Phenotypes and endotypes of rhinitis and their impact on management: a PRACTALL report.

Rhinitis is an umbrella term that encompasses many different subtypes, several of which still elude complete characterization. The concept of phenotyping, being the definition of disease subtypes on the basis of clinical presentation, has been well established in the last decade. Classification of rhinitis entities on the basis of phenotypes has facilitated their characterization and has helped practicing clinicians to efficiently approach rhinitis patients. Recently, the concept of endotypes, that is, the definition of disease subtypes on the basis of underlying pathophysiology, has emerged. Phenotypes/endotypes are dynamic, overlapping, and may evolve into one another, thus rendering clear-cut definitions difficult. Nevertheless, a phenotype-/endotype-based classification approach could lead toward the application of stratified and personalized medicine in the rhinitis field. In this PRACTALL document, rhinitis phenotypes and endotypes are described, and rhinitis diagnosis and management approaches focusing on those phenotypes/endotypes are presented and discussed. We emphasize the concept of control-based management, which transcends all rhinitis subtypes.

Association of CHRNA5-A3-B4 SNP rs2036527 with smoking cessation therapy response in African-American smokers.

Associations between CHRNA5-A3-B4 variants and smoking behaviors exist; however, the association with smoking abstinence is less understood, particularly that among African Americans. In 1,295 African Americans enrolled in two clinical trials, we investigated the association between CHRNA5-A3-B4 and smoking abstinence. The rs2056527(A) allele was associated with lower abstinence with active pharmacotherapy (during treatment: odds ratio (OR) = 0.42, P < 0.001; end of treatment (EOT): OR = 0.55, P = 0.004), or with nicotine gum alone (during treatment: OR = 0.31, P < 0.001; EOT: OR = 0.51, P = 0.02), but not significantly with bupropion, although similar directions and magnitudes were observed (during treatment: OR = 0.54, P = 0.05; EOT: OR = 0.59, P = 0.08). In addition, the rs588765(T) allele was associated with abstinence with gum during treatment (OR = 2.31, P < 0.01). The SNP rs16969968 occurred at a low frequency and was not consistently associated with abstinence. CHRNA5-A3-B4 variants were not associated with tobacco consumption, and adjustments for smoking behaviors did not alter the associations with smoking abstinence. Together, our data suggest that among African Americans, CHRNA5-A3-B4 variants are not associated with baseline smoking but can influence smoking abstinence during active pharmacotherapy.

CYP2B6 and bupropion's smoking-cessation pharmacology: the role of hydroxybupropion.

Bupropion is indicated to promote smoking cessation. Animal studies suggest that the pharmacologic activity of bupropion can be mediated by its major metabolite, hydroxybupropion. We measured plasma bupropion and its metabolite levels in a double-blind, placebo controlled, randomized smoking-cessation trial. Among the treatment-adherent individuals, higher hydroxybupropion concentrations (per µg/ml) resulted in better smoking-cessation outcomes (week 3, 7, and 26 odds ratio (OR) = 2.82, 2.96, and 2.37, respectively, P = 0.005-0.040); this was not observed with bupropion levels (OR = 1.00-1.03, P = 0.59-0.90). Genetic variation in CYP2B6, the enzyme that metabolizes bupropion to hydroxybupropion, was identified as a significant source of variability in hydroxybupropion formation. Our data indicate that hydroxybupropion contributes to the pharmacologic effects of bupropion for smoking cessation, and that variability in response to bupropion treatment is related to variability in CYP2B6-mediated hydroxybupropion formation. These findings suggest that dosing of bupropion to achieve a hydroxybupropion level of 0.7 µg/ml or increasing bupropion dose for CYP2B6 slow metabolizers could improve bupropion's cessation outcomes.

From old organisms to new molecules: integrative biology and therapeutic targets in accelerated human ageing.

