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Biomedical subjects

A Orr

Publications and source records attributed to A Orr.

At least 19 recordsLinked to original sources
PubMed1992

Rational design and molecular effects of a new topoisomerase II inhibitor, azatoxin.

Azatoxin [NSC 640737-M; 5.R,11aS-1H,6H,3-one-5,4,11,11a-tetrahydro-5-(3,5-dimethoxy-4-hydr oxyphenyl) oxazolo (3',4':1,6)pyrido-(3,4-b)indole] was rationally designed from a model for the pharmacophore of drugs with topoisomerase II inhibition activity. This pharmacophore has at least 2 domains: a quasiplanar polycyclic ring system proposed to bind between the DNA base pairs and a pendant substituent proposed to interact with the enzyme and/or to the DNA grooves. The present study shows that, in cell free systems, azatoxin induces a large number of double strand-breaks in linear Simian virus 40 and human c-myc DNA. These breaks yield cleavage patterns that are different from those of well established topoisomerase II inhibitors (epipodophyllotoxins, amsacrine, mitoxantrone). Azatoxin also inhibits the catalytic activity of purified topoisomerase II, and is a nonintercalator. The structure-activity relationship of 3 isomers and 6 derivatives of azatoxin shows a stringent stereochemical requirement for activity. The effects of azatoxin pendant ring substitution on topoisomerase II mediated DNA cleavage activity were similar to the relationship observed for etoposide.

DNA↗
PubMed1992

Differential effects of amsacrine and epipodophyllotoxins on topoisomerase II cleavage in the human c-myc protooncogene.

Amsacrine and demethylepipodophyllotoxins (etoposide and teniposide) are potent topoisomerase II inhibitors which have optimum activity in different cancers. To investigate whether these differences are due to different activity on cellular oncogenes, drug-induced topoisomerase II cleavage sites were mapped and sequenced in the human c-myc protooncogene. In the presence of purified murine L1210 topoisomerase II, amsacrine induces prominent cleavage in the P2 promoter (site 2499/2502). Footprinting experiments indicate that topoisomerase II binds to the entire promoter region (approximately 20 base pairs on the sides of the P2 site). In the case of teniposide or etoposide, cleavage is more diffuse and markedly less at the P2 site. Mapping of cleavage sites in human small cell lung carcinoma cells (NCI N417) also shows that cleavage in the P2 promoter region is induced preferentially by amsacrine but not by demethylepipodophyllotoxins. Thus, selective gene damage among topoisomerase II inhibitors may contribute to differential anticancer activity.

Amsacrine↗
PubMed1991

Distribution of topoisomerase II cleavage sites in simian virus 40 DNA and the effects of drugs.

The distributions of DNA cleavage sites induced by topoisomerase II in the presence or absence of specific drugs were mapped in the simian virus 40 genome. The drugs studied were 5-iminodaunorubicin, amsacrine (m-AMSA), teniposide (VM-26) and 2-methyl-9-hydroxyellipticinium; each produced a distinctive pattern of enhanced cleavage. Consistently intense cleavage, both in the presence and in the absence of drugs, occurred in the nuclear matrix-associated region. Since topoisomerase II is a major constituent of the nuclear matrix, and cleavage complexes include a covalent link between topoisomerase II and DNA, the findings suggest that topoisomerase II may function to attach DNA to the nuclear matrix. Cleavage usually occurred on both DNA strands with the expected four base-pair 5' stagger, and strong sites tended to occur within A/T runs such as have been associated with binding to the nuclear scaffold. Intense cleavage was present also in the replication termination region, but was absent from the vicinity of the replication origin. Cleavage intensities were found to change with time in a manner that depended both on the site and on the drug, suggesting that topoisomerase II can move along the DNA from a kinetically preferred site to a thermodynamically preferred site.

Amsacrine↗
PubMed1991

High level expression and purification of herpes simplex virus type 1 immediate early polypeptide Vmw110.

