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Biomedical subjects

P R Cook

Publications and source records attributed to P R Cook.

At least 19 recordsLinked to original sources
PubMed1992

Transcription by an immobilized RNA polymerase from bacteriophage T7 and the topology of transcription.

It is often assumed that a polymerase moves along the template as it synthesizes RNA. However, a polymerase that tracks along a helical strand will generate a transcript that is entwined about the template. No such interlocking results if the polymerase is immobile and the template moves past it. Therefore we investigated whether immobilization inhibits the RNA polymerase of T7 bacteriophage using a hybrid protein, in which the polymerase is connected through a peptide linker to an immobilizing domain, which in turn was attached through an antibody to protein A covalently linked to plastic beads. Polymerase could be released by cleaving the linker with a protease, factor Xa. Comparison of the activity of the bound and free enzymes showed that immobilization reduced the rate of initiation about fivefold. However, when re-initiation was eliminated by removing excess template, immobilization was found to have little effect on the rate of elongation. Perhaps the untwining problem is sidestepped in vivo by immobilizing the polymerase.

Base Sequence↗
PubMed1992

Evidence for cross-linking DNA by bis-intercalators with rigid and extended linkers is provided by knotting and catenation.

A new series of DNA bis-intercalators is reported in which acridine moieties are connected by rigid and extended pyridine-based linkers of varied length. Cross-linking of DNA by bis-intercalation is inferred from the unwinding and folding of linear DNA induced by the compounds; after ligation and removal of the bis-intercalator, superhelical circles, catenanes and knots that bear a residual imprint of the bis-intercalator are observed. These novel bis-intercalators are of interest because they can be used to probe the spatial organization of DNA, especially near sites of replication, recombination or topoisomerase action where two duplexes must be in close proximity. Preliminary results on the effects of the various compounds on the cloning efficiency of bacteria and replication by permeabilized human cells are also presented.

Acridine Orange↗
PubMed1992

Bisintercalators of DNA with a rigid linker in an extended configuration.

A new class of DNA bisintercalators is reported in which phenanthridinium or acridinium rings are connected by rigid and extended linkers of varied length. Cross-linking of DNA by bisintercalation is inferred from the unwinding and folding of linear DNA induced by the compound; after ligation and removal of the bisintercalator, superhelical circles, catenanes, and knots that bear an imprint of the bisintercalator are observed. These novel bisintercalators are of interest because they can be used to probe the organization of DNA in three-dimensional space, especially near sites of replication, recombination, or topoisomerase action, where two duplexes must be in close proximity.

Acridines↗
PubMed1992

The attachments of chromatin loops to the nucleoskeleton.

It is widely assumed by cell biologists that chromatin is looped by attachment to some nuclear skeleton. 'Structural' attachments might be mediated through specific sequences; these would be attached in most cells in an organism, underlying the basic structure of the mitotic chromosome and persisting throughout interphase. 'Functional' attachments might also exist, perhaps if active polymerases are attached to the skeleton and replication and transcription occur as DNA is reeled through them. Cells of different tissues--and even cells of the same tissue--would have different attachments of this type. Problems associated with demonstrating these two kinds of attachment are discussed. We find little good evidence for 'structural' attachments and explore the idea that 'functional' attachments are the only kind that exist: 'functional' attachments involving active transcription units might be stable enough to organize chromatin during both interphase and mitosis, but 'dynamic' enough to allow duplication of attached sequences without disrupting loops.

Animals↗
PubMed1992

Anaphylaxis in immunotherapy.

Although anaphylactic reactions resulting from immunotherapy rarely occur, when they do occur they can be life-threatening. The literature reveals more than 30 deaths attributed to immunotherapy but numerous reports of anaphylaxis. We reviewed a 5-year experience of immunotherapy in a University practice and their experience with systemic reactions is presented. More than 125,000 injections were given in this time period. Of the 550 patients receiving immunotherapy, 14% experienced anaphylactic reactions, the majority of which were mild.

Anaphylaxis↗
PubMed1991

Cellular gels. Purifying and mapping long DNA molecules.

Long DNA molecules of greater than 10(5) bp (0.1 Mbp) are easily broken by pipetting. Therefore, chromosomal DNA is generally isolated after embedding cells in a protective coat of agarose. The embedded DNA can then be cut into long pieces and fractionated on gels using pulsed fields, but these pieces are again easily broken if the resolved DNA molecules are recovered from the gels. We now describe a novel gel matrix, a 'cellular' gel, that permits the recovery of resolved fragments from gels in a form that enables facile manipulation without shear. This facilitates purification and restriction mapping of fragments of 0.1-1.0 Mbp. We illustrate the utility of the method by mapping chromosome III of baker's yeast, which has a length of approximately 0.36 Mbp. This method should facilitate purification and restriction mapping of yeast artificial chromosomes.