Understanding the basic biology of human ageing is a key milestone in attempting to ameliorate the deleterious consequences of old age. This is an urgent research priority given the global demographic shift towards an ageing population. Although some molecular pathways that have been proposed to contribute to ageing have been discovered using classical biochemistry and genetics, the complex, polygenic and stochastic nature of ageing is such that the process as a whole is not immediately amenable to biochemical analysis. Thus, attempts have been made to elucidate the causes of monogenic progeroid disorders that recapitulate some, if not all, features of normal ageing in the hope that this may contribute to our understanding of normal human ageing. Two canonical progeroid disorders are Werner's syndrome and Hutchinson-Gilford progeroid syndrome (also known as progeria). Because such disorders are essentially phenocopies of ageing, rather than ageing itself, advances made in understanding their pathogenesis must always be contextualised within theories proposed to help explain how the normal process operates. One such possible ageing mechanism is described by the cell senescence hypothesis of ageing. Here, we discuss this hypothesis and demonstrate that it provides a plausible explanation for many of the ageing phenotypes seen in Werner's syndrome and Hutchinson-Gilford progeriod syndrome. The recent exciting advances made in potential therapies for these two syndromes are also reviewed.

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.

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.

Hastening death: a comparison of two end-of-life decisions.

This study determined the relationship of psychosocial and background variables to elders' end-of-life (EOL) decision preferences. Responding to 5 EOL decision scenarios depicting terminally ill elders, 200 elders aged 60-90 indicated preferences regarding extending life (EL), refusing treatment (RT), and assisted suicide (AS). They were also assessed on religiosity, values, fear of death, locus of control, health, socioeconomic status, and age. Results of multinomial logistic regression indicated that EOL decisions of three groups (favoring EL, favoring RT, and favoring both AS and RT) were significantly influenced by religiosity, value for preservation of life, value for quality of life, fear of death, and locus of control belief. The importance of safeguarding older adults' autonomy in EOL decisions was stressed.

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.

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.

MDM2 and MDMX bind and stabilize the p53-related protein p73.

The p53 gene encodes one of the most important tumor suppressors in human cells and undergoes frequent mutational inactivation in cancers. MDM2, a transcriptional target of p53, binds p53 and can both inhibit p53-mediated transcription [1] [2] and target p53 for proteasome-mediated proteolysis [3] [4]. A close relative of p53, p73, has recently been identified [5] [6]. Here, we report that, like p53, p73alpha and the alternative transcription product p73beta also bind MDM2. Interaction between MDM2 and p53 represents a key step in the regulation of p53, as MDM2 promotes the degradation of p53. In striking contrast to p53, the half-life of p73 was found to be increased by binding to MDM2. Like MDM2, the MDM2-related protein MDMX also bound p73 and stabilized the level of p73. Moreover, the growth suppression functions of p73 and the induction of endogenous p21, a major mediator of the p53-dependent growth arrest pathway, were enhanced in the presence of MDM2. These differences between the regulation of p53 and p73 by MDM2/MDMX may highlight a physiological difference in their action.

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.

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.

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.

Associative and nonassociative tolerance: the effects of dose and interdose interval.

Two experiments examined the effects of dose and interdose interval (IDI) on associative and nonassociative tolerance to morphine analgesia in rats. Associative contingencies were manipulated by administering low (5 mg/kg) or high (20 mg/kg) doses of morphine explicitly paired or unpaired with a distinctive context. Nonassociative processes were manipulated by administering morphine at a short (6-h) or long (96-h) IDI. Tolerance was assessed as shifts in morphine dose-response curves on the tail-flick test. Animals in the long IDI conditions showed considerable context-specific tolerance. Tolerance in the short IDI conditions was not influenced by contextual contingencies at the immediate test (Experiment 1) and showed no retention over a 30-day interval (Experiment 2), suggesting this tolerance was nonassociative. The impact of massed exposure to morphine and context on the disruption of learning at the short IDI is discussed.

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.

Who binds wins: Competition for PCNA rings out cell-cycle changes.

PCNA, proliferating cell nuclear antigen, is a pivotal protein in DNA replication, DNA repair and possibly cell-cycle control. The protein has a trimeric ring structure that might slide along duplex DNA and form a platform for association with a variety of proteins, in particular holding the DNA polymerases in close association with their template. This article reviews evidence suggesting that the activity of PCNA in replication and repair is coordinated within the cell cycle by cooperative and competitive interactions with an extensive network of enzymes and regulatory proteins.

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.

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.