Herpes simplex virus type 1 (HSV-1) encodes five immediate early (IE) polypeptides. This paper reports the construction of a baculovirus vector which expresses large amounts of Vmw110, the product of IE gene 1. The expressed protein has been purified to near homogeneity and has a mobility on SDS polyacrylamide gels identical to that of Vmw110 produced during HSV-1 infection. Characterisation of its properties indicated that it forms dimers and perhaps higher order oligomers in solution and that the purified protein binds to both single stranded and double stranded calf thymus DNA cellulose columns. However, filter binding experiments were unable to detect any stable association of Vmw110 with DNA in solution.

Animals↗
PubMed1991

Local base sequence preferences for DNA cleavage by mammalian topoisomerase II in the presence of amsacrine or teniposide.

Several classes of antitumor drugs are known to stabilize topoisomerase complexes in which the enzyme is covalently bound to a terminus of a DNA strand break. The DNA cleavage sites generally are different for each class of drugs. We have determined the DNA sequence locations of a large number of drug-stimulated cleavage sites of topoisomerase II, and find that the results provide a clue to the possible structure of the complexes and the origin of the drug-specific differences. Cleavage enhancements by VM-26 and amsacrine (m-AMSA), which are representative of different classes of topoisomerase II inhibitors, have strong dependence on bases directly at the sites of cleavage. The preferred bases were C at the 3' terminus for VM-26 and A at the 5' terminus for m-AMSA. Also, a region of dyad symmetry of 12 to 16 base pairs was detected about the enzyme cleavage positions. These results are consistent with those obtained with doxorubicin, although in the case of doxorubicin, cleavage requires the presence of an A at the 3' terminus of at least one the pair of breaks that constitute a double-strand cleavage (Capranico et al., Nucleic Acids Res., 1990, 18: 6611). These findings suggest that topoisomerase II inhibitors may stack with one or the other base pair flanking the enzyme cleavage sites.

Amsacrine↗
PubMed1991

Purification of the DNA binding domain of herpes simplex virus type 1 immediate-early protein Vmw175 as a homodimer and extensive mutagenesis of its DNA recognition site.

The herpes simplex virus type 1 (HSV-1) Immediate-Early (IE) polypeptide Vmw175 is essential for the activation of transcription from viral early and late promoters. Vmw175 also reduces the activity of its own (IE-3) promoter in transfection assays. Both transactivation and repression mediated by Vmw175 require the integrity of a conserved domain of the polypeptide which has been shown to bind to specific DNA sequences. We have investigated the DNA sequence requirements for Vmw175 binding using a randomly mutated target. The results indicate that the binding site covers a region of 13 nucleotides divided into proximal and distal parts which are consistent with the consensus ATCGTNNNNNYSG. We have also expressed several different constructs encompassing the DNA binding domain of Vmw175 in bacteria, and obtained preparations of greater than 90% purity. The DNA binding domain is a dimer in solution, and binds DNA with a specificity similar to that of the intact protein, although the smallest DNA binding competent protein has a slightly reduced specificity.

Base Sequence↗
PubMed1991

The Vmw175 binding site in the IE-1 promoter has no apparent role in the expression of Vmw110 during herpes simplex virus type 1 infection.

The immediate-early (IE) genes of herpes simplex virus type 1 (HSV-1) are the first to be expressed during infection in tissue culture. Since they are transcribed at abnormally high levels in the absence of IE protein synthesis they appear to be subject to repression during normal infection. One of the major HSV-1 regulatory proteins, Vmw175 (the product of IE gene 3), is required for normal IE gene regulation since mutations which inactivate it lead to abnormally high levels of IE gene expression. The mechanism of repression of the IE-3 promoter requires both the ability of Vmw175 to bind to DNA and the presence of a Vmw175 recognition DNA binding sequence at the cap site of the IE-3 promoter. A similar Vmw175 DNA binding sequence has been defined within the IE-1 promoter. This paper describes the construction of a variant of HSV-1 with a mutation within the IE-1 Vmw175 DNA binding site. Although the mutation destroyed the ability of Vmw175 to bind to the site, and greatly reduced the ability of Vmw175 to repress the IE-1 promoter in transfection assays, the mutation had no effect on the levels of Vmw110 expression during normal HSV-1 infection.

Animals↗
PubMed1991

A transformation-specific polypeptide distinct from heat shock proteins is induced by herpes simplex virus type 2 infection.

