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Biomedical subjects

G Price

Publications and source records attributed to G Price.

At least 19 recordsLinked to original sources
PubMed2000

Major DNA replication initiation sites in the c-myc locus in human cells.

DNA replication initiation sites and initiation frequencies over 12. 5 kb of the human c-myc locus, including 4.6 kb of new 5' sequence, were determined based on short nascent DNA abundance measured by competitive polymerase chain reaction using 21 primer sets. In previous measurements, no comparative quantitation of nascent strand abundance was performed, and distinction of major from minor initiation sites was not feasible. Two major initiation sites were identified in this study. One predominant site has been located at approximately 0.5 kb upstream of exon 1 of the c-myc gene, and a second new major site is located in exon 2. The site in exon 2 has not been previously identified. In addition, there are other sites that may act as less frequently used initiation sites, some of which may correspond to sites in previous reports. Furthermore, a comparison of the abundance of DNA replication intermediates over this same region of the c-myc locus between HeLa and normal skin fibroblast (NSF) cells indicated that the relative distribution was very similar, but that nascent strand abundance in HeLa cells was approximately twice that in NSF relative to the abundance at the lamin B2 origin. This increased activity at initiation sites in the c-myc locus may mainly be influenced by regulators at higher levels in transformed cells like HeLa.

Cells, Cultured↗
PubMed2000

Reduction of anxiety during MR imaging: a controlled trial.

An anxiety reduction protocol was developed and evaluated for routine use with neurology and neuropsychiatry patients undergoing brain or spinal scans. Thirty five patients underwent standard procedures, with limited information given in advance. Twenty nine experimental patients received a booklet giving information about the scanning procedure and advice on cognitive strategies for anxiety reduction, a tape-recorded demonstration of scanner noise, a visit to the control room before entering the scanner, a device to signal for adjustment of music volume, precise timings of each scan, and a clock visible during scanning. Anxiety was measured before, during, and after scanning, using subjective ratings and a retrospective version of the Spielberger State Anxiety Scale. There was no difference in anxiety between groups immediately prior to the scan and immediately after entering the scanner. Patients in the experimental group were significantly less anxious during the scan than control patients, as measured by mean anxiety ratings made during the imaging procedure and by retrospective State Anxiety scores completed immediately after leaving the scanner. The results show that scan-related anxiety can be reduced by introducing these simple changes to MR imaging procedures, with minimal cost, no special training of staff, and no disruption of the running of the MR Unit.

Adult↗
PubMed1999

Identification of initiation sites for DNA replication in the human dnmt1 (DNA-methyltransferase) locus.

Vertebrates have developed multiple mechanisms to coordinate the replication of epigenetic and genetic information. Dnmt1 encodes the maintenance enzyme DNA-methyltransferase, which is responsible for propagating the DNA methylation pattern and the epigenetic information that it encodes during replication. Direct sequence analysis and bisulfite mapping of the 5' region of DNA-methyltransferase 1 (dnmt1) have indicated the presence of many sequence elements associated with previously characterized origins of DNA replication. This study tests the hypothesis that the dnmt1 region containing these elements is an origin of replication in human cells. First, we demonstrate that a vector containing this dnmt1 sequence is able to support autonomous replication when transfected into HeLa cells. Second, using a gel retardation assay, we show that it contains a site for binding of origin-rich sequences binding activity, a recently purified replication protein. Finally, using competitive polymerase chain reaction, we show that replication initiates in this region in vivo. Based on these lines of evidence, we propose that initiation sites for DNA replication are located between the first intron and exon 7 of the human dnmt1 locus.

Chromosome Mapping↗
PubMed1999

Separation-of-function mutations in Saccharomyces cerevisiae MSH2 that confer mismatch repair defects but do not affect nonhomologous-tail removal during recombination.

Yeast Msh2p forms complexes with Msh3p and Msh6p to repair DNA mispairs that arise during DNA replication. In addition to their role in mismatch repair (MMR), the MSH2 and MSH3 gene products are required to remove 3' nonhomologous DNA tails during genetic recombination. The mismatch repair genes MSH6, MLH1, and PMS1, whose products interact with Msh2p, are not required in this process. We have identified mutations in MSH2 that do not disrupt genetic recombination but confer a strong defect in mismatch repair. Twenty-four msh2 mutations that conferred a dominant negative phenotype for mismatch repair were isolated. A subset of these mutations mapped to residues in Msh2p that were analogous to mutations identified in human nonpolyposis colorectal cancer msh2 kindreds. Approximately half of the these MMR-defective mutations retained wild-type or nearly wild-type activity for the removal of nonhomologous DNA tails during genetic recombination. The identification of mutations in MSH2 that disrupt mismatch repair without affecting recombination provides a first step in dissecting the Msh-effector protein complexes that are thought to play different roles during DNA repair and genetic recombination.

