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L S Cox

Publications and source records attributed to L S Cox.

At least 19 recordsLinked to original sources
PubMed2001

Identification of an MCM4 homologue expressed specifically in the sexual stage of Plasmodium falciparum.

Mini-chromosome maintenance (MCM) proteins play an essential role in DNA replication initiation. We have isolated a novel gene encoding an MCM-like protein from the human malaria parasite Plasmodium falciparum using the vectorette technique. The gene has no introns and comprises an open reading frame encoding 1005 amino acid residues with a predicted Mr of 115 kDa. The encoded protein, termed PfMCM4, contains all conserved sequences in the MCM family and displays the highest homology to the Cdc54 (MCM4) of Saccharomyces cerevisiae. However, PfMCM4 possesses five unique amino acid inserts with sizes ranging from seven to 75 residues. Southern blotting of genomic DNA digests and chromosomal separations showed that the Pfmcm4 gene is present as a single copy per haploid genome and is located on chromosome 13. A 4000-nucleotide transcript of this gene is expressed specifically in the sexual erythrocytic stage, indicating that PfMCM4 may be involved in gametogenesis in which DNA is quickly replicated.

Amino Acid Sequence↗
PubMed2001

Evaluation of the brief questionnaire of smoking urges (QSU-brief) in laboratory and clinical settings.

A brief, 10-item version of the Questionnaire of Smoking Urges (QSU; Tiffany & Drobes, British Journal of Addiction 86:1467-1476, 1991) was administered to 221 active cigarette smokers in a laboratory setting (Study 1) and to 112 smokers enrolled in a comprehensive smoking cessation program (Study 2). In the laboratory setting, craving to smoke was evaluated in response to neutral and smoking-related stimuli. In the clinical setting, craving was assessed prior to cessation and again during treatment. Factor analyses revealed that a two-factor solution best described the item structure of the QSU-Brief across conditions. Factor 1 items reflected a strong desire and intention to smoke, with smoking perceived as rewarding for active smokers. Factor 2 items represented an anticipation of relief from negative affect with an urgent desire to smoke. The findings were consistent with the expressions of craving found in the 32-item version of the QSU (Tiffany & Drobes, 1991). Regression analyses demonstrated stronger baseline mood intensity and self-reported tendency to smoke to achieve pleasurable effects and to experience the desire to smoke when cigarettes are unavailable were predictive of general levels of craving report in active smokers in the laboratory and clinical setting. The findings supported a multidimensional conceptualization of craving to smoke and demonstrated the utility of a brief multidimensional measure of craving.

Adult↗
PubMed2000

Sexual stage-specific expression of a third calcium-dependent protein kinase from Plasmodium falciparum.

A third calcium-dependent protein kinase (CDPK) gene has been isolated from the human malaria parasite Plasmodium falciparum by vectorette technology. The gene consists of five exons and four introns. The open reading frame resulting from removal of the four introns encodes a protein of 562 amino acid residues with a predicted molecular mass of 65.3 kDa. The encoded protein, termed PfCDPK3, consists of four distinct domains characteristic of a member of the CDPK family and displays the highest homology (46% identity and 69% similarity) to PfCDPK2, the second CDPK of P. falciparum. The N-terminal variable domain is rich in serine/threonine and lysine and contains multiple consensus phosphorylation sites for a range of protein kinases. The catalytic domain possesses all conserved motifs of the protein kinase family except for the highly conserved glutamic acid residue in subdomain VIII, which is replaced by a glutamine residue. The sequence of the junction domain comprising 31 amino acid residues is less conserved. The calmodulin-like regulatory domain contains four EF-hand calcium-binding motifs, each consisting of a loop of 12 amino acid residues which is flanked by two alpha-helices. Southern blotting of genomic DNA digests showed that the Pfcdpk3 gene is present as a single copy per haploid genome. A 2900 nucleotide transcript of this gene is expressed specifically in the sexual erythrocytic stage, indicating that PfCDPK3 is involved in sexual stage-specific events. It is proposed that PfCDPK3 may serve as a link between calcium and gametogenesis of P. falciparum.

