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8039588
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Impact of recycling and environmental legislation.
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Social and political pressures are stimulating a rapid growth in environmental legislation and the framework of national and European directives is reviewed. The pressure for recycling and the incorporation of recycled material is at risk of conflict with safety for food contact packaging. Various recycling opportunities are reviewed, concluding that recycling must be directed only to where there is an environmental benefit; also that re-use must not jeopardize food safety. For direct food contact with foodstuffs, chemical recycling is the only confident way of ensuring product purity. Containment of recycled material behind a barrier layer leaves the question of barrier performance to undefined contaminants.
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8039587
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United States of America and European regulation of food packaging: finding common ground to reach a common goal.
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There are pronounced differences between the USA approach to the regulation of food packaging and the system being adopted in the European Economic Community (EEC). These differences have significant implications for the efficient regulation of packaging materials and for achieving the common goal of regulatory harmonization. The United States effects preclearance of packaging materials by generic regulation, a modified 'positive list' system with certain jurisdictional exclusions. Distinguishing characteristics of the USA system include exemptions for materials that are 'prior sanctioned', 'generally-recognized-as-safe' (GRAS) or 'not reasonably expected to become a component of food'. The USA also embraces the application of the concept of Estimated Dietary Intake and use limitations to take into account likely exposure and, thereby, delimit the requirements for toxicological data. The EEC, on the other hand, is moving towards adoption of a strict positive list system under which no substance may be used in making a package or packaging material unless it is on the positive list on the basis of a toxicological conclusion as to the general safety of the substance. This paper examines the logical and philosophical underpinnings of both systems and the potential for common sense application of de minimis, or regulatory threshold principles, and worldwide use of the Estimated Dietary Intake concept, to help bring about a measure of harmonization consistent with the safe and efficient regulation of food packaging materials.
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8039586
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Update on migration research and regulatory initiatives.
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The Food and Drug Administration's (FDA) current research programme on migration from food packaging materials focuses on high-temperature packaging applications. Results from two studies conducted by Arthur D. Little, Inc., under contract to the FDA, are presented as well as highlights of the FDA's research programme on migration from microwave heat susceptor packaging. Some regulatory policy issues affecting the FDA's future regulation of food packaging materials are also examined.
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8039585
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The role of the Scientific Committee for Food in evaluating plastics for packaging.
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One of the tasks of the Scientific Committee for Food (SCF) is to advise the Commission of the European Communities (CEC) on the safety-in-use of monomers, other starting substances and additives used in food packaging materials. This advice forms the basis of Community Directives for the regulation of food packaging materials by a system of positive lists of substances authorized for use. The SCF considers the available migration and toxicity data and classifies each substance into one of ten lists, reflecting whether or not there are adequate data and whether the data indicate the potential for adverse effects. This paper describes the SCF classification system and discusses the rationale behind the SCF approach to toxicity testing and evaluation of food packaging materials, with particular emphasis on the recent change which took place in 1990 when the Committee issued a new set of guidelines. These guidelines outlined a new tiered approach to toxicity testing, based on the principle that the greater the potential human exposure to a substance, the more toxicity data are required to make a sound health assessment.
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8039584
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Legislation, control and research in the Nordic countries on plastics for packaging food.
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The present legislation in the Nordic countries for food contact materials is expressed in general terms and contains few detailed requirements. At present Finland is implementing the EEC legislation, Sweden and Norway will probably do so shortly and Denmark has been a member of the EEC since 1973. Current food legislation in Sweden only covers materials or articles intended to come into contact with foodstuffs during processing or packaging in the food industry or by retailers. It does not apply to food packaging materials purchased for use at home or to household utensils. Upon request, the Toxicology Division at the Swedish National Food Administration (NFA) carries out evaluations of materials intended to come into contact with food. In addition, a voluntary organization--Normpack--is currently operating in Sweden. Normpack consists of manufacturers, dealers and users of food packaging materials, who have agreed to abide by certain common standards. In Norway, the Packaging Convention (Emballasjekonvensjonen--on safety of food packaging material from the health point view) serves a similar purpose. Research in this field is conducted at the National Food Agency of Denmark, The Danish Packaging and Transportation Research Institute (ETi) of the Danish Technological Institute (DTI), the Food Research Laboratory at the Technical Research Centre of Finland, MATFORSK, Norconserv and Statoil in Norway and the NFA, PackForsk and the Swedish Institute for Food Research (SIK) in Sweden. Previous studies have concerned plasticizers in PVC (polyvinyl chloride) cling film, overall migration studies on cling film, specific migration of vinyl chloride, styrene and acrylonitrile and off-flavours.(ABSTRACT TRUNCATED AT 250 WORDS)
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8039583
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Current research on food contact materials undertaken by the UK Ministry of Agriculture, Fisheries and Food.
