRL regions derived at least in part from the same parent (HSV-1 or HSV-2) as a result of previously undetected crossovers in these regions. Recombinants with a predominance of one orientation of L have TRL and IRL regions derived from different parents. Homology between a sequences alone at the L terminus and L-S joint is sufficient for normal inversion of L. Analysis of another recombinant, RE4, which fails to invert normally in both L and S, suggests that normal inversion of S is dependent upon the presence of TRS and IRS regions derived at least in part from the same parent. We conclude that segment inversion specifically depends upon the a sequence, that the process of DNA replication and maturation does not necessarily produce molecules with identical a sequences, and that direct ligation of termini may occur during DNA replication.

Base Sequence↗
PubMed1983

Either orientation of the L segment of the herpes simplex virus genome may participate in the production of viable intertypic recombinants.

We have analysed the genome structures of 90 recombinant viruses produced by co-infection of baby hamster kidney cells with the DNA of a herpes simplex virus type 1 temperature-sensitive mutant (tsD) and specific DNA fragments of wild-type herpes simplex virus type 2 at non-permissive temperature. Crossovers were located predominantly in regions of greatest intertypic homology, and we conclude that primary recombination occurred with the L segment of the genome in either orientation.

Animals↗
PubMed1983

Location and orientation of homologous sequences in the genomes of five herpesviruses.

Molecular hybridization experiments were carried out to investigate homologous regions in the genomes of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), equid herpesvirus 1 (EHV-1), pseudorabies virus (PRV) and varicella-zoster virus (VZV). Virion DNA probes from EHV-1, PRV and VZV hybridized to similar regions of the HSV genome, and the use of cloned DNA probes allowed heterologous genomes to be oriented with respect to homologous regions. The HSV-1 and HSV-2 genomes are colinear, the EHV-1 and VZV genomes are colinear with the IL or ISL genome arrangement of HSV, and the PRV genome is essentially colinear with the IL genome arrangement of HSV except that the region 0.1 to 0.4 fractional genome units appears to be inverted. A detailed analysis of sequences in the HSV-2 and PRV genomes to which the HSV-1 major capsid protein gene hybridized was carried out in order to demonstrate the application of molecular hybridization to the location of genes in heterologous genomes. The lesion in a DNA-positive temperature-sensitive mutant of PRV was mapped within the putative PRV major capsid protein gene. We conclude that the herpesviruses we have studied possess several highly conserved genes, and propose that they are similar in genetic organization despite presumably separate evolutionary histories.

Capsid↗
PubMed1982

Genomic location and lack of phosphorylation of the HSV immediate-early polypeptide IE 12.

Herpes simplex virus type 1 (HSV-1) induces an immediate-early (IE) polypeptide IE 12. An equivalent polypeptide coded by HSV-2 which migrated slightly more slowly on SDS-polyacrylamide gels was identified and designated IE 12.3. Analysis of the serotype of the IE polypeptide induced by five HSV-1/HSV-2 intertypic recombinants when correlated with their genome structures showed that IE 12/IE 12.3 mapped in the region spanning the TRs/Us junction. Unlike four other IE polypeptides induced by HSV-1 (IEs 175, 110, 68 and 63), IE 12 was not detectably phosphorylated.

Base Sequence↗
PubMed1982

Thymidine kinase deletion mutants of herpes simplex virus type 1.

Deletions in the cloned thymidine kinase (TK) gene of herpes simplex virus type 1 (HSV-1), strain 17 syn+, were produced by two methods. Removal of a 506 base pair fragment from between the unique SstI and Bg/II restriction endonuclease sites of pTK1 (HSV-1 BamHI p cloned in pAT153) and subsequent transformation of Escherichia coli resulted in the isolation of 50 deleted plasmids. Sequential digestion of pTK1 with Bg/II and nuclease BAL 31 followed by ligation and recleavage with Bg/II resulted in the isolation of 31 deleted plasmids. Three clones, pTK2, pTK3 and pTK4, obtained following Bg/II and SstI treatment of pTK1 were recombined with wild-type (wt) HSV-1 (17) syn+ DNA in baby hamster kidney (BHK) cells to produce TK- deletion mutants HSV-1 (17) TK 1301, HSV-1 (17) TK 1302 and HSV-1 (17) TK 1303 respectively. 5-Bromo-2'-deoxyuridine, 5-bromo-2'-deoxycytidine and 9-(2-hydroxyethoxymethyl)guanine were used to reduce the background of TK+ virus in heterogeneous recombinant stocks analysed for the presence of TK- recombinants. All recombinant clones isolated produced a small syncytial plaque morphology in BHK cells. The mutants HSV-1 (17) TK 1301 and HSV-1 (17) TK 1302 were TK-, failed to produce polypeptides of molecular weights 43000 and 19000 found in wt-infected cells and demonstrated one-step growth curves different from wt virus and the TK- mutant HSV-1 (17) dPyk-7. Superinfection studies with HSV-1 (17) TK 1301, HSV-1 (17) TK 1302, HSV-1 (MDK) and HSV-1 (17) dPyk-7 indicated that all TK- mutants except dPyK-7 produce a trans-acting gene product which can switch on the transforming HSV-1 TK gene.

Animals↗
PubMed1981

The relative effectiveness of .OH, H2O2, O2-, and reducing free radicals in causing damage to biomembranes. A study of radiation damage to erythrocyte ghosts using selective free radical scavengers.

