Cost-effectiveness analysis of medical management versus conservative surgery for early tubal pregnancy.

STUDY QUESTION: Is conservative surgery (laparoscopic salpingotomy) cost-effective, using fertility as the endpoint compared with medical management (Methotrexate) in women with an early tubal pregnancy? SUMMARY ANSWER: Conservative surgery appeared slightly, but not statistically significantly, more effective than medical management but also more costly. WHAT IS KNOWN ALREADY: Women with an early tubal pregnancy treated with medical therapy (Methotrexate) or conservative surgery (laparoscopic salpingotomy) have comparable future intrauterine pregnancy rates by natural conception. Also, cost-minimisation studies have shown that medical therapy was less expensive than conservative surgery, but there is no cost-effectiveness study comparing these two treatments with fertility as the endpoint. STUDY DESIGN, SIZE, DURATION: A multicentre randomised controlled trial-based (DEMETER study) cost-effectiveness analysis of conservative surgery compared with medical therapy in women with an early tubal pregnancy was performed. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Included women had an ultrasound that confirmed an early tubal pregnancy. They were randomly allocated to conservative surgery or to medical therapy. The study clinical outcome was the intrauterine pregnancy rate. The payer's perspective was considered. Costs of conservative surgery and medical therapy were compared. The analysis was performed according to the intention-to-treat principle. Missing variables were imputed using the fully conditional method. To characterise uncertainty and to provide a summary of it, a non-parametric bootstrap resampling was executed and cost-effectiveness accessibility curves were constructed. MAIN RESULTS AND THE ROLE OF CHANCE: At baseline, costs per woman in the conservative surgery group and in the medical therapy group were 2627€ and 2463€, respectively, with a statistically significant difference of +164€. Conservative surgery resulted in a marginally, but non-significant (P = 0.46), higher future intrauterine pregnancy rate compared to medical therapy (0.700 vs. 0.649); leading, after bootstrap, to an incremental cost-effectiveness ratio of 1299€ (95% CI = -29 252; +29 919). Acceptability curves showed that conservative surgery could be considered a cost-effective treatment at a threshold of 3201€ for one additional future intrauterine pregnancy. LIMITATIONS, REASONS FOR CAUTION: A limitation was that monetary valuation was carried out using 2016 euros while the DEMETER study took place from 2005 to 2009. Anyway, the results would not have been very different given the marginal changes in the health insurance reimbursement tariffs during this period. WIDER IMPLICATIONS OF THE FINDINGS: Conservative surgery can be considered a cost-effective treatment, if the additional cost of 3201€ per additional future intrauterine pregnancy is an acceptable financial effort for the payer. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: NCT 00137982.

Breast cancer and screening in persons with an intellectual disability living in institutions in France.

BACKGROUND: We aimed to describe, among a population of women with intellectual disabilities (ID) living in institutions in France, the characteristics in whom breast cancer (BC) was diagnosed and of those who participated in BC screening. METHODS: Study was performed in 2009 among a random, representative sample of women with ID living in institutions in France. Participants answered a questionnaire either directly by themselves, or with the help of an intermediary. RESULTS: In total, 978 women with ID aged over 18 years were included, and 14 were diagnosed with BC. The incidence observed in this sample of women with ID is similar to that of the general population (standardised incidence ratio, SIR 0.857, 95% confidence interval (CI) 0.42-1.53). Average age at diagnosis was 47.8 years, and the risk of developing BC before the age of 50 was 2.03% (0.4-3.66). This risk was not significantly different from that of the general population (2.4%, 1.0-3.78). Obesity was almost twice as frequent in women who had BC as compared to those without BC (43% vs. 22.5%, P = 0.0196). Among the 310 women aged >50 years and eligible for the national BC screening programme, 238 (77%) had already had at least one mammogram, and 199 had had it within the previous 2 years. Adherence to the screening programme was 64.2% (199/310) in the participating institutions. This rate was slightly higher than the national average of 62% for the same period. CONCLUSIONS: The results of this study show that BC is equally as frequent among women with ID living in institutions as in the general population, and occurs at around the same age. Obesity was significantly more frequent among women in whom BC was diagnosed in our study. Participation in BC screening is slightly higher among women with ID living in institutions than among the general population.