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Recent research funded by the UK Ministry of Agriculture, Fisheries and Food (MAFF) in the area of migration from food contact materials is reviewed and set within a framework of surveillance; evaluation of new technology; support of current regulations; and anticipation of future controls. Recent surveillance projects monitoring foods for migration of monomeric plasticizers (in particular di-(2-ethylhexyl)adipate), polymeric additives and mineral hydrocarbons are highlighted. Development of high temperature testing conditions for food contact materials has been carried out in support of regulations and proposals are made for the control of susceptors by analysis of release of volatiles. Migration of benzophenone from the printing ink of a paper board sleeve during microwave heating of a pre-cooked meal is described as a recent example of a migration situation that would not easily have been anticipated. Finally, the approach being adopted for investigating paper and board food contact materials for inorganic constituents, for volatile organic and for solvent-extractable organic components is outlined as an example of work being carried out in anticipation of future regulatory controls.
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8039582
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Evaluation of plastics for food packaging.
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One of the principal aims of the regulations for food contact materials and articles is the protection of the consumer. In order to fulfil this goal many analytical questions must be answered in the next few years. Of great help in the evaluation procedures can be theoretical predictions of migration based on empirical data for partition and diffusion coefficients. Using well established gas chromatographic methods for the detection of volatiles and new techniques involving supercritical gases for the analysis of minimally volatile constituents both specific and overall migration can be investigated. Further activities must be focused on the organoleptic properties and the average migration potential in different groups of materials and articles produced by the usual technology. Better correlations between rates of migration into food simulants and into real foodstuffs should be found in order to make the evaluation of plastics as realistic as possible.
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8039581
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Test methods to simulate high-temperature exposure.
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A study was carried out to compare the actual overall migration from packaging materials into food simulants during heating in a microwave oven, and the overall migration from these materials into food simulants applying time and temperature conditions stipulated in the current EC and Dutch legislation on food packaging. It was demonstrated that the requirement by some food packaging regulations to conduct migration tests in a conventional oven using test conditions that are more stringent than the conditions occurring in practice results in excessively severe and unreasonable test conditions. On the basis of the results obtained additional test conditions (e.g. 30 min and 1 h in combination with test temperatures exceeding 121 degrees C, and a test temperature of 130 degrees C) are proposed to be inserted in existing food packaging regulations to enable realistic migration testing of microwave packaging materials under conventional test conditions. It is concluded that the overall migration behaviour of packaging materials intended for microwave oven use, including microwave-active (susceptor) materials, can be judged on the basis of migration testing using conventional heating. The suitability of iso-octane as a volatile fatty food simulant for the determination of the overall migration under high-temperature test conditions was also investigated. For most samples investigated a good agreement was observed between the overall migration values obtained with olive oil applying both microwave and conventional heating, and those obtained with iso-octane under conventional conditions.
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8039580
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Testing polymeric coatings on metal and paper substrates.
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The predicted migration test requirements for food and beverage cans with internal lacquer coatings and plastics paperboard composites against future EC legislation, are described. In a project currently in hand at Pira International on behalf of the UK Ministry of Agriculture, Fisheries and Food, some of the problems which arise when migration testing food and beverage cans with the food simulant 3% (w/v) acetic acid solution are being investigated. The 3% (w/v) acetic acid simulant has a tendency to penetrate the lacquer coating resulting in corrosion of the metal substrate. Experiments in hand and planned on this problem, with some of the initial results, are presented. There are also problems with overall migration testing of food packaging consisting of plastics paperboard composites with the fatty food simulant (olive oil). With one particular problem there is at present no obvious answer.
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8039579
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Practical aspects of testing food contact materials for migration.