Amino Acid Sequence↗
PubMed2000

Effects of transdermal nicotine patches on abstinence-induced and cue-elicited craving in cigarette smokers.

The impact of a transdermal nicotine patch on smokers' craving for cigarettes and reactivity to smoking cues was investigated. Sixty-one smokers were assessed during 2 sessions separated by 6 hr. Cue reactivity to imaginal and in vivo smoking and nonsmoking stimuli was evaluated during both sessions. During the interval between sessions, participants were abstinent from cigarettes and wore either a nicotine transdermal (21 mg) or placebo patch. In both sessions, exposure to in vivo and imaginal smoking stimuli elicited cue-specific increases in craving, negative affect, vividness, heart rate, and skin conductance. The nicotine patch attenuated craving and other effects induced by abstinence from cigarettes but had no selective impact on craving or any other reaction elicited by smoking cues. These results are discussed in terms of models of craving and clinical implications of transdermal nicotine for craving reduction.

Administration, Cutaneous↗
PubMed1999

Context-specific morphine tolerance on the paw-pressure and tail-shock vocalization tests: evidence of associative tolerance without conditioned compensatory responding.

RATIONALE: Demonstrations of associative tolerance to the analgesic effects of morphine, not confounded by practice or novelty effects, have been restricted to the tail-flick and flinch-jump tests. OBJECTIVES: Experiment 1 investigated whether associative tolerance would be found on two other nociceptive assessment methods: the paw-pressure withdrawal and tail-shock vocalization thresholds. Experiment 2 tested the hypothesis that conditioned compensatory behavioral responses are the substrate of associative morphine tolerance in the paw-pressure, tail-shock, and tail-flick tests. METHODS: Rats were given eight morphine injections (20 mg/kg, i.p.) explicitly paired or unpaired with a distinctive context. Control animals were given saline injections over the course of conditioning. Animals were then tested after morphine (experiment 1) or placebo injections (experiment 2) in the context. RESULTS: There was evidence of context-specific tolerance across both testing methods, with a rightward shift of dose-response curves of paired relative to unpaired animals. No evidence of conditioned compensatory responding was found on any of the three testing methods. CONCLUSIONS: The data indicated that, although Pavlovian processes can play a major role in tolerance acquisition, there was little support for the thesis that the conditioned tolerance response is a behavioral effect that is opposite in direction to the direct effects of the drug.

Analgesics, Opioid↗
PubMed1999

Comparative study of spray booth filter system efficiency.

During recent years, greater emphasis has been placed on the control of particulate emissions from painting operations. This has gained more importance as more is learned about the potential release of toxic metals to the atmosphere from painting operations. This has led to queries about the efficiency of various painting arrestor systems to reduce particulate discharges to the atmosphere. Even more important is the capability of the arrestor systems to control PM10 emissions. In 1995, the U.S. Environmental Protection Agency initiated a study to evaluate various dry paint overspray arrestor systems. This study was designed to evaluate not only the total emissions control capability of the arrestor but also the PM10 control capability of the various system designs. Paint overspray arrestor systems using five different filtration concepts or materials were selected. They include systems constructed of fiberglass, paper, Styrofoam, and cardboard materials. These systems used filtration techniques incorporating the following filtration phenomena and designs: cyclone, baffle, bag systems, and mesh systems. The testing used an optical particle counting procedure to determine the concentration of particles of a given size fraction to penetrate a test arrestor system. The results of the testing indicated that there are significant differences in the efficiency of the tested system designs to capture and retain PM10. This paper summarizes the results of the research conducted to determine the capability of the arrestor systems to capture particulate of sizes down to approximately 1 micron in surface diameter.

Air Pollutants, Occupational↗
PubMed1997

Homologous regions of Fen1 and p21Cip1 compete for binding to the same site on PCNA: a potential mechanism to co-ordinate DNA replication and repair.

