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Biomedical subjects

D A Jackson

Publications and source records attributed to D A Jackson.

At least 55 records · Page 3Linked to original sources
PubMed1997

Phylogenetic analysis of heterothallic Neurospora species.

We examined the phylogenetic relationships among five heterothallic species of Neurospora using restriction fragment polymorphisms derived from cosmid probes and sequence data from the upstream regions of two genes, al-1 and frq. Distance, maximum likelihood, and parsimony trees derived from the data support the hypothesis that strains assigned to N. sitophila, N. discreta, and N. tetrasperma form respective monophyletic groups. Strains assigned to N. intermedia and N. crassa, however, did not form two respective monophyletic groups, consistent with a previous suggestion based on analysis of mitochondrial DNAs that N. crassa and N. intermedia may be incompletely resolved sister taxa. Trees derived from restriction fragments and the al-1 sequence position N. tetrasperma as the sister species of N. sitophila. None of the trees produced by our data supported a previous analysis of sequences in the region of the mating type idiomorph that grouped N. crassa and N. sitophila as sister taxa, as well as N. intermedia and N. tetrasperma as sister taxa. Moreover, sequences from al-1, frq, and the mating-type region produced different trees when analyzed separately. The lack of consensus obtained with different sequences could result from the sorting of ancestral polymorphism during speciation or gene flow across species boundaries, or both.

Genes, Fungal↗
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

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

Improved cyclic urea inhibitors of the HIV-1 protease: synthesis, potency, resistance profile, human pharmacokinetics and X-ray crystal structure of DMP 450.

BACKGROUND: Effective HIV protease inhibitors must combine potency towards wild-type and mutant variants of HIV with oral bioavailability such that drug levels in relevant tissues continuously exceed that required for inhibition of virus replication. Computer-aided design led to the discovery of cyclic urea inhibitors of the HIV protease. We set out to improve the physical properties and oral bioavailability of these compounds. RESULTS: We have synthesized DMP 450 (bis-methanesulfonic acid salt), a water-soluble cyclic urea compound and a potent inhibitor of HIV replication in cell culture that also inhibits variants of HIV with single amino acid substitutions in the protease. DMP 450 is highly selective for HIV protease, consistent with displacement of the retrovirus-specific structural water molecule. Single doses of 10 mg kg-1 DMP 450 result in plasma levels in man in excess of that required to inhibit wild-type and several mutant HIVs. A plasmid-based, in vivo assay model suggests that maintenance of plasma levels of DMP 450 near the antiviral IC90 suppresses HIV protease activity in the animal. We did identify mutants that are resistant to DMP 450, however; multiple mutations within the protease gene caused a significant reduction in the antiviral response. CONCLUSIONS: DMP 450 is a significant advance within the cyclic urea class of HIV protease inhibitors due to its exceptional oral bioavailability. The data presented here suggest that an optimal cyclic urea will provide clinical benefit in treating AIDS if it combines favorable pharmacokinetics with potent activity against not only single mutants of HIV, but also multiply-mutant variants.

Administration, Oral↗
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↗
PubMed1995

Subtype-specific regulation of muscarinic receptor expression and function by heterologous receptor activation.

Incubation of cultured embryonic chicken heart cells with the beta-adrenergic agonist isoproterenol resulted in a dose-dependent increase in the number of mAChR on the surface of intact cells. The isoproterenol-mediated increase in mAChR number was time dependent and reached a maximum by 48 h. Chick heart cells treated with isoproterenol exhibited a greater than 6-fold increase in the sensitivity for carbachol-mediated inhibition of adenylyl cyclase activity as compared to control. Stimulation of cultured heart cells for 24 h with isoproterenol resulted in a 25-35% increase in cm2 mRNA levels as compared to control cm2 mRNA levels. In contrast, the level of cm4 mRNA was not significantly affected by isoproterenol treatment. cm2 mRNA levels were maximally elevated by 15 h following isoproterenol stimulation and remained elevated for up to 72 h. Incubation of cells with isoproterenol in the presence of Rp-cAMP, an inhibitor of cAMP-dependent protein kinase, blocked the increase in the level of cm2 mRNA. Thus, prolonged activation of beta-adrenergic receptors results in an increase in mAChR number and muscarinic responsiveness in chick heart cells due to a cAMP-dependent protein kinase mediated increase in cm2 mRNA levels.