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Progress in three areas has provided analytical methods and understanding to assist in migration testing for compliance with European Community Directives on food contact materials. A simple migration test has been developed to indicate whether or not a food makes fatty contact with plastics. This test is then used to guide the initial choice of appropriate food simulants. The Karl Fischer technique for water determination has been used to eliminate the need to humidity condition plastics in overall migration testing. This results in more rapid and more reliable migration testing. Finally, the stability of 'positive-list' monomers and other starting substances has been examined in food simulants. This identifies those substances which, for reasons of reactivity, may not be expected to survive a migration test and for which, therefore, migration testing as such is inappropriate.
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8039578
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The role of the Community Bureau of Reference in harmonizing compliance with the laws of the Commission of the European Communities.
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While Community Directives provide the legal basis for the harmonization of national regulations (e.g. food quality, quality of plastics in contact with foodstuffs, etc.), their implementation sometimes requires measurements and analyses which are beyond the capabilities of many laboratories. The BCR Programme of the Commission of the European Communities has undertaken a series of actions in order to help with the implementation of Directive 90/128/EEC for plastics materials intended to come into contact with foodstuffs. The certification of the overall migration characteristics of a polyamide material in aqueous food simulants by total immersion is well advanced. This material will be available through the BCR Programme in 1993 and will allow the laboratories to check their correct application of the normalized method and will provide a basis for laboratory quality assurance. A project is in progress for the preparation of a reference material for the measurement of overall migration by total immersion in olive oil. The preparation of a bank of monomers in the positive list of the above Directive and a handbook of physical and spectroscopic data for these monomers has been supported. Projects are being prepared for supporting the development of methods for the analysis of more than thirty monomers with restrictions in the positive list of Directive 90/128/EEC.
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8039577
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The development of European standard (CEN) methods in support of European Directives for plastics materials and articles intended to come into contact with foodstuff.
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CEC Directives have been implemented for plastics materials and articles intended to come into contact with foodstuffs. These introduce limits upon the overall migration from plastics into food and food simulants. In addition, specific migration limits or composition limits for free monomer in the final article, have been set for some monomers. Agreed test methods are required to allow these Directives to be respected. CEN, the European Committee for Standardization, has created a working group to develop suitable test methods. This is 'Working Group 5, Chemical Methods of Test', of CEN Technical Committee TC 194, Utensils in contact with food. This group has drafted a ten part standard for determining overall migration into aqueous and fatty food simulants by total immersion, by standard cell, by standard pouch and by filling. This draft standard has been approved by CEN TC 194 for circulation for public comment as a provisional standard, i.e. as an ENV. Further parts of this standard are in preparation for determining overall migration at high temperatures, etc. Simultaneously, Working Group 5 is cooperating with the BCR (Community Bureau of Reference) to produce reference materials with certified values of overall migration. CEN TC 194 Working Group 5 is also drafting methods for monomers subject to limitation in Directive 90/128/EEC. Good progress is being made on the monomers of highest priority but it is recognized that developing methods for all the monomers subject to limitation would take many years. Therefore, collaboration with the BCR, the Council of Europe and others is taking place to accelerate method development.
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8039576
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An industry view of compliance with European Community legislation.
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This paper describes how from a situation within the European Community where each Member State had its own individual regulations for materials and articles in contact with food, the Community has developed a harmonized approach. The view is expressed that whilst this harmonization is desirable, the EC legislation as currently written is impractical. Flaws in the principles underlying EC controls are identified and whilst some parts of the harmonized regulations can form the basis for a workable scheme, substantial revision is needed. Proposals for improvement to the current Directives are suggested.
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8039575
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Guidelines of the Commission of the European Communities: a challenge for the control of packaging.
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Questions arising from the Commission of the European Communities Directives and guidelines regulating packaging materials are discussed in relation to whether compliance ensures safety in use and the consequent analytical problems. Difficulties may arise from interactions between food contact materials and food involving mass transfer (migration, off-odours, 'scalping', loss of aroma) or mass transfer and chemical interactions and the implications for safety assurance and regulation are addressed. The criteria for suitable low molecular weight fatty food simulants and conditions for migration testing are presented. In food surveillance, the usefulness of various methods of analysis differs for monomers and for additives. For monomers, IR spectroscopy can identify the polymer type and which specific monomers need controlling; for unknown mixtures of additives, preliminary functional group identification by techniques such as 1H-NMR is useful.
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8039574
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Enforcement of European Community legislation at the national level.
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The Dutch system for screening food contact materials, in which global migration no longer has any function, has been applied. An extensive investigation of baby soothers and bottle teats is described and the daily practice of the enforcement of the Dutch Packaging and Food Utensils Regulation is illustrated by an investigation of food contact materials sampled from the Dutch market.