Chromosomes, Fungal↗
PubMed1991

Double-blind bilateral paired comparison of 0.05% halobetasol propionate cream and its vehicle in patients with chronic atopic dermatitis and other eczematous dermatoses.

Six investigators evaluated 0.05% halobetasol propionate cream and its vehicle in 111 patients with chronic atopic dermatitis and several other eczematous dermatoses. Patients applied treatment twice daily to bilateral lesions for 14 days. Investigators graded pruritus, erythema, scaling, papulation, and lichenification using 4-point severity scales on days 0, 7, and 14. On day 14 patients provided an assessment of efficacy for both treatments. Statistically significant differences favoring halobetasol propionate over the vehicle were seen for all signs and symptoms (p less than 0.001). Substantial improvements were achieved by the active treatment by day 7 (p less than 0.001). Patients assessments of efficacy were significantly higher for halobetasol cream than for vehicle (p less than 0.001). No instances of systemic effects or skin atrophy were reported and adverse experiences were limited to burning or stinging and other minor, nonspecific complaints distributed uniformly between active treatment and vehicle. These results demonstrate that 0.05% halobetasol propionate cream is highly effective in the treatment of atopic dermatitis and other eczematous dermatoses.

Administration, Cutaneous↗
PubMed1990

Attachment of DNA to the nucleoskeleton of HeLa cells examined using physiological conditions.

Although it is widely believed that eukaryotic DNA is looped by attachment to a nucleoskeleton, there is controversy about its composition and which sequences are attached to it. As most nuclear derivatives are isolated using unphysiological conditions, the criticism that attachments seen in vitro are generated artifactually has been difficult to rebut. Therefore we have re-investigated attachments of chromatin to the skeleton using physiological conditions. HeLa cells are encapsulated in agarose microbeads and lysed using Triton in a 'physiological' buffer. Then, most chromatin can be electroeluted after treatment with a restriction enzyme to leave some at the base of the loops still attached. Analysis of the size and amounts of these residual fragments indicates that the loops are 80-90kbp long. The residual fragments are stably attached, with about 1kbp of each fragment protected from nuclease attack. This is very much longer than a typical protein-binding site of 10-20bp.

Chromatin↗
PubMed1990

Active RNA polymerase I is fixed within the nucleus of HeLa cells.

We have investigated whether active RNA polymerase I, the enzyme responsible for transcribing ribosomal RNA, is immobilized by attachment to a large subnuclear structure in HeLa cells. As unphysiological salt concentrations induce artifacts, we have used isotonic conditions throughout the preparative and analytic procedures. Cells are encapsulated in agarose microbeads and lysed in Triton and a 'physiological' buffer; then soluble proteins and RNA diffuse out through the agarose pores to leave encapsulated chromatin. This can be manipulated without aggregation but is accessible to molecular probes; it retains the replicational and transcriptional activities of the living cell. After treatment with a restriction endonuclease, most chromatin can be removed from beads by electrophoresis: then active ribosomal genes and polymerase I remain behind. Active ribosomal genes are very accessible to nuclease digestion whilst the rest are even more inaccessible than inactive globin genes. Our observations confirm the complex organization of rDNA within nucleoli and are compatible with transcription occurring at fixed sites. A model for transcription involving an attached polymerase is presented.

Cell Nucleus↗
PubMed1990

The size of chromatin loops in HeLa cells.

It is widely believed that the chromatin fibre is organized into loops during interphase, with the loop being implicated as an important unit of nuclear function. However, there remains little direct evidence for looping, with estimates of loop size varying widely. This has led to the suggestion that some loops, or even all of them, arise artefactually during isolation as chromatin aggregates so easily. We have now investigated the effect of isolation procedure on loop size using HeLa cells encapsulated in agarose to allow easy manipulation. Loop size in various derivatives (i.e. nuclei, nucleoids, matrices and scaffolds) critically depended on procedure; some (or all) of their loops are artefacts. The loop size in derivatives isolated using the most 'physiological' conditions was 86 kb; this remained unchanged throughout the cell cycle. This loop size is probably an average of a range of loops of between 5 and 200 kb.

Cell Cycle↗
PubMed1990

Vagal and sympathetic activity during spinal analgesia.