Increased tobacco and alcohol use among women with joint hypermobility: a way to cope with anxiety?

Joint hypermobility (JH) is a common trait of heritable disorders of the connective tissue which has been identified as a risk factor for anxiety states. Anxiety and other negative emotions lead some people to use tobacco and alcohol as a coping strategy. The purpose of this study was to examine JH in relation to the consumption of these psychoactive substances and the associated anxiety and coping strategies. A cross-sectional sample of 305 female college students completed self-administered questionnaires on JH, tobacco and alcohol use, state and trait anxiety, and coping strategies. The prevalence of JH in the final sample (N = 301) was 51.8 %. The percentage of smokers, smokers using tobacco to calm anxiety, at-risk drinkers, tobacco and alcohol users, and the number of standard drinks consumed per week were significantly higher among females with JH than among those without it. The percentage of participants with severe state anxiety was significantly higher in the JH group, as were the emotion-focused coping score. The results of the logistic regression analysis showed that the odds of being assessed with JH were greater in those who consume tobacco and alcohol. This study provides evidence that women with JH report higher levels of state anxiety than non-JH women and use emotion-focused coping (i.e., efforts to regulate affect) more than any other coping strategies to deal with stress. These factors may help explain the increase in tobacco and alcohol use observed in this population.

Impact of introducing a national policy for prenatal Down syndrome screening on the diagnostic invasive procedure rate in England.

OBJECTIVE: To evaluate the impact of the introduction of a Down syndrome screening policy in England, including implementation of the first-trimester combined screening test, on reducing prenatal invasive diagnostic procedure rates. METHODS: All English cytogenetic laboratories were asked to submit data from the fiscal years 2003/2004 until 2011/2012 on all samples received from prenatal invasive procedures, including those that were undertaken following a higher-risk Down syndrome screening result. RESULTS: There was a gradual decline in the number of invasive procedures undertaken subsequent to a positive Down syndrome screening result in England, from 36 968 in 2003/2004 to 11 446 in 2008/2009, with only a relatively small subsequent decrease, to 10 215, in 2011/2012. This corresponds to a 72% reduction in the number of referrals received by the cytogenetic laboratories over a 9-year period and correlates with the national policy of implementing the combined screening test in place of second-trimester screening, which has reduced the overall screen-positive rate to 3.1% from an initial level of 6.0%. CONCLUSIONS: Implementation of a national Down syndrome screening policy based on the combined screening test has significantly reduced the number of invasive tests performed. However, as the combined screening test has become the replacement for second-trimester testing and has been almost completely implemented it appears that improvements in screening using current approaches may have reached their limits.

[Hypodontia: therapeutic strategy elaborated from 30 cases]. / L'oligodontie: stratégie thérapeutique à partir de 30 cas.

INTRODUCTION: Hypodontia is defined as the absence of at least six permanent teeth due to the hypodevelopment of tooth germs. The prevalence is weak and varies according to the ethnic origin. This familial abnormality is due to various mutations or polymorphisms of genes. It may be associated with other dental abnormalities. In children, it is responsible for growth, mastication, and phonation disorders, hypo-alveolia and lingual interposition in the toothless sites. We report the management of 30 patients presenting with oligodontia. MATERIAL AND METHOD: The diagnosis was made on clinical and paraclinical data. The vertical and transversal facial dimensions, the three facial proportions, labial morphology, the number and localization of teeth, and occlusion were analyzed for future orthognathic and implant rehabilitation. The occlusion was studied transversally and vertically on dental casts. Rehabilitation with implant supported dental prostheses was chosen when conditions were met. The therapeutic protocol could include up to four chronological phases: the early prosthetic phase, the orthodontic phase, the surgical phase, and the implant and prosthetic phase. RESULTS: The 30 patients' age (13 male and 17 female patients) ranged from six to 20 years. An average of 15 dents were missing (6 to 28). Except for one case, all the third molars were missing. In 66% of the cases, the second molar was missing, in 33% for the first molar. In 66% of the cases, two lateral maxillary incisors were missing. There were no premolars in 50% of the patients. Agenesis of premolars and molars induced posterior bone insufficiency in the mandibula. Dysmorphosis was always present with vertical growth disorders. Six patients were not given orthodontic treatment because they did not have enough teeth. Orthognathic surgery was used in 14 cases (46.7%). Seventeen patients (56.7%) underwent apposition graft. In three patients, a high position of the alveolar nerve required nerve derivation. Implants were inserted in 14 patients (46.7%). The total number of implants was 155. DISCUSSION: Rehabilitation with implant supported dental prostheses is a good treatment for oligodontia. It requires an interdisciplinary approach (including a maxillo-facial surgeon, an orthodontist, a dentist, and a prosthesis specialist). The patient's motivation as well as that of his family is crucial.