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8039573
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Future European Community directives and petition procedures.
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The underlying principles of current European Community legislation in the area of food contact materials and articles are reviewed, and the prospects for extension to new areas are assessed. Future Directives are broadly described although it is not possible to timetable the programme for ultimate agreement and implementation. To assist in understanding Directives and for the practical purposes of enforcement Commission documents are outlined covering a 'Compendium of Methods of Analysis', a 'Practical Guide for users of EEC Directives on Food Packaging' and a 'Framework Document for Research Requirements in Support of Regulations'. Finally procedures for authorization of new substances at Community and national levels are described.
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8039570
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Rewards and dangers in researching domestic violence.
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The purpose of this article is to present a framework that reconciles the goals of advocates for victims of domestic violence with the goals of clinical researchers who study this phenomenon. I will be discussing the tension between those goals, and the way a research model can complement advocacy goals.
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8039569
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Communication deviances and clarity among the mothers of normally achieving and learning-disabled boys.
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The main purpose of the study was to reexamine the association between maternal communication deviances and learning disabilities in children. In this study, we adapted and extended the procedure used by Ditton, Green, and Singer (1987). A two-part experimental task was used: one in which the child could not request any clarification of mother's instructions, and another in which the mother and child could communicate. Both communication deviances and the clarity of mothers' communication were analyzed. The subjects were 60 mother-child pairs in which half of the children had learning disabilities and the other half were normally achieving children matched for age and parents' SES. The dyads were videotaped in a laboratory setting. The mothers of learning-disabled (LD) children were found to give less exact instructions and to present more ambiguous messages to the child than the mothers of non-LD children. Despite the more inaccurate input from their mother, the LD boys did not request clarification for ambiguous statements any more than did the NLD boys.
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8039568
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The development of a clinical rating scale for the McMaster model of family functioning.
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This article describes the development and validation of the McMaster Clinical Rating Scale (MCRS). The MCRS is a 7-item scale designed to be completed by a trained rater after completion of an in-depth interview of the family. We present data from four new studies and review previously published articles concerning the reliability, validity, and clinical utility of the MCRS. Adequate interrater reliability and rater stability were obtained. The MCRS was found to correlate significantly with the self-report Family Assessment Device and to discriminate between families in different phases of a depressive disorder.
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8039567
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Time and rhythm in couples.
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In this article, a theory is offered on the role of time in couple functioning and distress. The theory argues that no single set of temporal patterns is associated with couple distress or satisfaction. Rather, it advocates attention to the individual differences between couples in their understanding of the meaning of the temporal patterns in their relationship. The couple's narrative about the evolution and maintenance of these temporal patterns can be understood as revealing much about partners' experiences of the relationship in terms of the concepts of closeness and power. Interventions on the temporal dimension may be useful when the couple presents with an explicit problem in temporal patterns; when a particular temporal pattern prevents the couple from addressing other issues; and when the therapist wishes to reframe a problem in a manner that lowers conflict intensity. Along with the theory, a preliminary taxonomy of time problems in couples is presented as a guide to assessment. This is followed by clinical vignettes to illustrate how the theory can be used in couple therapy.
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8039566
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The misuse and use of science in family therapy.
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The wish to adopt ideas and metaphors from science can have a constricting effect on thinking about family therapy theory and practice. We describe three examples from the recent literature. The two problems describe: (a) borrowing the prestige and certainty of scientific ideas and metaphors and using them as cultural representations of reality, and (b) embracing certain philosophically comprehensive systems of thought. We then recommend some appropriate borrowing from the natural history tradition of science, and give some examples of ways in which that tradition has widened rather than narrowed the range of ideas that are used in family therapy.
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8039565
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Discourses in the mirrored room: a postmodern analysis of therapy.
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A postmodern approach is used to examine the discourses that circulate in the therapy room. Dominant discourses support and reflect the prevailing ideologies in the society. Three ready examples concern gender relations: the male sex drive discourse, the permissive discourse, and the marriage-between-equals discourse. I point out how the therapy room is a mirrored room that can reflect back only the discourses brought to it by the family and therapist. There is a predetermined content in the conversation of therapy: that provided by the dominant discourses of the language community and culture. I suggest that therapists need to develop a reflexive awareness if muted discourses are to enter the mirrored room.