Indices of vagal and sympathetic activity were studied in 30 elderly males, to elucidate their possible roles in causing hypotension during spinal analgesia. The technique of spinal analgesia and the regimen of intravenous fluids were standardised. An index of vagal activity was derived from the degree of heart rate variation (successive RR interval change) on ECG recordings. Sympathetic activity was evaluated by changes in the skin conductance (SCR) of 15 patients. Analgesia to pinprick reached a median dermatome level of T5-6 (range T2-T10) by 15 min. Hypotension was correlated with the level of analgesia, and was more likely when spinal analgesia was higher than T5. There was no correlation between vagal activity and the degree of hypotension. The depression of skin conductance responses was not correlated with the degree of hypotension nor with vagal activity. Vagal efferent activity, measured at the heart, does not seem to play a causative role in hypotension occurring during spinal analgesia.

Aged↗
PubMed1989

The nucleoskeleton and the topology of transcription.

Transcription is conventionally believed to occur by passage of a mobile polymerase along a fixed template. Evidence for this model is derived almost entirely from material prepared using hypotonic salt concentrations. Studies on subnuclear structures isolated using hypertonic conditions, and more recently using conditions closer to the physiological, suggest an alternative. Transcription occurs as the template moves past a polymerase attached to a nucleoskeleton; this skeleton is the active site of transcription. Evidence for the two models is summarised. Much of it is consistent with the polymerase being attached and not freely diffusible. Some consequences of such a model are discussed.

Models, Biological↗
PubMed1989

Happy mapping: a proposal for linkage mapping the human genome.

A theoretical approach for linkage mapping the genome of any higher eukaryote is described. It uses the polymerase chain reaction, oligonucleotides of random sequence and single haploid cells. Markers are defined and then the DNA of a single sperm is broken at random (eg by gamma-rays) and physically split into 3 aliquots. Each aliquot is screened for the presence of each marker. Closely-linked markers are more likely to be found in the same aliquot than unlinked markers. The entire process is repeated with further sperm and the frequency that any two markers co-segregate determined. Closely-linked markers co-segregate from most cells; unlinked markers do so rarely. A map can then be constructed from these co-segregation frequencies. A specific application for determining the order and distance between sets of closely-linked and previously-defined markers is also described.

Chromosome Mapping↗
PubMed1989

Blood-brain barrier to pertechnetate following drug-induced hypotension.

The integrity of the blood-brain barrier (BBB) was examined in rabbits, in terms of the partition of 99mTc-pertechnetate (99mTcO4-) between brain and blood, following intracarotid injection of hypertonic arabinose, hypertension (168 mm Hg and 10% inspired carbon dioxide), or hypotension (less than 20 mm Hg for 15 min). Corrections were made for changes in tissue blood contents, using chromium-51 as a red cell marker. In control animals the mean brain:blood ratio was 0.038 (range 0.027-0.052). Following arabinose there was a five-fold increase in mean BBB permeability (mean brain:blood ratio 0.192 (0.070-0.378)). There was no change after hypertension and carbon dioxide (mean ratio 0.034) or after hypotension (mean ratio 0.032), despite an increase in cerebral extracellular potassium. Examination of other tissues showed no change in the 99mTcO4- tissue/blood partition in heart muscle in any study but, following hypotension, ratios in the kidney (mean ratio 1.63) and, to a lesser extent, the liver (mean ratio 1.37) had increased, suggesting an abnormality of active transport under these conditions. We conclude that, while 99mTcO4- tissue/blood partitioning revealed osmotic disruption of the BBB, profound hypotension with evidence of brain cell damage did not change BBB permeability to the same marker. Hypotension may influence active transport of this ion in liver and kidney.

Animals↗
PubMed1988

Visualization of a filamentous nucleoskeleton with a 23 nm axial repeat.

Whether nucleoskeletons seen after extracting cells are preparative artefacts is controversial. Using an extraction method that preserves vital nuclear functions, we have visualized part of a nucleoskeleton by electron microscopy of thick resinless sections. Cells encapsulated in agarose microbeads are lysed using Triton in a physiological buffer; the agarose coat prevents aggregation and protects fragile cell contents. These extracted cells are accessible to small molecules and transcribe and replicate at rates close to those in vivo. After electroeluting most chromatin after treatment with HaeIII, a skeleton is uncovered which ramifies throughout the nucleus. Individual filaments are approximately 10 nm wide with an axial repeat of 23 nm, characteristic of intermediate filaments.

Cell Nucleus↗