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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
PubMed1996

The topology of transcription by immobilized polymerases.

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 polymerase immobilized in a large transcription factory. The evidence for immobilized polymerases is reviewed, and a model for transcription by such fixed enzymes is presented. According to the model, gene activation would involve reducing gene-factory distance and increasing the affinity of a promoter for a factory. Locus controlling regions and enhancers would attach to a factory and increase the chances that a promoter could bind to a polymerase; after transcriptional termination, the gene would detach from the factory. As some RNA processing occurs cotranscriptionally, processing sites are also likely to be associated with the factory.

DNA↗
PubMed1996

The localization of sites containing nascent RNA and splicing factors.

Sites of transcription in the HeLa nucleus are not diffusely spread throughout euchromatin but are concentrated in approximately 2000 discrete sites or "transcription factories." These sites can be immunolabeled after allowing permeabilized cells to elongate nascent RNA chains in the presence of BrUTP. Splicing factors are also concentrated in a few tens of nuclear domains known as "speckles"; they are also more diffusely spread throughout euchromatin. As there has been some controversy whether the speckles are sites of transcription, we investigated the relative distributions of transcription factories and the speckles (detected using Sm autoimmune antibodies). We used conditions that minimize the redistribution of both the nascent RNA and the antigens during permeabilization and fixation and imaged the sites with high resolution using cryosections and a "confocal" microscope. The speckles contained little-if any-nascent RNA and so were usually not transcriptionally active, but they were often surrounded by a few transcription sites. Whether they are active sites of processing of RNA or merely sites where the machinery is stored remains to be established.

Binding Sites↗
PubMed1996

The structure of 4-way DNA junctions: specific binding of bis-intercalators with rigid linkers.

During replication and recombination, two DNA duplexes lie side by side. We have developed reagents that might be used to probe structure during these critical processes; they contain two intercalating groups connected by a rigid linker that forces those groups to point in opposite directions. If their stereochemistry proves appropriate, such structure-specific agents should intercalate specifically into adjacent duplexes in the Y- and X-shaped structures (i.e. 3- and 4-way junctions, now known as 3H and 4H junctions) found at replication and recombination sites. We prepared DNA structures in which four duplexes were arranged in all possible combinations around 2- and 4-way junctions and then probed the accessibility to DNase I of all their phosphodiester bonds. In the absence of any bis-intercalators, 7-9 nucleotides (nt) in each of the strands in 4-way junctions were protected from attack; protected regions were significantly offset to the 3' side of the junction in continuous strands, but only slightly offset, if at all, in exchanging strands. All the intercalators decreased accessibility throughout the structure, but none did so at specific points in the two adjacent arms of 4-way junctions. However, one bis-intercalator--but not its sister with a shorter linker--strikingly increased access to a particular CpT bond that lay 9 nt away from the centre of some 4-way junctions without reducing access to neighbouring bonds. Binding was both sequence and structure specific, and depended on complementary stereochemistry between bis-intercalator and junction.

Acridines↗
PubMed1996

Sequences attaching loops of nuclear and mitochondrial DNA to underlying structures in human cells: the role of transcription units.

DNA sequences attaching loops of nuclear and mitochondrial DNA to underlying structures in HeLa cells have been cloned and 106 representative clones sequenced; 10 clones containing random genomic fragments served as controls. As chromatin is prone to rearrangement, care was taken to isolate sequences using 'physiological' conditions that did not create additional attachments. Comparison (by Southern blotting) of the concentration of each cloned sequence in 'total' and 'attached' fractions of DNA showed that most clones did contain attached sequences, but even highly-attached sequences were not attached in all cells in the population. Results demonstrated that 28% of clones were derived from three specific parts of the mitochondrial genome and 22% from different parts of the alu repeat. In addition, 41% of clones contained unique nuclear sequences; these contained no more of the motifs found attached to nuclear scaffolds or matrices (ie SARs or MARs) than would be expected from their base composition. No other attachment motif(s) could be identified by sequence analysis. However, Northern blotting showed that all the mitochondrial clones and 76% of clones containing unique sequences were transcribed; the degree of attachment correlated with transcriptional activity. These results are consistent with transcription being responsible for ever-changing attachments in both nuclei and mitochondria.

Base Sequence↗
PubMed1996

4-Picoline-2,2':6',2"-terpyridine-platinum(II) - a potent intercalator of DNA.

4-Picoloine-2,2':6',2"-terpyridine-platinum(II) is shown in a ligation assay to unwind and so intercalate into DNA. Circular dichroism is used to determine an equilibrium binding constant of approximately 2 x 10(7) M(-1) for the most stable binding mode of 4-picoline-2,2':6',2"-terpyridine-platinum(II) to poly[d(A-T)2] with a site size of about 4 base pairs, and about 1 x 10(6) M(-1) for a second binding mode with a site size of about 2 base pairs. Fluorescence spectroscopy provides further evidence for the strong equilibrium binding constant of 4-picoline-2,2':6',2"-terpyridine-platinum(II) in that it displaces ethidium bromide bound to DNA. The double positive charge on 4-picoline-2,2':6',2"-terpyridine-platinum(II), together with the intercalative binding mode is probably responsible for the large binding constant.