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8039563
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[Some philosophical assumptions of the debate on the ethics of environmental epidemiology].
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The aim of this paper is to analyse the existing debate on the ethical problems concerning environmental epidemiological research. The analysis is mostly focused on the philosophical assumptions of this debate. We try to show that, beside the traditional deontological considerations, it is important to formulate other considerations on different issues. We try to demonstrate, on the basis of some reflections on the structure of environmental epidemiology and on science in general, that there is room for subjective choices of researchers in the production of data. We finally show how some of the subjective choices are grounded in moral options and how those moral values can belong to different moral theories.
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8039561
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[Comorbidity and the physician's decision: the case of undertreatment in care of breast tumors].
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When a physician has to come to a decision while caring for a given patient, he is supposed to take into account several kind of information related to the disease, illness or patients' personal characteristics. Although it is wellknown that the framework in which such decision has to be taken is complex, most of the scientific knowledge is based on pieces of evidence that derive from studies where complexity is avoided by applying restriction rules. The goal of this approach is to enhance the internal validity while preserving the generalizability of the findings. But in some cases this approach raises more doubts than certainty. It is the case of the process to assemble practice guidelines to reduce the gap between the best care and what is observed into practice using the available knowledge (i.e., available literature or experts' recommendations). As a variety of relevant clinical and patients' personal characteristics are not available at all or little is known on their impact on patients global health status, variables that actually do drive the practice are not included in guidelines that are intended to change it. This paper introduces the conceptual model of such debate and assesses the impact of co-morbidity--a variable seldom taken into account in effectiveness, quality and appropriateness studies- on the kind of medical care given to a sample of 1019 patients with early stage breast cancer. Empirical results and future implications are eventually discussed.
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8039560
|
[Behavior associated with HIV-1 infection in drug addicts in Rome, 1990-1992].
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Two cross-sectional surveys have been conducted in Rome in 1990 and 1992 to investigate prevalence and temporal differences of risk behaviours among drug injectors. A total of 487 drug injectors in 1990, and 450 in 1992 have been interviewed both in the street and in treatment services. Twenty-four percent of the subjects interviewed in 1990 reported having used second-hand syringes in the preceding 6 months, as compared to 14% in 1992; in the two years 29% and 13%, respectively, reported having passed a second-hand syringe to other drug injectors. Fifty-six percent (46% in 1992) of primary partners of drug injectors interviewed were not drug users themselves, while the prevalence of non drug using occasional partners was 34% and 43% in the two surveys. In 1990 condom use with primary partner was reported by 48% of drug injectors, and by 41% in 1992; condom use with occasional partners was 56% and 64% in the two years. The differences in sharing behaviours were observed for HIV-1 positive subjects, while HIV-1 negatives reported the same prevalence of use of second-hand syringes in 1990 and 1992; no statistically significant differences have been found for sexual behaviours among the HIV-1 positives, while the HIV-1 negatives reported a lower prevalence of condom use with primary partner. The observed differences in the two years remain also adjusting for the socio-demographic characteristics of the two populations in a multiple logistic regression model. Prevalence of HIV-1 related risk behaviours among drug injectors is still too high.(ABSTRACT TRUNCATED AT 250 WORDS)
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8039559
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[Integration of geographic and analytical approach in environmental epidemiology].
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Some developments of research in environmental epidemiology are reviewed, with special emphasis on the limitations of currently adopted methods. At the light of causal criteria commonly used in the evaluation of epidemiologic studies, an approach based on the integration of geographic and analytic studies is suggested, in order to obtain results relevant in terms of public health.
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8039558
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[The public-private mix in hospital care in the Lombardy region].
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1) to compare complexity and severity of the case-mix in the public and private sector, overall and across individual hospitals, and to examine their relative efficiency, by contrasting DRG and/or stage specific average length of stay (ALOS); 2) to assess the impact of a new contractual scheme based on a preassigned number of beddays for a restricted list of specific conditions. Discharge data on 940.670 admissions to 101 public hospitals and 185.161 admissions to 55 private hospitals in the Regione Lombardia in 1990, assigned to HCFA-DRGs, 8th version and to stages and substages of principal and unrelated diagnostic categories, based on Disease Staging. The spread of the case-mix is higher in the private sector, which also shows a higher concentration of admissions across hospital for specific medical and surgical conditions. The proportion of more advanced stages of disease is higher in the public sector, for most of the most frequent diagnostic categories. Obstetric care, including abortion, is the largest single public sector activity, while it is virtually not existent in the private sector. Elective surgical procedures, including ENT, cataract and varicose veins surgery make up a substantial proportion of the private hospitals' case load. DRGs-specific ALOS is longer in public hospitals for the most frequent surgical DRGs, mainly due to their preoperative LOS. The net impact of the proposed contractual scheme will save substantial proportion of beddays for most of the conditions considered, except cataract and varicose veins surgery.