[The tobacco cessation consultation of the Salah-Azeiz Tunis cancer center: one year results]. / La consultation d'aide au sevrage tabagique de l'institut de cancérologie Salah-Azeiz de Tunis : résultats à un an.

BACKGROUND: To reduce tobacco smoking is, in Tunisia, a public health priority. The tobacco cessation consultation is one of the interventions to fight against the tobacco epidemic; it is a new activity developed in the Salah-Azeiz Tunis cancer centre. The objective of this work is to evaluate the impact of the consultation on the smokers after one year of activities and to analyse the prognostic factors of tobacco cessation in order to improve the efficacy of such an activity in the future, in Tunisia. METHODS: The cessation methods were based on cognitive and behavioral therapies associated with nicotine patch, delivered free of charge. During one year (July 2003 to June 2004), 340 smokers were attended. Data on their smoking status and psychological characteristics were collected using a standardized questionnaire. RESULTS: The patients were mostly men (79%), these smokers did not present important anxiety-depressive troubles, 68% thought that they were able to quit easily. The majority (57%) smoked more than 20 cigarettes a day and was nicotine-dependent (Fagenström >7). Half of this population had already intended to stop, they declared to smoke mostly because of stress. The global cessation rate after a median follow-up of 32 weeks is 27%. The cessation rate lasting six months for 83 patients followed regularly was 23%. The good prognostic factors of cessation, after multivariate analysis, were (when taking out from the model the regular follow-up), to be confidant on the possibility to stop (OR=0.87 [0.78-0.97]). On the contrary, a high score (>7) for Fagerström test (OR=1.9 [1.1-3.4), the use of smoking to fight against stress (OR=1.08 [1.0-1.2) and a smoking environment at home (OR=4.5 [1.1-18.9]) were prognostic of a failure in quitting smoking. CONCLUSION: These results show that the cessation rate, which is still too low as compared with the literature, could be increased by a better follow-up of smokers associated with an information campaign on the existing possibilities to quit smoking in Tunisia.

[Smoking cessation in patients with lung cancer]. / Indications et méthodes d'aide au sevrage tabagique chez un patient fumeur atteint de cancer bronchique.

Smoking cessation is an important part of the management of patients with lung cancer. Continued smoking has been found to diminish treatment efficacy, to exacerbate side effects and to have a detrimental effect on survival. Smoking increases postoperative pulmonary complications and tolerance and efficacy of medical treatment (chemotherapy, targeted therapy, radiotherapy) are diminished. Moreover, the quality of life of current smokers is lower and the risk of a second primary malignancy is increased. Hospitalization is a good opportunity to propose smoking cessation. Clinical practice guidelines recommend the use of combined behavioral and pharmacological therapies. The efficacy of smoking cessation programs for cancer patients has been demonstrated. There is a clear dose-response relationship between number of contacts, intensity level of person-to-person contact and total amount of contact time. Multidisciplinary approaches increase abstinence rates. First line phamacotherapies (nicotine replacement therapy and sustained-release antidepressant bupropion) have been found to be safe and effective. Varenicline is a new drug for smoking cessation but it remains to be evaluated in oncology patients.

[Prevention of skin cancer programs: analysis of the impact of randomized trials]. / Programmes de prévention des cancers cutanés. Analyse de l'impact des études randomisées.