Following genomic damage, the cessation of DNA replication is co-ordinated with onset of DNA repair; this co-ordination is essential to avoid mutation and genomic instability. To investigate these phenomena, we have analysed proteins that interact with PCNA, which is required for both DNA replication and repair. One such protein is p21Cip1, which inhibits DNA replication through its interaction with PCNA, while allowing repair to continue. We have identified an interaction between PCNA and the structure specific nuclease, Fen1, which is involved in DNA replication. Deletion analysis suggests that p21Cip1 and Fen1 bind to the same region of PCNA. Within Fen1 and its homologues a small region (10 amino acids) is sufficient for PCNA binding, which contains an 8 amino acid conserved PCNA-binding motif. This motif shares critical residues with the PCNA-binding region of p21Cip1. A PCNA binding peptide from p21Cip1 competes with Fen1 peptides for binding to PCNA, disrupts the Fen1-PCNA complex in replicating cell extracts, and concomitantly inhibits DNA synthesis. Competition between homologous regions of Fen1 and p21Cip1 for binding to the same site on PCNA may provide a mechanism to co-ordinate the functions of PCNA in DNA replication and repair.

Amino Acid Sequence↗
PubMed1997

Multiple pathways control cell growth and transformation: overlapping and independent activities of p53 and p21Cip1/WAF1/Sdi1.

Many tumour therapies act by inducing a cellular damage response pathway mediated by the tumour suppressor protein p53. Alternative outcomes of p53 induction include apoptosis or transient cell-cycle arrest, both thought to require the transcriptional activity of wild-type p53. Current research highlights the action of a p53-activated gene, p21Cip1/WAF1/Sdi1, which encodes a cyclin-kinase inhibitor important in mediating p53-dependent cell-cycle arrest, while programmed cell death in response to DNA damage requires transcriptionally active p53 but not activation of p21Cip1/WAF1/Sdi1. This review examines the roles of p53 and p21Cip1/WAF1/Sdi1 in controlling cell proliferation, in the light of a new study on expression of p53 and p21Cip1/WAF1/Sdi1 in squamous cell carcinoma of the larynx.

Apoptosis↗
PubMed1996

Two pathways for base excision repair in mammalian cells.

Abasic sites (apurinic/apyrimidinic, AP sites) are the most common DNA lesions generated by both spontaneous and induced base loss. In a previous study we have shown that circular plasmid molecules containing multiple AP sites are efficiently repaired by Chinese hamster extracts in an in vitro repair assay. An average patch size of 6.6 nucleotides for a single AP site was calculated. To define the exact repair patch, a circular DNA duplex with a single AP site was constructed. The repair synthesis carried out by hamster and human cell extracts was characterized by restriction endonuclease analysis of the area containing the lesion. The results indicate that, besides the repair events involving the incorporation of a single nucleotide at the lesion site, repair synthesis occurred also 3' to the AP site and involved a repair patch of approximately 7 nucleotides. This alternative repair pathway was completely inhibited by the presence in the repair reaction of a polyclonal antibody raised against human proliferating cell nuclear antigen. These data give the first evidence that mammalian cell extracts repair natural AP sites by two distinct pathways: a single nucleotide gap filling reaction targeted at the AP site and a proliferating cell nuclear antigen-dependent pathway that removes a short oligonucleotide containing the abasic site and 3'-flanking nucleotides.

Animals↗
PubMed1995

Characterisation of the interaction between PCNA and Gadd45.