Alleles↗
PubMed1998

Mixed-lineage kinase 2-SH3 domain binds dynamin and greatly enhances activation of GTPase by phospholipid.

Mixed-lineage kinase 2 (MLK2) is a cytoplasmic protein kinase expressed at high levels in mammalian brain. The MLK2 structure is composed of a Src homology 3 (SH3) domain, two leucine zippers, a basic motif, a Cdc42/Rac interactive binding motif and a large C-terminal domain rich in proline, serine and threonine residues. To begin to define the role of MLK2 in mammalian brain, we used an MLK2-SH3 domain-glutathione S-transferase fusion protein (GST-MLK2-SH3) to isolate MLK2-binding proteins from rat brain extract. This analysis revealed that the major MLK2-SH3-domain-binding protein in rat brain is the GTPase dynamin. By using two different forms of the dynamin proline-rich domain as affinity ligands, the binding site for MLK2-SH3 was mapped to the C-terminal region of dynamin between residues 832 and 864. In GTPase assays, the addition of MLK2-SH3 stimulated the activity of purified dynamin I by 3-fold over the basal level, whereas the addition of a known dynamin activator, phosphatidylserine (PtdSer), stimulated a 6-fold increase. When MLK2-SH3 was added to the assay together with PtdSer, however, dynamin GTPase activity accelerated by more than 23-fold over basal level. An MLK2 mutant (MLK2-W59A-SH3), with alanine replacing a conserved tryptophan residue in the SH3 domain consensus motif, had no effect on dynamin activity, either alone or in the presence of PtdSer. In the same assay the SH3 domain from the regulatory subunit of phosphatidylinositol 3'-kinase stimulated a similar synergistic acceleration of dynamin GTPase activity in the presence of PtdSer. These results suggest that synergy between phospholipid and SH3 domain binding might be a general mechanism for the regulation of GTP hydrolysis by dynamin.

Animals↗
PubMed1998

Anoxia during kainate status epilepticus shortens behavioral convulsions but generates hippocampal neuron loss and supragranular mossy fiber sprouting.

In rats, this study determined the impact of systemic hypoxia during late kainate-induced status epilepticus on hippocampal neuron loss and mossy fiber sprouting. Non-fasted Sprague Dawley rats were prepared as follows: Naive controls (n=5); rats placed 2 min in a hypoxia chamber (hypoxia only; n=6); rats that seized for more than 6 h from kainic acid (KA-status; 12 mg/kg; i.p.; n=7); and another KA-status group placed into the hypoxia chamber 75 min after the convulsions started (KA-status/hypoxia; n=16). All rats, except for half of the KA-status/hypoxia animals, were perfused 2 weeks later (short-term). The other 8 KA-status/hypoxia rats were perfused after 2 months (long-term). Hippocampal sections were studied for neuron densities and aberrant mossy fiber sprouting at three ventral to dorsal levels. Fascia dentata (FD) mossy fiber sprouting was quantified as an increase in the inner minus outer molecular layer (IML-OML) gray value (GV) difference. Behaviorally, KA-status/hypoxia rats had a shorter duration of convulsive status epilepticus than KA-status animals without anoxia. Hippocampal sections showed that compared to controls: (1) hypoxia-only rats showed no differences in ventral neuron densities and neo-Timm's stained IML-OML GVs; (2) KA-status rats had decreased CA3 densities and a non-significant increase in ventral IML-OML GV differences; and (3) KA-status/hypoxia short-term animals showed decreased hilar, CA3 and CA1 densities and increased ventral IML-OML GV differences. Compared to KA-status/hypoxia short-term rats, long-term animals showed no differences in ventral hippocampal neuron densities, but middle and dorsal sections demonstrated increased IML-OML GV differences and animals were observed to have spontaneous limbic epilepsy. These results indicate that rats exposed to kainate-induced status epilepticus for over 1 h and then a hypoxic insult had a shorter duration of convulsive status, decreased hippocampal neuron densities and greater FD mossy fiber sprouting than controls and the amount of neuronal damage and sprouting was slightly more than animals subjected to 6 h of kainate-induced status. This supports the hypothesis that a physiologic insult during status can shorten the convulsive episode, but still produce hippocampal pathology with a number of clinical and pathologic similarities to human mesial temporal lobe epilepsy (MTLE).

Animals↗
PubMed1998

Conformation of replicated segments of chromosome fibres in human S-phase nucleus.