INTRODUCTION: Exposure to ultraviolet sun rays is an important risk factor for the development of skin cancer. Confronted with the increase in the incidence of severe forms (melanoma), primary prevention plays a major part, together with the development of campaigns promoting individual and collective protection against ultraviolet rays. OBJECTIVE: The aim of this trial was to identify the factors of success or failure of skin cancer prevention programs and to analyze their impact. METHOD: Articles published in the literature from 1982 to 2002 were selected from the Medline databank using the following key words: "skin cancer, melanoma, evaluation, prevention and education, review, program, campaign and randomized controlled trial". For the final analysis, only the randomized trials with control group were retained. RESULTS: All the prevention programs increased short, median or long term knowledge. Conversely, the trials were sometimes contradicting with regard to the change in attitude. No methodologically correct trial clearly reported any change in behavior, the majority of them only collected intent behavior. CONCLUSION: Despite the methodological weaknesses of most of the trials published, this review of the literature underlined certain points. The most efficient programs appear to be those targeting children, the training sessions of which are long and/or repeated, with active individual participation. Programs based on the deleterious consequences of sun exposure on physical appearance appeared to produce better results in terms of any change in attitude and intent behavior.

[Sun exposure and sun protection behavior and attitudes among the French population]. / Connaissances, attitudes et comportements des adultes vis-à-vis du soleil en France.

INTRODUCTION: The number of new skin cancers has constantly increased in France over the past two decades. The role of sun exposure can partly explain this phenomenon and justifies the development of information and prevention campaigns to change peoples' attitude towards sun bathing. To be effective, we need to know how much information and what attitudes the population currently has with regard to the sun. Although several studies in France have targeted children, little data is available regarding adults. This trial was aimed at pinpointing the knowledge, attitude and behavior of adults regarding sun exposure. MATERIAL AND METHODS: Data were collected during a randomized multicenter study on the prevention and early diagnosis of cutaneous tumors, conducted in 26 Health Centers from 1998 to 2000. Standardized questionnaires were handed to those consulting to assess their knowledge, attitudes and behavior towards sun bathing. The population was composed of 41 143 adults aged over 30, consulting one of the 26 Health Check-up units. Analysis of the data was made using SAS v 6.12 and STATA 7.0 software. Logistic regression identified the explicative factors of knowledge and behavior. All the statistical analyses were considered significant above a threshold of alpha<5%. RESULTS: A total of 33 021 persons filled-in the self-questionnaire. Forty-nine percent were women and 51% were men, with a mean age of 50 years. Geographically, 25% lived in the North-East, 16% in the North-West, 25 p.cent in the South-East and 34% in the South-West. Thirty percent claimed that they tanned without burning and 2% of the population studied had often suffered from sun burn, generally when they were adult. The risks related to sun burn were known, because 92% knew that the sun increased the risk of skin aging and 89% knew that it increased the risk of skin cancer. Regarding sun screens, knowledge was not so good; 42% thought that all products were the same and 53% that they allowed one to sun bathe longer. This knowledge was better in those with fair skins, in those who had a history of sun burn, in women and in those who lived in the northern areas of France. Conversely, knowledge decreased with age and was limited in those aged over 60. Regarding behavior, those with fair skin and who reddened under the sun without tanning, protected themselves more. The women declared they protected themselves more than the men, but they used less sun protective measures (clothing, hats...), other than sun screens, than men. Subjects aged over 60 protected themselves more than younger subjects. Lastly, a personal cutaneous history increased protective behavior, and those from northern France protected themselves more than those from the South. DISCUSSION: This analysis of 33 021 adults aged over 30 shows the good global knowledge of the consequences of sun bathing, but also the lack of knowledge on the interest of using external sun protection and the role of physical means of protection. Attitudes varied depending on gender, age and phototype and also depending on the area where they lived. Despite good knowledge, the most frequent behavior of adults aged over 30 is still not appropriate with differences depending on age, gender and area, which must prompt more appropriate targeting of prevention campaigns according to the populations concerned.

[Health Professionals' Interventions for promoting smoking cessation during pregnancy: a review of the evidence]. / Conduite à tenir pour aider les femmes enceintes à arrêter de fumer: rôles des professionnels de santé.