A tumour-specific polypeptide designated U90 is one of a set of polypeptides which are encoded by the host cell and are specific for the transformed cell state, being immunoprecipitated by the sera of tumour-bearing animals. The interest in these tumour-specific polypeptides centres on the finding that they are also recognized by antisera raised against herpes simplex virus type 2 (HSV-2)-infected cells, implying some role for HSV-2 in tumorigenesis. The peptide map of HSV-2-induced U90 is indistinguishable from that of U90 present in uninfected tumour cells, including mouse cells transformed by human papillomavirus type 16. In tumour cells, U90 is located principally in the plasma membrane fraction and cannot be induced by heat shock, glucose starvation, or treatment with tunicamycin or calcium ionophore. U90 is not related to either the heat shock protein of Mr 90,000 (HSP90) or the glucose-related polypeptide of Mr 94,000 (GRP94) as determined by peptide mapping and the use of monospecific, monoclonal and antipeptide antibodies. This suggests that U90 is a novel transformation-specific protein which can be induced by infection with HSV-2.

Animals↗
PubMed1991

Monitoring asthma.

The authors outline the management of asthma once the 'best lung function' is determined and answer some questions that may arise in general practice during the long-term monitoring of asthmatic patients. The answers represent the opinion of the authors, but are referenced where applicable.

Adult↗
PubMed1990

Use of suprathreshold test data to predict the results of quantitative testing in the nasal periphery.

We compared the results of suprathreshold testing of the nasal periphery with those of quantitative assessment of the same area. One eye each from 81 patients in four categories (normal [21 subjects], low-risk ocular hypertensive [20 subjects], high-risk ocular hypertensive [20 subjects] and early glaucoma [20 subjects]) was tested with the nasal suprathreshold points of the Octopus G1 program and the Sargon peripheral field-nasal (PFN) program. Simple algorithms were developed to generate from the nasal suprathreshold points of the G1 program two "qualitative indices", mean defect-qualitative (MD-Q) and loss variance-qualitative (LV-Q); these are analogous to the indices mean defect and loss variance respectively, which were calculated from the PFN program data. MD-Q and LV-Q were found to be well correlated with their quantitative counterparts. They provide a uniform method for interpreting the nasal suprathreshold points of the G1 program and help identify patients in whom further quantitative testing is likely to yield useful information.

Adult↗
PubMed1989

Legal AIDS: implications of AIDS and HIV for British and American law.

In its approach to AIDS and HIV the law has to protect two conflicting interests; it must recognise the right of the public to be protected against the disease and it must recognise the right of the individual not to be unfairly restricted by having or being at risk of the disease. Consequently the law must make some compromise which while protecting public health also protects the individual so that the individual will feel free to come forward for available treatment. In this way prevention of spread of the disease is encouraged. How this compromise is or might be affected by British and American law is examined in several areas, including medico-legal matters, criminal and tort law, employment, insurance and education.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Efficacy and safety of once-daily levobunolol for glaucoma therapy.

We studied the ocular hypotensive efficacy and safety of 0.5% levobunolol hydrochloride and 0.5% timolol maleate administered topically once daily for 3 months in 91 patients (46 in the levobunolol group and 45 in the timolol group) with primary or secondary open-angle glaucoma or ocular hypertension. In this randomized double-masked parallel clinical study, intraocular pressure (IOP) was successfully controlled in 78% of the patients who received levobunolol and 89% of those who received timolol. The overall mean decrease in IOP was 5.6 mm Hg (decrease of 23%) in the levobunolol group and 6.7 mm Hg (26%) in the timolol group, a nonsignificant difference. In both groups the overall mean IOP during treatment was significantly lower than the pretreatment value (p less than 0.001). For both treatment groups changes in heart rate and blood pressure were minimal. We conclude that both 0.5% levobunolol and 0.5% timolol administered once daily are effective and safe in lowering IOP in most patients with ocular hypertension or open-angle glaucoma.

Administration, Topical↗
PubMed1988

The value of indices in the central and peripheral visual fields for the detection of glaucoma.