Binding Sites↗
PubMed1996

Mimosine arrests the cell cycle after cells enter S-phase.

L-Mimosine (beta-N-[3-hydroxy-4-pyridone]-alpha-aminopropionic acid)--a rare amino acid derived from Mimosa and Leucaena plants--arrests cells reversibly late during G1 phase or at the beginning of S-phase. If mimosine were to arrest cells immediately before S-phase, it would provide a superb tool for the investigation of the initiation of DNA synthesis. Therefore, we reexamined the point of action of mimosine. Mitotic HeLa cells were released into 200 microM mimosine and grown for approximately 10 h to block them, before the cells were permeabilized and the amino acid removed by washing them thoroughly. On addition of the appropriate triphosphates, DNA synthesis-measured by the incorporation of [32P]dTTP--began immediately; as it is known that such permeabilized cells cannot initiate DNA synthesis but can only resume elongating previously initiated chains, mimosine must arrest after DNA synthesis has begun. Moreover, cells grown in mimosine assembled functional replication factories--detected by immunolabeling after incorporation of biotin-dUTP--that were typical of those found early during S-phase. Disappointingly, it seems that mimosine--like aphidocolin--blocks only after cells enter S-phase.

Amino Acids↗
PubMed1996

Paranasal sinus computed tomography scan findings in patients with cystic fibrosis.

Seventy paranasal sinus computed tomography scans of patients with cystic fibrosis were compared with those of age-matched control groups of randomly selected chronic sinusitis patients without cystic fibrosis to determine whether differences in disease patterns existed. In patients older than 10 years, frontal sinus agenesis and maxilloethmoid sinus opacification were significantly more prevalent in patients with cystic fibrosis than in chronic sinusitis patients without cystic fibrosis. Medial bulging of the lateral nasal wall was significantly greater in patients with cystic fibrosis than in chronic sinusitis patients without cystic fibrosis in patients older than 5 years. On the basis of these findings, a diagnostic triad of radiologic findings for cystic fibrosis detection is presented, as well as its clinical implications.

Adolescent↗
PubMed1996

Active RNA polymerases are localized within discrete transcription "factories' in human nuclei.

Nascent transcripts in permeabilized HeLa cells were elongated by approximately 30-2,000 nucleotides in Br-UTP or biotin-14-CTP, before incorporation sites were immunolabelled either pre- or post-embedding, and visualized by light or electron microscopy. Analogues were concentrated in approximately 2,100 (range 2,000-2,700) discrete sites attached to a nucleoskeleton and surrounded by chromatin. A typical site contained a cluster (diameter 71 nm) of at least 4, and probably about 20, engaged polymerases, plus associated transcripts that partially overlapped a zone of RNA polymerase II, ribonucleoproteins, and proteins rich in thiols and acidic groups. As each site probably contains many transcription units, these results suggest that active polymerases are confined to these sites, which we call transcription 'factories'. Results are consistent with transcription occurring as templates slide past attached polymerases, as nascent RNA is extruded into the factories.

Binding Sites↗
PubMed1996

Memory for musical tempo: additional evidence that auditory memory is absolute.

We report evidence that long-term memory retains absolute (accurate) features of perceptual events. Specifically, we show that memory for music seems to preserve the absolute tempo of the musical performance. In Experiment 1, 46 subjects sang two different popular songs from memory, and their tempos were compared with recorded versions of the songs. Seventy-two percent of the productions on two consecutive trials came within 8% of the actual tempo, demonstrating accuracy near the perceptual threshold (JND) for tempo. In Experiment 2, a control experiment, we found that folk songs lacking a tempo standard generally have a large variability in tempo; this counters arguments that memory for the tempo of remembered songs is driven by articulatory constraints. The relevance of the present findings to theories of perceptual memory and memory for music is discussed.

Adolescent↗
PubMed1996

Seasonal allergic rhinitis.

Seasonal allergic rhinitis is the most well understood of the types of allergic rhinitides. The disease is characterized by sneezing, itching, rhinorrhea, and nasal congestion upon exposure to pollen allergens in susceptible individuals. These symptoms can cause considerable misery in affected patients. Recent advances in the diagnosis and treatment of this disorder as well as new insights into the pathophysiology of allergic rhinitis enable practitioners to provide more effective therapy.

Allergens↗
PubMed1996

Allergic rhinosinusitis in the pediatric population.