Animals↗
PubMed1995

Nuclear organization: uniting replication foci, chromatin domains and chromosome structure.

In higher eukaryotes, 'replication factories' coordinate DNA synthesis within local clusters of chromatin domains. Recent experiments (1, 2) have confirmed the complexity of these clusters and established that the organization of sites labelled during S phase persists throughout the cell cycle. This implies that domain clusters are critical elements of an hierarchy that is fundamental to both nuclear and chromosome structure.

Animals↗
PubMed1995

S-phase progression in synchronized human cells.

In S-phase human cells, active DNA polymerases are clustered at morphologically discrete sites--replication factories. As S-phase proceeds, characteristic patterns of DNA synthesis correlate with the appearance of replication factories at the corresponding nuclear sites. The coordination of different phases of this replication program was investigated. Aphidicolin was used to synchronize HeLa cells at the beginning of S-phase and S-phase progression followed on removing the drug. Characteristic features of the S-phase program were not affected by the duration of treatment, implying that each phase of synthesis must complete before the next can begin. Prolonged exposure did not result in the progressive activation of all potential origins. Permeabilized cells labeled in vitro with biotin--dUTP usually displayed the typical early S-phase pattern, but often with sites of reduced activity. A minority of cells contained larger, aphidicolin-induced replication sites consistent with the fusion of adjacent factories. These quickly reverted to normal, once cells resumed growth--emphasizing the dynamic nature of nuclear organization. No apparent biochemical defects were observed when short drug treatments were used. Cells synchronized in G1 and incubated in aphidicolin for 2-4 h contained replication complexes distributed with the characteristic early S-phase pattern. Most DNA polymerases were blocked at authentic sites of initiation and resumed synthesis at the in vivo rate, once aphidicolin was removed. Conditions optimal for the isolation of early S-phase origins of replication are described.

Aphidicolin↗
PubMed1995

Pathways mediating abdominal phasic flexor muscle activity in crayfish with chronically cut nerve cords.

1. Nerve cord transection abolishes the ability of crayfish (Procambarus clarkii) to produce tailflips in response to gradually applied tactile or proprioceptive stimulation of the abdomen, but this ability eventually returns. To determine the time-course of this return and to analyze its underlying neural pathways, we made behavioral observations, electromyographic recordings from abdominal phasic flexor muscles, and intracellular recordings from motoneurons in crayfish with cord lesions between the thorax and the abdomen. 2. Abdominal stimulation activated the phasic flexor muscles in the rostral 5 abdominal segments and their homologs in the 6th segment, the posterior telson flexor muscles. Nearly one-quarter of cord-transfected animals responded to the stimuli with phasic flexor muscle activity by one week after the lesion, and almost 90% were responsive by 3 weeks. 3. Regeneration of axons across the lesion played little or no role in the recovery of phasic flexor muscle responsiveness. In addition, the lateral giant axons were not activated by the gradually applied stimuli that triggered phasic flexor muscle contractions. These results suggest that non-giant pathways confined to the abdominal nervous system become functional following chronic cord transection. 4. Retransection of the nerve cord below the original lesion showed that smaller subsets of the abdominal cord, including a single ganglion, could develop the ability to generate phasic flexor muscle contractions in response to gradually applied stimuli. 5. Phasic flexor motoneurons in cord-transected animals could be excited by stimulation of afferents throughout the abdomen. The sensory pathways producing this activation appear to project through the nerve cord without much cross-over between left and right sides.

Abdominal Muscles↗
PubMed1995

Choline enhances scopolamine-induced acetylcholine release in dorsal hippocampus of conscious, freely-moving rats.

We examined the effects of exogenous choline (30, 60, 120 mg/kg, i.p.) on basal and scopolamine-evoked acetylcholine (ACh) release in awake animals, using in vivo microdialysis. After collection of 3-4 baseline dialysate samples (15 min each), rats received either saline or choline chloride and 4 additional samples were collected. All animals then received scopolamine hydrochloride (0.5 mg/kg, i.p.) and 6 additional samples were collected. Basal ACh release in animals receiving choline did not differ from that in rats given saline, nor from ACh release prior to choline administration. Scopolamine alone increased average ACh levels in dialysates from 1.22 +/- 0.54 to 11.18 +/- 3.07 pmol/15 min (mean +/- SD; p = 0.001); administration of 60 mg/kg or 120 mg/kg of choline chloride significantly enhanced maximal scopolamine responses by about 55%. These results suggest that supplemental choline enhances evoked ACh release in hippocampus of freely-moving rats.