Active and passive exposure to tobacco smoke during pregnancy is the most serious and preventable cause of adverse maternal, fetal, and infant outcomes in France. The clinical and economic benefits of cessation have been documented. The objective of this article is to review the evidence base addressing smoking cessation in pregnant women. The article describes how best to assist the pregnant smoker in clinical practice or hospital to quit during pregnancy. The following low intensity interventions designed to be integrated into routine prenatal care are detailed: expired air CO measures, practice of the evidence-based 5 A's smoking cessation intervention for pregnant women, use of pregnant woman's self-help guide to quit smoking, relapse prevention, health professionals' training, participation to community program.

[Standards, Options and Recommendations for the management of stage I or II primary bronchial cancers treated exclusively with radiotherapy]. / Standards, Options et Recommandations pour la prise en charge des patients atteints d'un cancer bronchique primitif de stade I ou II traités par irradiation exclusive.

CONTEXT: The 'Standards, Options and Recommendations' (SOR) project, started in 1993, is a collaboration between the Federation of the French Cancer Centres (FNCLCC), the 20 French cancer centres and specialists from French public universities, general hospitals and private clinics. The main objective is the development of clinical practice guidelines to improve the quality of health care and outcome for cancer patients. The methodology is based on literature review and critical appraisal by a multidisciplinary group of experts, with feedback from specialists in cancer care delivery. OBJECTIVES: To develop clinical practice guidelines according to the definitions of the Standards, Options and Recommendations project for the management of stage I and II non small cell lung carcinoma treated by radiotherapy alone. METHODS: Data were identified by searching Medline and personal reference lists of members of the expert groups. Once the guidelines were defined, the document was submitted for review to independent reviewers, and to the medical committees of the 20 French cancer centres. RESULTS: The main recommendations for the management of stage I and II non small cell lung carcinoma treated by radiotherapy alone are: 1) The curative external irradiation with a continual course is an alternative to surgery only in the case of medically inoperable tumors or because the patient refuses surgery; 2) The external irradiation of the primary tumor only without the mediastinum could be proposed in peripheral stage IA. In proximal stage IA and IB, external irradiation should be carried out only as part of prospective randomised controlled trials comparing a localised irradiation of the primary tumor with a large irradiation of the mediastinum and the primary tumor. The treated volume must include the macroscopic tumoral volume with or without the microscopic tumoral volume and with a security margin from 1.5 to 2 cm; 3) There is a benefit to delivering a total dose in the primary tumor higher than 60 Gy in so far as the proposed irradiation, taking into account the respiratory function, does not increase the likelihood of severe adverse events due to radiation; and 4) The change in fractionation, the radiochemotherapy combination, the endobronchial brachytherapy with high dose rate alone or with external irradiation could be proposed only as part of prospective controlled trials for tumors classified as stage IB or II.

[Standards, options and recommendations for the management of locally advanced non small cell lung carcinoma]. / Standards, otions et recommandationspour la prise en charge thérapeutique des patients atteints d'un cancer bronchopulmonaire primitif non à petites cellules localement avancé.

CONTEXT: The "Standards, Options and Recommendations" (SOR) project, started in 1993, is a collaboration between the Federation of the French Cancer Centres (FNCLCC), the 20 French Cancer Centres and specialists from French Public Universities, General Hospitals and Private Clinics. The main objective is the development of clinical practice guidelines to improve the quality of health care and outcome for cancer patients. The methodology is based on literature review and critical appraisal by a multidisciplinary group of experts, with feedback from specialists in cancer care delivery. OBJECTIVES: To develop clinical practice guidelines according to the definitions of the Standards, Options and Recommendations project for the management of locally advanced non small cell lung carcinoma. METHODS: Data were identified by searching Medline and the personal reference lists of members of the expert groups. Once the guidelines were defined, the document was submitted for review to independent reviewers and to the medical committees of the 20 French Cancer Centres. RESULTS: The main recommendations are: 1) The management of the locally advanced non small cell lung carcinoma has two main goals: firstly to obtain local control of the disease (or to at least delay local progression in order to improve the survival or relapse free survival), and secondly to prevent the development of metastases. 2) There is a consensus that locally advanced non small cell lung carcinoma should be irradiated. External beam radiotherapy should be of optimal quality and delivered at a minimal dose of 60 Gy by standard fractionation. For patients with a poor life expectancy, this can be delivered as a split-course or hypofractionated scheme. 3) Treatment for patients with a performance status of 0-1 should consist of short duration induction chemotherapy (with a least two drugs one of which must be cisplatin), combined sequentially with conventional radiotherapy. 4) Surgery is contraindicated in extensive N3 disease. Combined radio-chemotherapy (adjuvant or neoadjuvant) is not indicated outside clinical trials. Surgery is justified in stage N2 disease as good local control can be achieved. T4-N0 disease should be treated surgically with curative intent.