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8039557
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[The health care market: what possible competition?].
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The subject of competition in health care exceeds the scientific journals being discussed in almost each public debate on the health care system. In this paper the elements characterising economic competition will be defined in comparison with the particular characteristics of the health care market. The peculiar features of that market require attention to be paid to the equity of the system, to the competitive mechanisms which are not completely evaluated and to the failures of the public bureaucracy still operating. The principal models of competition found in the literature are illustrated and some experiences as UK and Sweden are shown. Some elements of competition are proposed for the Italian National Health Service (INHS) with some rules for the possible implementation.
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8039555
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The 21-aminosteroid U-74389G protects the antioxidant enzymes in the ischemia/reperfusion-induced rat brain damage.
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The antioxidant enzymatic system in the ischemia/reperfusion induced brain injury in rats after U-74389G administration was evaluated. Ischemia/reperfusion caused a decrease in the activities of superoxide dismutase and glutathione reductase, as well as of total and free sulfhydryl groups, while thiobarbituric acid-reactive substances became elevated. Administration of U-74389G lead to restoring to normal values of all above parameters. Protective effect of the drug in ischemia/reperfusion induced brain injury has been suggested.
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8039554
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In vitro metabolism of 3,3',4,4'-tetrachlorobiphenyl in relation to ethoxyresorufin-O-deethylase activity in liver microsomes of some wildlife species and rat.
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A qualitative study was performed of the capacity of hepatic microsomes of several wildlife species to metabolize 3,3',4,4'-tetrachlorobiphenyl (TCB). Hepatic microsomes of species environmentally exposed to polychlorinated biphenyls (PCBs): harbour porpoise (Phocoena phocoena), harbour seal (Phoca vitulina), common tern (Sterna hirundo), and hepatic microsomes from species experimentally exposed to PCBs: eider duck (Somateria mollissima), rainbow trout (Salmo gairdneri), flounder (Platichthys flesus), and Wistar rat, were incubated with 14C-labelled TCB ([14C]TCB). The mammals and birds were able to metabolize TCB at a rate that correlated with their ethoxyresorufin-O-deethylase (EROD) activity. No [14C]TCB metabolism was observed in the fish, despite elevated EROD activity in the trout. HPLC analysis of diisopropylether extracts of the microsomal incubations indicated the presence of 4-OH-, 5-OH-, and 6-OH-tetrachlorobiphenyl metabolites and a yet unidentified metabolite. The ratio of the different hydroxy metabolites formed varied for the various species.
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8039553
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A model for the bioaccumulation of chlorobiphenyl congeners in marine mammals.
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The behaviour of chlorobiphenyls in marine mammals is best described by a pharmacokinetic model where the blood acts as the central transport compartment between the external environment and a number of peripheral organs, each maintaining a dynamic balance with the concentrations in the blood. Thus, blood samples can be a useful tool in monitoring programmes of chlorobiphenyl concentrations. Differences in the chlorobiphenyl patterns between seals and fish could be explained by the structure-biotransformation relationship developed in an experimental study. A harbour porpoise (Phocoena) seemed also able to metabolize chlorobiphenyl congeners with vicinal hydrogen atoms in the meta and para positions and two ortho-Cl atoms. Because the ratios between persistent and metabolizable congeners differed between specimens, it was not possible to derive 'dioxin type' toxic equivalents from concentrations of congeners occurring at much higher concentrations by calculation of their ratios.
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8039552
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Kinetics of Cd2+ in plasma, liver and kidneys after single intravenous injection of Cd-metallothionein-II.