The relative effectiveness of oxidizing (.OH, H2O2), ambivalent (O2-) and reducing free radicals (e- and CO2-) in causing damage to membranes and membrane=bound glyceraldehyde-3-phosphate dehydrogenase of resealed erythrocyte ghosts has been determined. The rates of damage to membrane-bound glyceraldehyde-3-phosphate dehydrogenase (R(enz)) were measured and the rates of damage to membranes (R(mb)) were assessed by measuring changes in permeability of the resealed ghosts to the relatively low molecular weight substrates of glyceraldehyde-3-phosphate dehydrogenase. Each radical was selectively isolated from the mixture produced during gamma-irradiation, using appropriate mixtures of scavengers such as catalase, superoxide dismutase and formate. .OH, O2- and H2O2 were approximately equally effective in inactivating membrane-bound glyceraldehyde-3-phosphate dehydrogenase, while e- and CO2- were the least effective. R(enz) values of O2- and H2O2 were 10-times and of .OH 15-times that of e-. R(mb) values were quite similar for e- and H2O2 (about twice that of O2-), while that of .OH was 3-times that of O2-. Hence, with respect to R(mb): .OH greater than e- = H2O2 greater than O2-, and with respect to R(enz): .OH greater than O2- = H2O2 much greater than e-. The difference between the effectiveness of the most damaging and the least damaging free radicals was more than 10-fold greater in damage to the enzyme than to the membranes. Comparison between H2O2 added as a chemical reagent and H2O2 formed by irradiation showed that membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase were relatively inert to reagent H2O2 but markedly susceptible to the latter.

Catalase↗
PubMed1981

One functional copy of the long terminal repeat gene specifying the immediate-early polypeptide IE 110 suffices for a productive infection of human foetal lung cells by herpes simplex virus.

The HSV-1/HSV-2 intertypic recombinant Bx1 (28-1) is heterotypic for the repeat sequences flanking the long unique region of the genome (IRL and TRL) and expresses both the immediate-early polypeptide IE 110 of HSV-1 and its functionally equivalent polypeptide IE 118 of HSV-2. The genome structures of five subclones of this recombinant and the immediate-early polypeptides they induce have been analysed. Subclone 14 lacked most of the IRL sequence, including the region from which part of the mRNA for IE 110 is transcribed, and expressed only the HSV-2 IE 118. Subclone 22 lacked almost all of TTL including the gene for IE 118 and induced only the HSV-1 IE 110. Since both subclones produced viable progeny in HFL cells it follows that expression of only one copy of the equivalent genes in TRL and IRL, here coding for the distinguishable polypeptides IE 110 or IE 118, is sufficient for successive complete cycles of virus replication.

Cells, Cultured↗
PubMed1981

Nucleotide sequences of the joint between the L and S segments of herpes simplex virus types 1 and 2.

The a sequence of herpes simplex virus (HSV) is present as a direct repeat at the genomic termini and also in inverted orientation at the joint between the L and S segments. DNA sequences have been determined for the joint regions of the genomes of HSV-1 and HSV-2, and relative to these sequences the genomic termini are in both cases located close to a short direct repeat of 17 to 21 base pairs (bp) at the b-a and a-c junctions. The HSV-1 joint region contains three separate tandem direct reiterations of short sequences, (12, 16 and 17 bp in strain 17) and we conclude that size heterogeneity in the a and c sequences is due to variable copy numbers of these repeated units. It is likely that a considerable part of the HSV-1 joint region does not code for polypeptide.

Base Sequence↗
PubMed1981

Analysis of the genome of equine herpesvirus type 1: arrangement of cleavage sites for restriction endonucleases EcoRI, BglII and BamHI.

The genome of an Australian isolate of equine herpesvirus type 1 (equine abortion virus) has been analysed using the restriction endonucleases EcoRI, BglII and BamHI, and a physical map constructed. Terminal fragments were identified by exonuclease treatments, and linkage of fragments was deduced by a combination of single- and double-digest experiments and cross-blot hybridizations. The genome has a mol. wt. of 100 x 10(6) and is comprised of a short unique region bounded by repetitive sequences, which is present in both orientations in approximately equal amounts in the DNA population, and a long unique region existing in only one orientation.

Bacterial Proteins↗
PubMed1981

Actions of gamma-radiation on resealed erythrocyte ghosts. A comparison with intact erythrocytes and a study of the effects of oxygen.

With respect to both permeability and inactivation of membranous GAPDH, ghosts were more susceptible than erythrocytes to free radicals produced in the gamma-irradiation of aqueous solutions. The rate of increase in the permeability of irradiated ghosts was immeasurably greater than that of irradiated erythrocytes, while the rate of inactivation of GAPDH was 21-fold greater. The sensitivity of ghosts to radiation damage was affected strongly by the presence of oxygen during irradiation. In the presence of air, the rates of increase of permeability and inactivation of GAPDH were 2.8- and 1.5-fold of those in the presence of N2. The use of buffer saturated with oxygen accelerated the aerobic rates of increase of permeability and inactivation of GAPDH by 60- and 2.7-fold. These results indicate that inactivation of GAPDH is somewhat sensitive to oxygen, particularly at high concentration of oxygen. Nevertheless, in air or under nitrogen, the rate of enzymic inactivation was almost an order of magnitude greater than that of increase of permeability, indicating that the former is much more sensitive to irradiation. The major mechanism of the oxygen effect observed is the ability of oxygen to increase the branching of the free radical chain reactions which propagate damage after initiation within the membrane.

Cell Membrane Permeability↗