Recent statistical analysis of the folding of G0/G1 chromosomes using fluorescence in situ hybridization (FISH) allowed development of a random walk/giant loop model of chromosome structure. According to this model there are two levels of organization of G0/G1 chromosome fibres. On the first level, the fibres are arranged in giant loops several Mbp in size, and within each loop the fibres are randomly folded. On the second level, the loop attachment sites form a chromosome backbone that also shows random folding. Newly replicated segments of mammalian chromosomes may be directly visualized at high resolution in S-phase nuclei using immunofluorescent methods and appear as worm-like fibres. In our earlier study, we analysed conformation of the fibres in human cells blocked for 16 h at the G1/S boundary with 5-fluorodeoxyuridine (FdU) and then released into S-phase by the addition of a DNA precursor. However, long treatment of cells with FdU induces very short replicons and may promote apoptosis. In this study we analysed conformation of the fibres in normally proliferating human cells that had not been blocked with FdU for a long time. It has been found that replicated chromosome fibres visualized just after 2 h of incubation of the cells with a non-radioactively labelled DNA precursor behave as flexible polymer chains without major constraints, and that their local conformation in the range of several microns of their contour length may be considered as random. Confocal analysis of human X chromosomes visualized in HeLa cells using FISH with a specific painting probe shows that in S-phase the chromosomes occupy distinct nuclear territories and their apparent size does not differ from that in non-S-phase cells. This observation indicates that the second level of chromosome organization also exists in S-phase chromosomes. It appears, therefore, that the random walk/giant loop model developed earlier for G0/G1 chromosomes is also valid for S-phase chromosomes.

Cell Nucleus↗
PubMed1997

Differential DNA replication origin activities in human normal skin fibroblast and HeLa cell lines.

A modification of the extrusion method for the isolation of nascent DNA from mammalian cells and a PCR-based assay has been used in order to compare the in vivo activities of DNA replication origins in different cell lines. Conventional PCR was firstly applied to detect the chromosomal activities of several known (origins associated with c-myc, hsp70, beta-globin, immunoglobulin mu-chain enhancer) and putative DNA replication origins (autonomously replicating sequences obtained from enriched libraries of human origins of DNA replication from normal and transformed cells) in four human cell lines (HeLa, NSF, WI-38 and SK-MG-1). Then, in nascent DNA samples from normal skin fibroblast (NSF) and HeLa cells, abundance of DNA sequences in the regions of five of these origins was determined by competitive PCR. Our results suggest that autonomously replicating sequences NOA3, S14, S3 and F15 are associated with functional chromosomal origins of replication. Quantitative comparison of origin activities demonstrates that origins associated with c-myc and NOA3 are approximately twice as active in HeLa cells as in NSF cells. The described approach can facilitate the identification of origins which may be differentially active in normal cells and transformed cells or in different cell types.

Cell Line, Transformed↗
PubMed1995

Complete nucleotide sequence, expression, and chromosomal localisation of human mixed-lineage kinase 2.

Protein kinases play pivotal roles in the control of many cellular processes. In a search for protein kinases expressed in human epithelial tumour cells, we discovered two members of a novel protein kinase family [Dorow, D. S., Devereux, L., Dietzsch, E. & de Kretser, T. A. (1993) Eur. J. Biochem. 213, 701-710]. Due to the unique mixture of structural domains within their amino acid sequences, we named the family mixed-lineage kinases (MLK). We initially isolated clones encoding partial cDNAs of MLK1 and 2 from a human colonic cDNA library. The MLK2 cDNA was subsequently used to screen a human brain cDNA library and we have now cloned and sequenced a 3454-bp cDNA encoding the full-length MLK2 protein. The predicted MLK2 polypeptide has 954 amino acids and contains a src homology 3 (SH3) domain, a kinase catalytic domain, a double leucine zipper and basic domain, and a large C-terminal domain. The 22-amino-acid N-terminal region has four glutamic acid residues immediately following the initiator methionine. Beginning at amino acid 23, the 55-amino-acid SH3 domain contains a 5-amino-acid insert in a position corresponding to inserts of 6 and 15 residues in the SH3 domains of n-src and the phosphatidylinositol 3'-kinase. Adjacent to the SH3 domain is a kinase catalytic domain with conserved motifs associated with both serine/threonine and tyrosine specificity. Beginning nine residues C-terminal to the catalytic domain, there are two leucine/isoleucine zippers separated by a 13-amino-acid spacer sequence and followed by a stretch of basic residues. The polybasic sequence contains a motif that is similar to nuclear localisation signals from several proteins. The C-terminal domain is composed of 491 amino acids of which 17% are serine or threonine and 16% are proline. This domain also has a biased ratio of basic to acidic amino acids with a calculated pI of 9.38. When used as a probe to examine mRNA expression in human tissues, a MLK2 cDNA hybridised to a species of 3.8 kb that was expressed at highest levels in RNA from brain and skeletal muscle. The 3454-bp cDNA was also used for fluorescence in situ hybridisation to localise the MLK2 gene to human chromosome 19 q13.2.