We assessed 81 patients in four groups (normal, low- and high-risk ocular hypertension, and early glaucoma) with the standard Octopus G1 central visual field program in addition to two quantitative programs, PFN (peripheral field-nasal) and PFT (peripheral field-temporal), designed for this study to test the nasal and temporal periphery, respectively. Indices were calculated for each program for each subject in all groups. We then examined the behavior of the indices across the separate visual field areas within each group as well as the behavior of the indices of each field area among the different groups. We found that quantitative testing of the peripheral nasal visual field provided valuable information in the detection of glaucomatous visual dysfunction additional to that provided by quantitative testing of the central visual field. Quantitative testing of the temporal periphery was less valuable.

Adult↗
PubMed1988

Identification of the gene encoding the 65-kilodalton DNA-binding protein of herpes simplex virus type 1.

Hybrid arrest of in vitro translation was used to localize the region of the herpes simplex virus type 1 genome encoding the 65-kilodalton DNA-binding protein (65KDBP) to between genome coordinates 0.592 and 0.649. Knowledge of the DNA sequence of this region allowed us to identify three open reading frames as likely candidates for the gene encoding 65KDBP. Two independent approaches were used to determine which of these three open reading frames encoded the protein. For the first approach a monoclonal antibody, MAb 6898, which reacted specifically with 65KDBP, was isolated. This antibody was used, with the techniques of hybrid arrest of in vitro translation and in vitro translation of selected mRNA, to identify the gene encoding 65KDBP. The second approach involved preparation of antisera directed against oligopeptides corresponding to regions of the predicted amino acid sequence of this gene. These antisera reacted specifically with 65KDBP, thus confirming the gene assignment.

DNA-Binding Proteins↗
PubMed1988

Hypomethylation of host cell DNA synthesized after infection or transformation of cells by herpes simplex virus.

Infection of rat embryo cells with herpes simplex virus type 2 caused undermethylation of host cell DNA synthesized during infection. DNA made prior to infection was not demethylated, but some of its degradation products, including methyl dCMP, were incorporated into viral DNA. The use of mutant virus showed that some viral DNA synthesis appears to be required for the inhibition of methylation. Inhibition of methylation cannot be explained by an absence of DNA methyltransferase as the activity of this enzyme did not change during the early period of infection. Inhibition of host cell DNA methylation may be an important step in the transformation of cells by herpesviruses, and various transformed cell lines tested showed reduced levels of DNA methylation.

Animals↗
PubMed1985

Cellular proteins expressed in herpes simplex virus transformed cells also accumulate on herpes simplex virus infection.

The cell proteins expressed in rat embryo cells transformed by herpes simplex virus (HSV) have been analysed by immunoprecipitation assays to determine those polypeptides which can be identified by immunoprecipitation with the sera of tumour-bearing animals and also with antisera to herpes simplex infected cells. Cell polypeptides commonly recognised by both these sera have been further characterised using a monoclonal antibody directed against a cellular polypeptide which accumulates on HSV-2 lytic infection. This monoclonal antibody recognises in HSV-transformed cells polypeptides of mol. wts. 90 000, 40 000 and 32 000. Further studies show that the accumulation of these polypeptides in HSV-transformed cells is not HSV specific but is a common feature of transformation or of cells which have been immortalised. We suggest that cellular polypeptides accumulating as a result of HSV infection may be of importance in the initiation of transformation by HSV, i.e., at the level of immortalisation of cells.

Adenoviruses, Human↗
PubMed1985

[3H]Choline labeling of cerebellothalamic neurons with observations on the cerebello-thalamo-parietal pathway in cats.

[3H]Choline injected into the ventral lateral thalamic nucleus (VL) labeled cell bodies of the deep cerebellar nuclei and adjacent vestibular nuclei by retrograde axoplasmic transport. Injections in caudal and dorsal parts of VL labeled cells in ventral parts of the dentate nucleus and interpositus posterior nucleus. Injections in rostral and ventral parts of VL labeled cells in the interpositus anterior nucleus and dorsal parts of the dentate nucleus. A few labeled cell bodies were found throughout the rostrocaudal extent of the fastigial nucleus and in adjacent parts of the vestibular nuclei. A combined injection of [3H]choline and [3H]amino acids labeled cells in the deep cerebellar nuclei and axon terminals in layer I of the middle suprasylvian gyrus (areas 5, 7).

Animals↗