Biomedical subjects
Publications and source records attributed to P R Cook.
Recent evidence suggests that active RNA polymerases are concentrated in discrete 'factories' where they work together on many different templates. The evidence that such factories specialize in the transcription of particular groups of genes is reviewed.
Models for replication and transcription often display polymerases that track like locomotives along their DNA templates. However, recent evidence supports an alternative model in which DNA and RNA polymerases are immobilized by attachment to larger structures, where they reel in their templates and extrude newly made nucleic acids. These polymerases do not act independently; they are concentrated in discrete "factories," where they work together on many different templates. Evidence for models involving tracking and immobile polymerases is reviewed.
Mammalian nuclei contain three different RNA polymerases defined by their characteristic locations and drug sensitivities; polymerase I is found in nucleoli, and polymerases II and III in the nucleoplasm. As nascent transcripts made by polymerases I and II are concentrated in discrete sites, the locations of those made by polymerase III were investigated. HeLa cells were lysed with saponin in an improved 'physiological' buffer that preserves transcriptional activity and nuclear ultrastructure; then, engaged polymerases were allowed to extend nascent transcripts in Br-UTP, before the resulting Br-RNA was immunolabelled indirectly with fluorochromes or gold particles. Biochemical analysis showed that approximately 10 000 transcripts were being made by polymerase III at the moment of lysis, while confocal and electron microscopy showed that these transcripts were concentrated in only approximately 2000 sites (diameter approximately 40 nm). Therefore, each site contains approximately five active polymerases. These sites contain specific subunits of polymerase III, but not the hyperphosphorylated form of the largest subunit of polymerase II. The results indicate that the active forms of all three nuclear polymerases are concentrated in their own dedicated transcription sites or 'factories', suggesting that different regions of the nucleus specialize in the transcription of different types of gene.
Individual chromosomes are not directly visible within the interphase nuclei of most somatic cells; they can only be seen during mitosis. We have developed a method that allows DNA strands to be observed directly in living cells, and we use it to analyze how mitotic chromosomes form. A fluorescent analogue (e.g., Cy5-dUTP) of the natural precursor, thymidine triphosphate, is introduced into cells, which are then grown on the heated stage of a confocal microscope. The analogue is incorporated by the endogenous enzymes into DNA. As the mechanisms for recognizing and removing the unusual residues do not prevent subsequent progress around the cell cycle, the now fluorescent DNA strands can be followed as they assemble into chromosomes, and segregate to daughters and granddaughters. Movies of such strands in living cells suggest that chromosome axes follow simple recognizable paths through their territories during G2 phase, and that late replicating regions maintain their relative positions as prophase chromosomes form. Quantitative analysis confirms that individual regions move little during this stage of chromosome condensation. As a result, the gross structure of an interphase chromosome territory is directly related to that of the prophase chromosome.
Endoscopic partial inferior turbinoplasty with a microdebrider has been introduced as an alternative to other inferior turbinectomy techniques for the treatment of nasal airway obstruction. Between June 1994 and December 1997, 100 patients underwent endoscopic partial inferior turbinoplasty. Concurrent septoplasty was performed in 81 patients, and functional endoscopic sinus surgery was performed in 43 patients. Synechiae formation in 12 patients was the most common complication and resolved in nearly all cases. Postoperative improvement in nasal patency occurred in 93% of the patients. In the 48 patients for whom preoperative subjective scores were available, a significant difference was noted when comparing preoperative and postoperative subjective and outcome scores of nasal patency (P < 0.0001). Patients with underlying allergy and chronic sinusitis tended to have lower outcome scores. Postoperative rhinomanometry was obtained in 21 patients and revealed a normalized mean total nasal resistance of 0.23 Pa/mL/second. This improved to 0. 17 Pa/mL/second with topical decongestion (P = 0.0029), revealing the retention of the turbinate mucosa's vasoactive capability. We conclude that endoscopic partial inferior turbinoplasty provides a good alternative to other inferior turbinectomy techniques and is associated with excellent outcomes and minimal morbidity.