Novel auxin transport inhibitors phenocopy the auxin influx carrier mutation aux1.

The hormone auxin is transported in plants through the combined actions of diffusion and specific auxin influx and efflux carriers. In contrast to auxin efflux, for which there are well documented inhibitors, understanding the developmental roles of carrier-mediated auxin influx has been hampered by the absence of specific competitive inhibitors. However, several molecules that inhibit auxin influx in cultured cells have been described recently. The physiological effects of two of these novel influx carrier inhibitors, 1-naphthoxyacetic acid (1-NOA) and 3-chloro-4-hydroxyphenylacetic acid (CHPAA), have been investigated in intact seedlings and tissue segments using classical and new auxin transport bioassays. Both molecules do disrupt root gravitropism, which is a developmental process requiring rapid auxin redistribution. Furthermore, the auxin-insensitive and agravitropic root-growth characteristics of aux1 plants were phenocopied by 1-NOA and CHPAA. Similarly, the agravitropic phenotype of inhibitor-treated seedlings was rescued by the auxin 1-naphthaleneacetic acid, but not by 2,4-dichlorophenoxyacetic acid, again resembling the relative abilities of these two auxins to rescue the phenotype of aux1. Further investigations have shown that none of these compounds block polar auxin transport, and that CHPAA exhibits some auxin-like activity at high concentrations. Whilst results indicate that 1-NOA and CHPAA represent useful tools for physiological studies addressing the role of auxin influx in planta, 1-NOA is likely to prove the more useful of the two compounds.

Inhibitors of the carrier-mediated influx of auxin in suspension-cultured tobacco cells.

Active auxin transport in plant cells is catalyzed by two carriers working in opposite directions at the plasma membrane, the influx and efflux carriers. A role for the efflux carrier in polar auxin transport (PAT) in plants has been shown from studies using phytotropins. Phytotropins have been invaluable in demonstrating that PAT is essential to ensure polarized and coordinated growth and to provide plants with the capacity to respond to environmental stimuli. However, the function of the influx carrier at the whole-plant level is unknown. Our work aims to identify new auxin-transport inhibitors which could be employed to investigate its function. Thirty-five aryl and aryloxyalkylcarboxylic acids were assayed for their ability to perturb the accumulation of 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (1-NAA) in suspension-cultured tobacco (Nicotiana tabacum L.) cells. As 2,4-D and 1-NAA are preferentially transported by the influx and efflux carriers, respectively, accumulation experiments utilizing synthetic auxins provide independant information on the activities of both carriers. The majority (60%) of compounds half-inhibited the carrier-mediated influx of [14C]2,4-D at concentrations of less than 10 microM. Most failed to interfere with [3H]NAA efflux, at least in the short term. Even though they increasingly perturbed auxin efflux when given a prolonged treatment, several compounds were much better at discriminating between influx and efflux carrier activities than naphthalene-2-acetic acid which is commonly employed to investigate influx-carrier properties. Structure-activity relationships and factors influencing ligand specificity with regard to auxin carriers are discussed.

AUX1 regulates root gravitropism in Arabidopsis by facilitating auxin uptake within root apical tissues.

Plants employ a specialized transport system composed of separate influx and efflux carriers to mobilize the plant hormone auxin between its site(s) of synthesis and action. Mutations within the permease-like AUX1 protein significantly reduce the rate of carrier-mediated auxin uptake within Arabidopsis roots, conferring an agravitropic phenotype. We are able to bypass the defect within auxin uptake and restore the gravitropic root phenotype of aux1 by growing mutant seedlings in the presence of the membrane-permeable synthetic auxin, 1-naphthaleneacetic acid. We illustrate that AUX1 expression overlaps that previously described for the auxin efflux carrier, AtPIN2, using transgenic lines expressing an AUX1 promoter::uidA (GUS) gene. Finally, we demonstrate that AUX1 regulates gravitropic curvature by acting in unison with the auxin efflux carrier to co-ordinate the localized redistribution of auxin within the Arabidopsis root apex. Our results provide the first example of a developmental role for the auxin influx carrier within higher plants and supply new insight into the molecular basis of gravitropic signalling.