Acetylcholine↗
PubMed1995

Molecular analysis of the regulation of muscarinic receptor expression and function.

Several systems are being used to determine the molecular and cellular basis for the regulation of expression and function of the muscarinic receptors. Treatment of chick heart cells in culture results in decreased levels of mRNA encoding the cm2 and cm4 receptors. This probably results from decreased gene transcription which requires concomitant mAChR-mediated inhibition of adenylyl cyclase and mAChR-mediated stimulation of phospholipase C. Site-directed mutagenesis was used to demonstrate that the single tyrosine residue in the carboxyl-terminal cytoplasmic tail of the m2 receptor is involved in agonist-induced down-regulation but not sequestration. Activation of heterologous receptors in chick heart cells can also regulate mAChR mRNA levels. A cAMP-regulated luciferase reporter gene, has been used to demonstrate that the m4 receptor preferentially couples to Gi alpha-2 or Go alpha over Gi alpha-1 or Gi alpha-3 to mediate inhibition of adenylyl cyclase activity. Finally, in order to determine the role of individual receptor subtypes in muscarinic-mediated responses in vivo, we are beginning to use the method of targeted gene disruption by homologous recombination to generate mice deficient in specific receptor subtypes.

Adenylate Cyclase Toxin↗
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

Maternal behaviour and socio-economic influences on the bacterial content of infant weaning foods in rural northern Thailand.

The bacterial contamination of infant weaning foods was examined in the context of a longitudinal study of lactation and infant growth, the Chiang Mai Lactation Study. Sixty-two mother-infant pairs were selected by random sampling from a rural area outside the city of Chiang Mai and studied for 48 hours in their homes on six occasions over the first year of life. Data on food hygiene practices and maternal factors were related to the total bacterial count per gram and coliform content of weaning foods. Bottle feeding, premastication, and mashing were significantly related to an increased bacterial content of weaning foods, while boiling foods to make soups, preparing in and feeding from a banana leaf, and using boiled water to prepare foods all reduced their bacterial content. Storage also increased the bacterial contamination in foods and foods were more highly contaminated in the rainy season. Maternal age and education were also related to some feeding practices. By promoting the feeding of traditional, but less contaminated weaning foods, an intervention is put forward which would aim to reduce weaning food contamination and thereby reduce incidence of diarrhoea in this area of Northern Thailand.

Adult↗
PubMed1995

Out-of-body experience in a patient emerging from anesthesia.

Most PACU nurses do not have the opportunity to talk to their patients about their anesthetic experiences after surgery. This case study discusses one patient's experience as told to the author (an anesthesia nurse clinician) during a postoperative visit.

Adult↗
PubMed1995

H-reflexes in the upper extremity and the effects of voluntary contraction.

We report a simple technique for obtaining H-reflexes from the extensor carpi radialis (ECR) muscle. In 50 normal test subjects, above-elbow stimulation of the radial nerve evoked ECR H-reflexes, in all 100 arms with facilitation, and in only 8 without facilitation. H-waves were less common in extensor digitorum communis and rare in brachioradialis; these could have been volume-conducted ECR responses. Median-nerve stimulation at the elbow evoked H-reflexes in 92 of the 100 flexor carpi radialis (FCR) muscles; 44 of these required no facilitation. Background EMG (facilitation) activity or an overlapping M-wave often obscured the H-wave onset: among the onsets considered reliable in FCR (67/92) and ECR (84/100), mean latencies were 16.2 msec and 16.5 msec respectively, and correlated well with subject height and arm length. There was no significant difference in latency obtained with and without facilitation for the FCR H-reflexes and between techniques for the ECR H-reflexes. Right-left differences in latency ranged from 0 to 0.9 msec for ECR and 0 to 0.6 msec for FCR H-reflexes.

Adult↗