Food allergies are an important cause of morbidity in the pediatric population. We used a questionnaire to assess the value of in vitro testing in evaluating children for food allergy. The patient population was divided into two groups: those who tested positive (Class I or greater) for food allergy (combined food/inhalant allergy or food allergy alone) and those who tested negative for food allergy (inhalant only or negative test). Although we recognize that a significant proportion of food allergies may not be IgE-mediated, for the purposes of this study we considered a patient food allergy--positive if in vitro testing was positive and food allergy--negative if testing was negative. Thirty-eight of 77 patients tested (49%) were positive for food allergies. Those with food allergies did not have pretreatment scores significantly different from those of patients negative for food allergies (55 vs. 65, p = 0.11). Similarly, we detected no statistically significant difference between the symptom-improvement scores of the two groups (37 vs. 40, p = 0.57). We conclude that in vitro testing for food allergies is warranted in children with allergic symptoms, for several reasons: The prevalence of IgE-mediated food allergy in children may be higher than previously thought, and children with food allergies experience the same severity of symptoms as those without. It must be remembered that in vitro testing only detects IgE levels, and therefore a negative test does not mean that the patient does not have food allergies. However, when positive, in vitro testing gives the physician the opportunity to modify the treatment plan, helping avoid time-consuming and demanding oral food challenges. Our questionnaire also showed that food allergy and non-food allergy patients had similar pretreatment and posttreatment symptom scores.
Various complexes that contain the core subunits of RNA polymerase II associated with different transcription factors have been isolated from eukaryotes; their precise molecular constitution depends on the purification procedure. We estimated the numbers of various components of such complexes in an HeLa cell by quantitative immunoblotting. The cells were lysed with saponin in a physiological buffer; approximately 140,000 unengaged polymerases (mainly of form IIA) were released. Only approximately 4,000 of these soluble molecules sedimented in glycerol gradients as holoenzyme-sized complexes. About 180,000 molecules of polymerases (approximately 110,000 molecules of form IIO) and 10,000 to 30,000 molecules of each of TFIIB, TFIIEalpha, TFIIEbeta, TFIIF-RAP74, TFIIF-RAP30, and TFIIH-MAT1 remained tightly associated with the nuclear substructure. Most proteins and run-on activity were retained when approximately 50% of the chromatin was detached with a nuclease, but approximately 45,000 molecules of bound TATA binding protein (TBP) were detached. Similar results were obtained after cross-linking living cells with formaldehyde. The results provide little support for the existence of a large pool of soluble holoenzyme; they are consistent with TBP-promoter complexes in nuclease-sensitive chromatin being assembled into preinitiation complexes attached to the underlying structure.
The resolution of conventional light microscopy is limited to approximately 200 nm in the x- and y-axes and >500 nm in the z-axis. A simple way of improving z-axis resolution is to analyze thin sections of 100-200 nm. The utility of such an approach is illustrated by reference to transcription sites imaged in cryosections of human nuclei. Cells are permeabilized, allowed to extend nascent transcripts in Br-UTP, fixed, cryosectioned, and Br-RNA-immunolabeled with fluorochromes and gold particles. As expected, physical sectioning improves resolution and brings other advantages. First, sections allow improved antibody access and better immunolabeling. Second, more sites (with a more representative range of intensities) can now be resolved against lower backgrounds, facilitating quantitative analysis. Third, problems associated with chromatic aberration when two differently colored images of the same objects are collected can be sidestepped by refocusing between image collection. Fourth, exactly the same sites can be imaged by light and electron microscopy, allowing direct comparison between the two techniques. Immunogold labeling and electron microscopy provided the most accurate counts of site number. The results confirm that nascent transcripts in the nucleoplasm are confined to several thousand sites, or "factories," with diameters of approximately 40 nm. (J Histochem Cytochem 47:471-480, 1999)
PTF (PSE-binding transcription factor) activates transcription of snRNA and related genes. We investigated its distribution in HeLa nuclei by immunofluorescence, and found it spread throughout the nucleoplasm in small foci. In some cells, PTF is also concentrated in one, or very few, discrete regions (diameter approximately 1.3 micron) that appear during G1 phase and disappear in S phase. Oct1, a transcription factor that interacts with PTF, is also enriched in these domains; RNA polymerase II, TBP and Sp1 are also present. Each domain typically contains 2 or 3 transcription 'factories' where Br-UTP is incorporated into nascent transcripts. Accordingly, we have christened this region the Oct1/PTF/transcription (OPT) domain. It colocalizes with some, but not all, PIKA domains. It is distinct from other nuclear domains, including coiled bodies, gemini bodies, PML bodies and the perinucleolar compartment. A small region on chromosome 6 (band 6p21) containing only approximately 30 Mbp DNA, and chromosomes 6 and 7, associate with the domain significantly more than other chromosomes. The domains may act like nucleoli to bring particular genes on specific chromosomes together to a region where the appropriate transcription and processing factors are concentrated, thereby facilitating the expression of those genes.
