Biomedical subjects
Publications and source records attributed to P R Cook.
Recent experiments suggest that active polymerases are concentrated in large structures, 'factories', within eukaryotic nuclei. Data concerning the structure of these factories is reviewed.
Nurses need critical thinking skills to provide competent care. For interventions such as drug administration, a system of critical thinking assists nurses to accomplish tasks safely and efficiently. The use of the "think and apply" model provides nurses with a practical way to integrate critical thinking skills into practice.
Current models for RNA synthesis involve an RNA polymerase that tracks along a static template. However, research on chromatin loops suggests that the template slides past a stationary polymerase; individual polymerases tie the chromatin fibre into loops and clusters of polymerases determine the basic structure of the interphase and metaphase chromosome. RNA polymerase is then both a player and a manager of the chromosome loop.
During S phase, DNA replication begins at numerous sites throughout the genome. Textbooks would have us believe that each replication fork tracks along the immobile DNA until it runs into the adjacent fork, but recent results question this view. Various studies show that replication forks are concentrated in immobile 'factory' units throughout the nucleus. Each factory contains as many as 40 different replication forks and associated polymerases. These findings suggest that newly synthesized DNA is extruded as each template moves like a conveyor through the factory.
A total of 283 consecutive patients with chronic sinusitis underwent functional endoscopic sinus surgery. There were 72 allergic patients and 211 nonallergic patients. Data were collected on the effect of immunotherapy on middle meatotomy patency, synechiae formation, and recurrent polyps in allergic patients. Data supported the following conclusions: (1) Immunotherapy given either before or after surgery does not statistically influence middle meatotomy patency, synechiae formation, or recurrence of polyps after functional endoscopic sinus surgery. However, the data do suggest, for all three outcome parameters, that allergic patients who undergo immunotherapy do better than those who do not undergo immunotherapy and, with the exception of recurrent polyps, do as well as nonallergic patients. (2) The prevalence of preoperative polyps is the same for allergic and nonallergic patients in this study, but polyp recurrence is higher in allergic patients. (3) Approximately 40% of allergic patients who began preoperative immunotherapy stopped immunotherapy after surgery because their allergic symptoms resolved or were minimal. A comment regarding this observation is provided.
Anaphylaxis may be defined as a systemic, immunoglobulin E-mediated (Gell-Coombs type I) hypersensitivity reaction triggered by exposure to an antigen in a previously sensitized patient. Anaphylaxis may occur in a variety of circumstances; however, when it occurs as the result of immunotherapy, it is of great concern to the practicing allergist. When describing or reporting anaphylaxis relating to immunotherapy, most allergists speak in terms of the types of reactions, local vs. systemic. Germane to this discussion is the use of the term systemic reaction, which can mean anything from mild allergy symptoms resulting from an allergy injection to bradycardia and hypotension (shock). In this article we report serious or significant systemic reactions, which are characterized by any of the following symptoms: urticaria, sneezing or nasal obstruction, throat tightness or congestion, wheezing, and shock (bradycardia or hypotension). There were no fatalities reported from the survey group. The overall reaction rate was 0.3%.
Twenty asthma patients who underwent functional endoscopic sinus surgery for chronic sinusitis were studied. Medical records and questionnaire data for these 20 patients were studied regarding the impact of sinus disease and functional endoscopic sinus surgery on their asthma. We found that 95% reported that their asthma was worsened by their sinus disease (95% confidence interval, 0.74 to 0.99+), and 85% reported that functional endoscopic sinus surgery improved their asthma (0.60 to 0.97). Of the 13 patients who used both inhalers and systemic medication, 53.8% were able to eliminate some of their medication (0.21 to 0.79). Furthermore, 61.5% of these patients had a concomitant reduction in their inhaler use (0.28 to 0.85). All patients (six) who used only inhalers experienced a reduction in their inhaler use (0.54 to 1.00), and two patients were able to eliminate their inhalers completely. One of two patients who were steroid dependent was able to discontinue steroids after surgery. Of patients who used steroids intermittently (13), 53.8% were able to eliminate the use of steroids after surgery (0.21 to 0.79). Patients who required preoperative hospital admissions (4) and emergency room or urgent physician office visits (18) had a 75.0% and 81.3% (p < 0.001) reduction in visits, respectively, after surgery. Because 43% of the cost of asthma is the result of hospitalizations and emergency department/urgent physician office visits, a significant impact on health care costs can be realized with functional endoscopic sinus surgery in this patient population.