We have examined the interaction between the DNA replication and repair protein PCNA, and the growth arrest and DNA damage induced protein Gadd45. An anti-Gadd45 polyclonal antibody co-immunoprecipitates PCNA but in reciprocal experiments, an anti-C terminal anti-PCNA antibody failed to co-immunoprecipitate Gadd45. We used a yeast two hybrid assay to demonstrate that human Gadd45 interacts with both human and S. pombe PCNA. We have determined that the N-terminal 94 amino acids of Gadd45 bind to PCNA, and using a series of N-terminal and C-terminal deletions of human PCNA we have mapped two potential Gadd45 binding sites. Deletion of the last 6 amino acids of PCNA ablated interaction, suggesting a role in Gadd45 binding. This explains the inability of an anti-C terminal PCNA antibody to co-immunoprecipitate Gadd45. Using a peptide ELISA approach, we showed that Gadd45 protein binds strongly to three regions of PCNA (residues 1-20, 61-80, and 196-215) and weakly to residues 121-170. The crystal structure of PCNA provides insight into our genetic and immunochemical data. Our results confirm an interaction between PCNA and Gadd45, define regions of both molecules involved in this interaction, and are consistent with a potential stoichiometry of 2 Gadd45 molecules to each PCNA monomer. These data provide support for the notion that PCNA-Gadd45 interactions co-ordinate cell cycle and DNA repair.

Amino Acid Sequence↗
PubMed1995

A direct effect of activated human p53 on nuclear DNA replication.

p53 is a transcriptional activator and repressor, but recent evidence suggests that some of its many biological functions may not be dependent on transcription. To determine whether p53 exerts a direct influence on nuclear DNA replication, purified human p53 was added to a transcription-free DNA replication extract from Xenopus eggs. Full-length human p53 that inhibits SV40 DNA replication in vitro had no effect on nuclear DNA synthesis in the Xenopus system. In contrast, a C-terminal truncated form of p53 (p53 delta 30), which is constitutively active for DNA binding and similar to an alternately spliced form found in vivo, showed a concentration-dependent inhibition of DNA replication in both the soluble SV40 system and eukaryotic nuclei. This inhibition occurred primarily at initiation of DNA synthesis. Oxidation of p53 delta 30, which eliminates DNA binding activity, also abrogated the protein's ability to inhibit nuclear DNA synthesis. The p53 binding DNA consensus sequence enhanced rather than competed away inhibitory activity of p53 delta 30. Therefore, p53 that is constitutively active for DNA binding can inhibit nuclear DNA replication in the absence of transcription. This inhibition may require binding of p53 to DNA, in addition to interactions between p53 and proteins of the replication complex.

Animals↗
PubMed1995

A small peptide inhibitor of DNA replication defines the site of interaction between the cyclin-dependent kinase inhibitor p21WAF1 and proliferating cell nuclear antigen.

BACKGROUND: p21WAF1 is a potent inhibitor of the cell-cycle regulatory cyclin-dependent kinases (Cdks). It acts on Cdks in the G1 and S phases of the cell cycle, and also binds to proliferating cell nuclear antigen (PCNA), blocking DNA replication in vitro. Transcription of p21WAF1 can be induced by the human tumour suppressor protein p53, suggesting that the action of p21WAF1 may be important in cancer prevention. We have investigated the interaction between p21WAF1 and PCNA using a genetic two-hybrid screen and with arrays of synthetic peptides derived from the p21WAF1 protein sequence. RESULTS: We have established that the carboxy-terminal region of p21WAF1 interacts with PCNA in a yeast two-hybrid screen. Interaction with p21WAF1 involves the central loop of PCNA, which connects the two domains of the PCNA monomer. The interaction was finely mapped using peptides derived from the entire sequence of the p21WAF1 protein, and the critical residues were found to be QTSMTDFY (amino acids 144-151 of p21WAF1). Remarkably, a 20-residue peptide containing this sequence inhibited replication of simian virus 40 (SV40) DNA in vitro and could capture PCNA from whole cell extracts, demonstrating that small molecules can retain the biological activity characteristic of the whole protein. Sequential alanine-scan mutations of the peptide demonstrated that its ability to block replication correlates with its affinity for binding PCNA. CONCLUSIONS: We have shown that PCNA and the cell-cycle regulator p21WAF1 interact in vivo, and that this interaction requires the central loop of PCNA and an eight amino-acid motif from the carboxyl terminus of p21WAF1.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Tumour suppressors, kinases and clamps: how p53 regulates the cell cycle in response to DNA damage.