Short-Lived and Phosphorylated Proteins Contribute to Carrier-Mediated Efflux, but Not to Influx, of Auxin in Suspension-Cultured Tobacco Cells

Auxin is transported across the plasma membrane of plant cells by diffusion and by two carriers operating in opposite directions, the influx and efflux carriers. Both carriers most likely play an important role in controlling auxin concentration and distribution in plants but little is known regarding their regulation. We describe the influence of modifications of the transmembrane pH gradient and the effect of agents interfering with protein synthesis, protein traffic, and protein phosphorylation on the activity of the auxin carriers in suspension-cultured tobacco (Nicotiana tabacum L.) cells. Carrier-mediated influx and efflux were monitored independently by measuring the accumulation of [14C]2,4-dichlorophenoxyacetic acid and [3H]naphthylacetic acid, respectively. The activity of the influx carrier decreased on increasing external pH and on decreasing internal pH, whereas that of the efflux carrier was only impaired on internal acidification. The efflux carrier activity was inhibited by cycloheximide, brefeldin A, and the protein kinase inhibitors staurosporine and K252a, as shown by the increased capability of treated cells to accumulate [3H]naphthylacetic acid. Kinetics and reversibility of the effect of brefeldin A were consistent with one or several components of the efflux system being turned over at the plasma membrane with a half-time of less than 10 min. Inhibition of efflux by protein kinase inhibitors suggested that protein phosphorylation was essential to sustain the activity of the efflux carrier. On the contrary, the pharmacological agents used in this study failed to inhibit [14C]2,4-dichlorophenoxyacetic acid accumulation, suggesting that rapidly turned-over proteins or proteins activated by phosphorylation are not essential to carrier-mediated auxin influx. Our data support the idea that the efflux carrier in plants constitutes a complex system regulated at multiple levels, in marked contrast with the influx carrier. Physiological implications of the kinetic features of this regulation are discussed.

The rolB Gene of Agrobacterium rhizogenes Does Not Increase the Auxin Sensitivity of Tobacco Protoplasts by Modifying the Intracellular Auxin Concentration.

Phenotypical alterations observed in rolB-transformed plants have been proposed to result from a rise in intracellular free auxin due to a RolB-catalyzed hydrolysis of auxin conjugates(J.J. Estruch, J. Schell, A. Spena [1991] EMBO J 10: 3125-3128).We have investigated this hypothesis in detail using tobacco (Nicotiana tabacum) mesophyll protoplasts isolated from plants transformed with the rolB gene under the control of its own promoter (BBGUS 6 clone) or the cauliflower mosaic virus 35S promoter (CaMVBT 3 clone). Protoplasts expressing rolB showed an increased sensitivity to the auxin-induced hyperpolarization of the plasma membrane when triggered with exogenous auxin. Because this phenotypical trait was homogeneously displayed over the entire population, protoplasts were judged to be a more reliable test system than the tissue fragments used in previous studies to monitor rolB gene effects on cellular auxin levels. Accumulation of free 1-[3H]-naphthaleneacetic acid (NAA) was equivalent in CaMVBT 3, BBGUS 6, and wild-type protoplasts, Naphthyl-[beta]-glucose ester, the major NAA metabolite in protoplasts, reached similar levels in CaMVBT 3 protoplasts, reached similar levels in CaMVBT 3 and normal protoplasts and was hydrolyzed at the same rate in BBGUS 6 and normal protoplasts. Furthermore, NAA accumulation and metabolism in BBGUS 6 protoplasts were independent of the rolB gene expression level. Essentially similar results were obtained with indoleacetic acid. Thus, it was concluded that the rolB-dependent behavior of transgenic tobacco protoplasts is not a consequence of modifying the intracellular auxin concentration but likely results from changes in the auxin perception pathway.