Eighteen patients were operated on by functional endoscopic sinus surgery who had no ostiomeatal unit obstruction on computed tomography scan and had unremarkable paranasal sinuses. These patients also had no apparent ostiomeatal unit obstruction on diagnostic nasal endoscopy. Data were collected on these patients regarding the impact of very limited functional endoscopic sinus surgery on their principal complaint of recurrent sinusitis with facial pain/headache thought to be of sinogenic origin. Sixteen patients (88.9%) had a reduction in the number of sinus infections requiring antibiotic therapy. This reduction was significant at p < 0.0001. Twelve of 14 patients whose facial pain/headache was believed to be of sinogenic origin had a significant reduction in severity (95% confidence interval, 49.2% to 95.3%). We discuss the role of reversible nasal mucosal disease in the pathophysiology of recurrent rhinosinusitis in this patient population. This was a very small, select group of patients who had specific complaints and had had medical treatment failures. This therapy is not recommended for every patient, but only a select few with classic complaints of sinus headaches or recurrent sinusitis and negative computed tomography scans.
HeLa cells synchronized at different stages of the cell cycle were permeabilized and incubated with analogues of nucleotide triphosphates; then sites of incorporation were immunolabeled with the appropriate fluorescent probes. Confocal microscopy showed that sites of replication and transcription were not diffusely spread throughout nuclei, reflecting the distribution of euchromatin; rather, they were concentrated in 'foci' where many polymerases act together. Transcription foci aggregated as cells progressed towards the G1/S boundary; later they dispersed and became more diffuse. Replication was initiated only at transcription sites; later, when heterochromatin was replicated in enlarged foci, these remained sites of transcription. This illustrates the dynamic nature of nuclear architecture and suggests that transcription may be required for the initiation of DNA synthesis.
Sites of transcription of ribosomal RNA in HeLa cells were visualized by electron microscopy. Cells were either incubated with Br-uridine, or permeabilized and then incubated with BrUTP, before sites containing Br-RNA were immunolabeled with gold particles. Short incubations ensured that most incorporated analogue remained at synthetic sites. Fibrillar centres were unlabelled except at their periphery; label was concentrated over certain regions of the surrounding dense fibrillar component. These results suggest that the dense fibrillar component is the site of rRNA transcription. After dispersing the granular component and the dense fibrillar component by a hypotonic treatment, removal of most chromatin and preparation of resinless sections, fibrillar centres remained fixed to a nucleoskeleton. These structural and functional features are incorporated into a model for rRNA transcription.
RNA polymerases have been implicated in the initiation of replication in bacteria. The conflicting evidence for a role in initiation in eukaryotes is reviewed.
The repair of damage induced in DNA by ultraviolet light involves excision of the damage and then repair synthesis to fill the gap. We investigated the sites of repair synthesis using MRC-5 fibroblasts and HeLa cells in G1 phase. Cells were encapsulated in agarose microbeads to protect them during manipulation, irradiated, incubated to allow repair to initiate, and permeabilized with streptolysin O to allow entry of labelled triphosphates; [32P]dTTP was incorporated into acid-insoluble material in a dose-dependent manner. Incubation with biotin-16-dUTP allowed sites of incorporation to be indirectly immunolabeled using a FITC-conjugated antibody; sites were not diffusely spread throughout nuclei but concentrated in discrete foci. This is similar to sites of S phase activity that are attached to an underlying nucleoskeleton. After treatment with an endonuclease, most repaired DNA electroeluted from beads with chromatin fragments; this was unlike nascent DNA made during S phase and suggests that repaired DNA is not as closely associated with the skeleton. However, the procedure destroyed repair activity, so repaired DNA might be attached in vivo through a polymerase that was removed electrophoretically. Therefore this approach cannot be used to determine decisively whether repair sites are associated with a skeleton in vivo.
The repair of damage induced in DNA by ultraviolet light involves excision of the damaged sequence and synthesis of new DNA to repair the gap. Sites of such repair synthesis were visualized by incubating permeabilized HeLa or MRC-5 cells with the DNA precursor, biotin-dUTP, in a physiological buffer; then incorporated biotin was immunolabeled with fluorescent antibodies. Repair did not take place at sites that reflected the DNA distribution; rather, sites were focally concentrated in a complex pattern. This pattern changed with time; initially intense repair took place at transcriptionally active sites but when transcription became inhibited it continued at sites with little transcription. Repair synthesis in vitro also occurred in the absence of transcription. Repair sites generally contained a high concentration of proliferating cell nuclear antigen but not the tumour-suppressor protein, p53.