The human tumour suppressor protein p53 is critical for regulation of the cell cycle on genotoxic insult. When DNA is damaged by radiation, chemicals or viral infection, cells respond rapidly by arresting the cell cycle. A G1 arrest requires the activity of wild-type p53, as it is not observed in cells lacking functionally wild-type protein, and at least some component of S phase and G2/M arrests is also thought to be p53-dependent. p53 functions as a transcription factor which binds specific DNA sequences, and recently major downstream targets have been identified, including p21Cip1, an inhibitor of the cell cycle kinases that also blocks the replicative but not the repair function of DNA polymerase delta auxiliary factor, PCNA. Current interest focuses on developing novel cancer therapies based on our knowledge of the activity of p53 and p21Cip1 in the cell cycle.

Alternative Splicing↗
PubMed1994

Weaving a pattern from disparate threads: lamin function in nuclear assembly and DNA replication.

The major residual structure that remains associated with the nuclear envelope following extraction of isolated nuclei or oocyte germinal vesicles with non-ionic detergents, nucleases and high salt is the lamina (Fawcett, 1966; Aaronson and Blobel, 1975; Dwyer and Blobel, 1976). The nuclear lamina is composed of intermediate filament proteins, termed lamins (Gerace and Blobel, 1980; Shelton et al., 1980), which polymerise to form a basket-weave lattice of fibrils, which covers the entire inner surface of the nuclear envelope and interlinks nuclear pores (Aebi et al., 1986; Stewart and Whytock, 1988; Goldberg and Allen, 1992). At mitosis, the nuclear envelope and the lamina both break down to allow chromosome segregation. As a consequence, each structure has to be rebuilt during anaphase and telophase, allowing cells an opportunity to reposition chromosomes (Heslop-Harrison and Bennett, 1990) and to reorganise looped chromatin domains (Franke, 1974; Franke et al., 1981; Hochstrasser et al., 1986), which may in turn control the use of subsets of genes. Because of the position that it occupies, its dynamics during mitosis and the fact that it is an essential component of proliferating cells, the lamina has been assigned a number of putative roles both in nuclear metabolism and in nuclear envelope assembly (Burke and Gerace, 1986; Nigg, 1989). However, to date there is little clear cut evidence that satisfactorily explains the function of the lamina in relation to its structure. In this Commentary we will describe some of the recent work that addresses this problem and attempt to provide a unified model for the role of lamins in nuclear envelope assembly and for the lamina in the initiation of DNA replication.

Amino Acid Sequence↗
PubMed1994

Xenopus p53 is biochemically similar to the human tumour suppressor protein p53 and is induced upon DNA damage in somatic cells.

Xenopus p53 cDNA, homologous to the human tumour suppressor p53, has previously been cloned from oocyte and gastrula libraries. In this report, we describe a polyclonal antibody 2674 raised against Xenopus p53 (Xp53) expressed in bacteria, that recognises proteins of approximately 52, 46 and 35 kDa present in Xenopus oocytes, parthenogenically activated eggs and in somatic tissue culture cells. We report here purification of Xp53 from insect cells infected with Xp53-baculovirus, and this protein is shown to be phosphorylated by casein kinase II but has low sequence-specific DNA binding activity. Using similar purification conditions, we have isolated endogenous Xp53, showing that Xenopus eggs contain high levels of p53 protein. Xp53 from eggs binds to the p53-specific DNA-binding consensus sequence. Two dimensional gel analysis indicates that Xp53 from eggs may exist in various states of phosphorylation. u.v.-induced DNA damage of somatic Xenopus cells results in accumulation of Xp53. We suggest that the high levels of putative Xp53 detected in eggs may represent maternal stockpiles of a protein necessary to protect rapidly dividing cells from the effects of DNA damage.

Animals↗