Allergic disease is very common in the U.S. population and even more common in children seen by otolaryngologists. Allergy causes or contributes to virtually all of the diseases in the respiratory tract that otolaryngologists, particularly otolaryngic allergists, evaluate and treat. The signs and symptoms of allergic disease in the pediatric patient are reviewed to improve the appreciation of the role of allergy in pediatric otolaryngology. Recent research developments, which are reviewed in detail in this article, underscore the importance of allergic, inflammatory response in rhinosinusitis. The key to long-term therapeutic success, particularly in pediatric rhinosinusitis, is management of the underlying allergic diathesis.

Child↗
PubMed1996

Paranasal sinus disease in patients with cystic fibrosis.

The enhanced understanding of the pathophysiology of cystic fibrosis (CF) has advanced significantly the management of this disorder, resulting in both an increased longevity and an improved quality of life. Sinonasal disease is an important aspect of CF. The goal of this article is to provide a current synopsis of CF sinonasal disease with an emphasis on disease symptomatology, physical signs, characteristic radiographic features, and outcome-based management options. When indicated, functional endoscopic sinus surgery has been shown to be a safe and effective treatment option for children with CF.

Adolescent↗
PubMed1995

The structural basis of nuclear function.

Most models for transcription and replication involve polymerases that track along the template. We review here experiments that suggest an alternative in which polymerization occurs as the template slides past a polymerase fixed to a large structure in the eukaryotic nucleus--a "factory" attached to a nucleoskeleton. This means that higher-order structure dictates how and when DNA is replicated or transcribed.

Animals↗
PubMed1995

Effect of partial middle turbinectomy on nasal airflow and resistance.

We report the first prospective study of the effect of partial middle turbinectomy on nasal airflow and resistance as measured objectively by active anterior rhinomanometry. Our study group consisted of 31 consecutive patients who underwent functional endoscopic sinus surgery with simultaneous partial middle turbinate resections. We found that all patients had significant improvement in nasal airflow (p < 0.001) and significant decrease in nasal resistance (p < 0.001). Thus we found no deleterious effect on nasal function. Additionally, we reviewed the literature on retrospective series in which patients had received partial middle turbinectomies and found no evidence that nasal function was impaired after surgery. We conclude that partial middle turbinectomy may be performed without adversely altering nasal function, as measured by active anterior rhinomanometry.

Adolescent↗
PubMed1995

Symptom outcome after functional endoscopic sinus surgery in patients with cystic fibrosis: a prospective study.

Twenty-nine consecutive patients with cystic fibrosis were offered functional endoscopic sinus surgery. Twenty-six of these patients underwent surgery. A symptom questionnaire was obtained from the patient or parent before surgery. At the end of the study this symptom questionnaire was again administered to the same individual, and 21 completed it. There were 14 male and 7 female patients, with a mean age of 12.3 years and a median age of 8.7 years. Mean follow-up was 34.3 months. Results for the following symptoms were significant: nasal airway obstruction was improved (p < 0.0002), olfactory function was improved (p < 0.0037), purulent nasal discharge was decreased (p < 0.001), and activity level was increased (p < 0.001). Other parameters are also reported in the text. In summary, this study prospectively studies the effects of functional endoscopic sinus surgery on symptoms in patients with cystic fibrosis. The study points out several significant areas of symptom improvement and supports the selective use of functional endoscopic sinus surgery in patients with cystic fibrosis. Indications for surgery are provided.

Activities of Daily Living↗
PubMed1995

Lamin proteins form an internal nucleoskeleton as well as a peripheral lamina in human cells.

The nuclear lamina forms a protein mesh that underlies the nuclear membrane. In most mammalian cells it contains the intermediate filament proteins, lamins A, B and C. As their name indicates, lamins are generally thought to be confined to the nuclear periphery. We now show that they also form part of a diffuse skeleton that ramifies throughout the interior of the nucleus. Unlike their peripheral counterparts, these internal lamins are buried in dense chromatin and so are inaccessible to antibodies, but accessibility can be increased by removing chromatin. Knobs and nodes on an internal skeleton can then be immunolabelled using fluorescein- or gold-conjugated anti-lamin A antibodies. These results suggest that the lamins are misnamed as they are also found internally.

Antibodies, Monoclonal↗
PubMed1995

A chromomeric model for nuclear and chromosome structure.

The basic structural elements of chromatin and chromosomes are reviewed. Then a model involving only three architectural motifs, nucleosomes, chromatin loops and transcription factories/chromomeres, is presented. Loops are tied through transcription factors and RNA polymerases to factories during interphase and to the remnants of those factories, chromomeres, during mitosis. On entry into mitosis, increased adhesiveness between nucleosomes and between factories drives a 'sticky-end' aggregation to the most compact and stable structure, a cylinder of nucleosomes around an axial chromomeric core.

Cell Nucleolus↗