Recent scientific studies have demonstrated the efficacy of various forms of immunotherapy for the treatment of allergic diseases. Traditional subcutaneous immunotherapy, sublingual, oral, and intranasal immunotherapy have been shown to significantly reduce symptoms and favorably modulate the immune response. Outcome studies that use patient response data from standardized surveys represent the next challenge to all practicing allergists.
Allergy is a significant problem in as much as 60% of patients seeking care at an otolaryngologist's office. This raises the issue of the most effective means of diagnosing allergy, especially in pediatric patients, who often may not tolerate skin testing. The RAST and CAP (Pharmacia) tests have been shown to be quite comparable with skin testing in diagnosing allergy. This study was undertaken to assess the effectiveness of treatment of children with allergies who had undergone in vitro testing. This was done by obtaining subjective symptom scores both before and after treatment. These were obtained from patients with different in vitro results, including some with negative testing. Overall, 77 of 155 mailed questionnaires were returned. Results showed overall improvement of 38.81 points in 12 symptoms (p = 0.001; n = 62). Each symptom was rated on a visual scale of 1 to 10. A small portion of the patient population received surgical treatment (tympanostomy tube placement with or without adenoidectomy) in addition to medical treatment and avoidance. These patients did not have significantly higher pretreatment scores and did not have a higher improvement rate for all symptoms. We believe this study shows the validity of in vitro testing and treatment of allergy in children.
Using HeLa cells, we have developed methods to determine 1) the number of RNA polymerases that are active at any moment, 2) the number of transcription sites, and 3) the number of polymerases associated with one transcription unit. To count engaged polymerases, cells were encapsulated in agarose, permeabilized, treated with ribonuclease, and the now-truncated transcripts extended in [32P]uridine triphosphate; then, the number of growing transcripts was calculated from the total number of nucleotides incorporated and the average increment in length of the transcripts. Approximately 15, 000 transcripts were elongated by polymerase I, and approximately 75,000 were elongated by polymerases II and III. Transcription sites were detected after the cells were grown in bromouridine for <2.5 min, after which the resulting bromo-RNA was labeled with gold particles; electron microscopy showed that most extranucleolar transcripts were concentrated in approximately 2400 sites with diameters of approximately 80 nm. The number of polymerases associated with a transcription unit was counted after templates were spread over a large area; most extranucleolar units were associated with one elongating complex. These results suggest that many templates are attached in a "cloud" of loops around a site; each site, or transcription "factory," would contain approximately 30 active polymerases and associated transcripts.
Some SR proteins are associated with eukaryotic transcripts as they move from synthetic sites (transcription "factories"), through downstream sites, to nuclear pores. Downstream sites can also be isolated as large nuclear ribonucleoprotein particles of approximately 200 S (diameter approximately 50 nm). In ultrathin sections of HeLa nuclei, indirect immunogold labeling with a specific antibody gives many small clusters of approximately 10 gold particles (diameter 50-80 nm). We gauged errors in estimating the diameter of underlying structures marked by immunogold probes (lengths approximately 20 nm). We examined systematically how probe dimensions affected cluster diameter. Probes contained one to three immunoglobulin molecules, sometimes a protein A molecule, and a gold particle of 5-15 nm. We found that (a) immunolabeling particles were tightly packed, (b) reducing particle size by 5 nm reduced cluster diameter by 10 nm, (c) reducing the number of immunoglobulins in the immunolabeling sandwich from three to two reduced cluster diameter by approximately 4 nm, (d) replacing the last immunoglobulin in a sandwich with protein A increased diameter by approximately 7 nm and led to a peripheral concentration of particles, and (e) increasing the number of layers in the sandwich increased sensitivity. Assuming that underlying structures had diameters of 50 nm, we find that errors ranged from -20% to +50%.