Sites of replication in synchronized HeLa cells were visualized by light and electron microscopy; cells were permeabilized and incubated with biotin-16-dUTP, and incorporation sites were immunolabelled. Electron microscopy of thick resinless sections from which approximately 90% chromatin had been removed showed that most DNA synthesis occurs in specific dense structures (replication factories) attached to a diffuse nucleoskeleton. These factories appear at the end of G1-phase and quickly become active; as S-phase progresses, they increase in size and decrease in number like sites of incorporation seen by light microscopy. Electron microscopy of conventional thin sections proved that these factories are a subset of nuclear bodies; they changed in the same characteristic way and contained DNA polymerase alpha and proliferating cell nuclear antigen. As replication factories can be observed and labelled in non-permeabilized cells, they cannot be aggregation artifacts. Some replication occurs outside factories at discrete sites on the diffuse skeleton; it becomes significant by mid S-phase and later becomes concentrated beneath the lamina.
HeLa cells in early S phase were encapsulated in agarose microbeads, permeabilized, and incubated with biotin-11-dUTP in a "physiological" buffer. Sites of DNA synthesis were then immunolabeled. As others have found, approximately 150 focal sites of synthesis were visible in each nucleus by light microscopy; they also contained DNA polymerase alpha and proliferating cell nuclear antigen. Electron microscopy of thick resinless sections from which approximately 90% of the chromatin had been removed revealed a similar number of dense, morphologically discrete ovoid bodies strung along a nucleoskeleton. The ovoids remained morphologically and functionally intact despite the removal of most of the chromatin. After 2.5 min of incubation with biotin-11-dUTP, the incorporated analog was associated only with ovoids; after 5 min it began to spread into the adjacent chromatin, which became extensively labeled after 1 hr. This provides visual evidence for polymerization "factories" fixed to a skeleton, with replication occurring as the template moves through them.
We have devised a simple method for ordering markers on a chromosome and determining the distances between them. It uses haploid equivalents of DNA and the polymerase chain reaction, hence 'happy mapping'. Our approach is analogous to classical linkage mapping; we replace its two essential elements, chromosome breakage and segregation, by in vitro analogues. DNA from any source is broken randomly by gamma-irradiation or shearing. Markers are then segregated by diluting the resulting fragments to give aliquots containing approximately 1 haploid genome equivalent. Linked markers tend to be found together in an aliquot. After detecting markers using the polymerase chain reaction, map order and distance can be deduced from the frequency with which markers 'co-segregate'. We have mapped 7 markers scattered over 1.24 Mbp using only 140 aliquots. Using the 'whole-genome' chain reaction, we also show how the approach might be used to map thousands of markers scattered throughout the genome. The method is powerful because the frequency of chromosome breakage can be optimized to suit the resolution required.
HeLa cells were encapsulated in agarose microbeads, permeabilized and incubated with Br-UTP in a 'physiological' buffer; then sites of RNA synthesis were immunolabelled using an antibody that reacts with Br-RNA. After extending nascent RNA chains by < 400 nucleotides in vitro, approximately 300-500 focal synthetic sites can be seen in each nucleus by fluorescence microscopy. Most foci also contain a component of the splicing apparatus detected by an anti-Sm antibody. alpha-amanitin, an inhibitor of RNA polymerase II, prevents incorporation into these foci; then, using a slightly higher salt concentration, approximately 25 nucleolar foci became clearly visible. Both nucleolar and extra-nucleolar foci remain after nucleolytic removal of approximately 90% chromatin. An underlying structure probably organizes groups of transcription units into 'factories' where transcripts are both synthesized and processed.
A speculative model for reverse transcription of a viral RNA template into proviral dsDNA is presented. It has two essential features that are not included in current models: (i) the functional complex is dimeric, with two polymerization/RNase H sites and (ii) two templates are initially attached to the complex at their 3' ends. The model also has the optional features that (iii) the complex is rotationally symmetrical and (iv) attached to the virion core. The model attempts to explain why the viral genome is dimeric, the specificity of the 'jumps' between the ends of templates and how recombination occurs so readily. It also suggests novel targets for drug therapy during retroviral infection, for example, in AIDS.