Amino Acid Sequence↗
PubMed1995

The lymph node pathology of Omenn's syndrome.

Omenn's syndrome is a rare, usually fatal immunologic disorder of infancy characterized by recurrent infections, skin lesion, lymphadenopathy, peripheral blood lymphocytosis, and eosinophilia. Histologic evaluation of a lymph node revealed total effacement of the microscopic architecture resulting from a diffuse proliferation of interdigitating reticulum cells and a depletion of B lymphocytes. The lymph node lacked a distinct cortex and had no follicle formation. The most striking feature was a diffuse hyperplasia of S-100-protein-positive nonphagocytic reticulum cells with large, pale Langerhans-like nuclei. Ultrastructural examination identified these cells to be interdigitating reticulum cells. The lymphocytes were small and predominantly of the CD8 cytotoxic/suppressor cell type.

Antigens, CD↗
PubMed1994

A reproducible method for identification of human genomic DNA autonomously replicating sequences.

We demonstrate a method for the isolation of autonomously replicating sequences from pools of clones obtained from genomic DNA libraries constructed using affinity purification of cruciform DNA. The selection of autonomously replicating sequences was based on their differential ability to replicate as episomes after transfection of pools of plasmid clones into human HeLa cells. Two separate libraries containing affinity-purified cruciform DNA were used, one prepared from DNA of log phase primary human genital fibroblasts and the other prepared from DNA of log phase SW48 colon adenocarcinoma cells. Representative samples of the entire phage libraries were converted to phagemid clones by filamentous helper phage-mediated mass excision to produce pBluescript libraries in Escherichia coli. Clones were grown up individually and the bacteria pooled into groups of 48 for recovery of plasmid DNA. Plasmid pools of 48 independent clones (120 micrograms total) were then transfected by calcium phosphate coprecipitation onto log phase HeLa cells, which were allowed to grow for 3 days before recovery of plasmid by Hirt lysis. The recovery of plasmid from each transfection was estimated to range from 10 to 60 ng. DpnI digestion was then used to digest plasmids which had not been replicated and therefore retained a bacterial methylation pattern which was sensitive to digestion. We estimated from agarose electrophoresis gels that 40-200 pg of recovered plasmid DNA per transfected pool of DNA was resistant to DpnI and therefore was capable of transforming competent E. coli cells. The DpnI-resistant fraction yielded from one to seven independent clones from each pool, with genomic DNA inserts ranging in size from 0.35 to 3.4 kb.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA↗
PubMed1993

Patterns of smoking during pregnancy in Canterbury.

AIMS: To examine the prevalence and patterns of smoking in pregnancy with the object of improving smokefree programmes in the region. METHODS: A postal questionnaire on smoking in pregnancy was sent to all 1916 mothers giving singleton births in the Canterbury region over a five month period. There was a 71.7% response rate, however, smokers were significantly under represented. Data from nonresponders was obtained from obstetric records. RESULTS: Of the total sample, 30% smoked during their last pregnancy. There were significant differences between responders and nonresponders. The responders contained only 60% of all smokers. Nonresponders had twice the incidence of smoking, were more likely to identify as Maori, were younger and had lower birth weights. Nonresponders contained 40% of all smokers. Of the responders, 333 mothers smoked during at least some part of pregnancy: 113 (34%) quit, 168 (50%) cut down, and 48 (15%) made no change. Most (90%) of those who did quit did so during the first trimester. Lighter smokers (less than 10 per day) were more likely to quit or cut down. But smoking rates subsequently increased after the birth. CONCLUSIONS: In Canterbury, 30% of pregnant women smoke. Although 64% indicated a wish to quit and 30% to cut down, this contrasted with what they actually achieved: 34% quit and 50% cut down. Pregnancy influences smoking patterns and is an opportune time for smokefree promotion.

Adult↗
PubMed1993

Naltrexone treatment attenuates the inhibitory effect of nicotine treatment on serum LH in rats.

The hypothesis that endogenous opioids might have a role in mediating the suppressive effects of nicotine on serum LH concentrations in rats was investigated. Naltrexone treatment prevented the inhibitory effect of high doses of nicotine on serum LH concentrations. Nicotine treatment also prevented the stimulatory effect of naltrexone on serum LH concentrations. These results suggest that the inhibitory effects of nicotine on serum LH concentrations involve an opioidergic component.

Adrenalectomy↗