The route taken by transcripts from synthetic sites in the nucleus to the cytoplasm has been under scrutiny for years, but details of the pathway remain obscure. A new high-resolution method for mapping the pathway is described; HeLa cells are grown in Br-U so that the analogue is incorporated into RNA and exported to the cytoplasm, before Br-RNA is localized by immuno-electron microscopy. After exposure to low concentrations of Br-U for short periods, cells grow normally. Br-RNA is first found in several thousand extra-nucleolar transcription sites or factories (diameter 50-80 nm), before appearing in several hundred new downstream sites (diameter 50-80 nm) each minute; subsequently, progressively more downstream sites become labelled. These sites can be isolated on sucrose gradients as large nuclear ribonucleoprotein particles of approximately 200 S. Later, Br-RNA is seen docked approximately 200 nm away from approximately 20% nuclear pores, before exiting to the cytoplasm. Individual downstream sites are unlikely to contain individual transcripts; rather, results are consistent with groups of transcripts being shipped together from synthetic sites to pores. A subset of SR proteins are excellent markers of this pathway; this subset is concentrated in tens of thousands of sites, which include transcription, downstream and docking sites. Growth in high concentrations of Br-U for long periods is toxic, and Br-RNA accumulates just inside nuclear pores.
Allergen immunotherapy is a safe form of therapy with a very low incidence of systemic allergic reactions and fatalities. Over the past few years, as a result of several investigations, risk factors have been identified, although some disagreement remains. Asthmatics, patients on beta-blockers and highly sensitive patients are groups at increased risk for systemic reactions. Reactions are more common among individuals receiving extracts of pollens, particularly grass and ragweed. Most authors have also reported that reactions are more common in season. Chest tightness or wheezing, urticaria, pruritus and throat congestion were frequent symptoms of severe systemic reactions. The recommendations of Greenburg et al (Table 1) and Davis et al (Table 2) serve as valuable guidelines. Otolaryngologic allergists have found that home maintenance immunotherapy is a safe treatment option (in low-risk patients). In any case, immunotherapy should be supervised by a physician well trained in its use and indications, and should be administered by personnel trained in the treatment of medical emergencies, specifically those related to allergy. This training should include cardiac resuscitation. In addition, consideration should be given to premedicating patients with antihistamines or corticosteroids and to measuring peak flows prior to injection. Because asthmatics are a very high-risk group, it is recommended that these patients not undergo immunotherapy at home. In patients who have had multiple reactions or in those with severe asthma, consideration should be given to discontinuing injections. Finally, optimal or moderate allergen dosing may provide the needed balance between therapeutic efficacy and safety.
Botryomycosis is a chronic bacterial granulomatous disease often involving the skin and subcutaneous tissue. Head and neck involvement is rare. Botryomycosis presents with clinical and histological features similar to actinomycosis or mycetoma, but the causative organism is usually Staphylococcus aureus. Microscopically the organisms appear to be encapsulated in granules, which are thought to protect them from the effects of standard courses of antibiotics. Botryomycosis usually requires surgical intervention for cure. Major debilitating surgery has been required for most patients, because the infection has been unresponsive to seemingly appropriate medical therapy. We present an 8-month-old male with periorbital botryomycosis. Surgical specimens for diagnosis were obtained, but complete resection would have created debilitating functional and cosmetic defects. The lesion failed to respond to nafcillin alone or combination therapy with hyperbaric oxygen, but showed slow, steady improvement with long-term clindamycin. The patient has been disease free for more than 4 years, with minimal scarring and no functional impairment. Prolonged medical therapy for botryomycosis may be a viable alternative to the traditionally recommended surgical resection, thereby reducing cosmetic and functional morbidity.
There has been a recent explosion of new in vitro tests for the diagnosis of allergies. At present there is no general agreement on which type of in vitro test is best. Recently our hospital switched in vitro testing from the modified radioallergosorbent system (mRAST) to the Pharmacia CAP system (CAP). While changing in vitro testing techniques, 47 patients were tested with both the mRAST and CAP tests. Comparisons were made between the mRAST and CAP results of Alternaria tenuis and Dermatophagoides pteronyssinus allergens. These results were then compared with the results of patients who also underwent intradermal skin testing based on end point titration techniques.
One hundred eighty-two patients were evaluated after functional endoscopic sinus surgery. The goal was to establish whether any anatomical finding correlated with symptoms and to find any historical predictors of symptomatic failure. Of all physical findings reviewed, only scarring of middle meatal antrostomy and scarring of the ethmoids approached significance in predicting poor outcome. Surprisingly, of the historical factors reviewed, only gastroesophageal reflux disease was statistically significant as a predictor of poor symptomatic outcome.