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To explore the kinetics of Cd2+ in the body, rats received a single intravenous injection of CdCl2 or Cd-saturated metallothionein-II at 0.3 mg Cd/kg body weight. Cd2+ in the two agents was biexponentially eliminated from plasma: rapidly in the first 5 min, and gradually later. Compared with CdCl2, Cd-saturated metallothionein-II showed lower Cd2+ concentrations in plasma during the first 30 min; larger values for parameters concerning distribution of Cd2+, its total body clearance and half-life time in the beta phase. Cd2+ uptake in the liver was higher with CdCl2, and, conversely, in the kidneys it was higher with Cd-saturated metallothionein-II. In Cd-saturated metallothionein-II, the renal content of Cd2+ reached a maximum (8 micrograms Cd2+/g tissue) on day 1, gradually decreasing thereafter; there was a higher area under the Cd2+ content-time curve, and a lower mean residence time of Cd2+; the kidneys showed severe necrosis and defluxion of proximal tubular cells at days 1 and 5, although there were regenerative and reversion signs on day 5. These findings suggested that, in the case of Cd-saturated metallothionein-II, Cd2+ being taken into the cells of proximal tubules was excluded predominantly due to cellular death and the resultant defluxion.
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8039551
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Modulation of stress proteins by Cd2+ in a human T cell line.
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We previously showed in a human T cell line (CEM-C12 cells) that Cd2+ induced gene expression of stress proteins, metallothionein-IIA and heat shock protein 70 in a time- and dose-dependent manner. In the present study, CEM-C12 cells were pretreated for 24 h with 1 microM Cd2+ and then challenged with toxic concentrations of this metal. We found that maximal expression of the metallothionein-IIA and heat shock protein 70 genes was increased and this maximal level occurred at higher Cd2+ toxic concentrations. Actinomycin D chase experiments indicated that Cd2+ pretreatment did not modify metallothionein-IIA mRNA stability. The modulatory effect of Cd2+ pretreatment was dose-dependent from 100 pM to 1 microM. Such pretreatment also enhanced resistance to Cd2+ toxicity. Finally, verapamil, a calcium/potassium channel blocker displaced the dose-response curve for Cd2+ toxicity as well as metallothionein-IIA and heat shock protein 70 gene expression to higher Cd2+ concentrations.
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8039550
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Cell cycle phase-dependent cytotoxicity of actinomycin D in HeLa cells.
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The effects of brief actinomycin D treatment (0.1 microgram/ml, 0.5 h) on inhibition of cell growth and colony formation were studied in synchronized HeLa cells. Cells in late S and G2 phases were found to be maximally sensitive to inhibition of cell growth and colony formation after short exposure to actinomycin D. Cells in G1 and early S phases were less responsive to brief actinomycin D treatment, although there was a slowdown of cell growth between 24 and 48 h after removal of actinomycin D, recovery of cell growth was observed late (> 48 h) after drug removal. Cells in mitosis were maximally resistant to brief actinomycin D treatment, and continued to grow as did the control cells without drug. The effects of actinomycin D on inhibition of cell growth and colony formation were abolished by novobiocin but not by aphidicolin present during a brief actinomycin D treatment of cells at various cell cycle stages. Our results suggest that the effect of actinomycin D is cell cycle phase-dependent and may be involved in the action of topoisomerase II. Furthermore, actinomycin D at a low dose (0.1 microgram/ml, 0.5 h) induced a slight G1 block while a brief exposure to high dose actinomycin D (1.0 microgram/ml, 0.5 h) caused a slowdown in the rate of cell progression through S and G2/M phases. Similar S and G2/M phase block was seen in cells that had been briefly treated with actinomycin D (0.1 microgram/ml; 0.5 h) during late S and G2 phases.
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8039548
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Acetylcholinesterase protection and the anti-diisopropylfluorophosphate efficacy of E2020.
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The reversible noncovalent inhibitor of acetylcholinesterase (R,S)-1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]-methylpiperidine hydrochloride (E2020) was shown to inhibit electric eel acetylcholinesterase with high affinity in a mixed competitive-non-competitive way (Ki = 8.2 nM; Ki' = 13 nM). The pretreatment of electric eel acetylcholinesterase with E2020 dose-dependently prevented the inactivation of the enzyme by 40 microM diisopropylfluorophosphate. The EC50 for this protective effect (95% confidence limits) was 85 (76-96) nM, whereas under the same conditions E2020 IC50 was 12.3 (9.6-16) nM. E2020 injected together with atropine sulfate (17.4 mg/kg) into mice at doses in the range of 1.04-6.24 mg/kg 15 min before diisopropylfluorophosphate, caused a dose-dependent increase in diisopropylfluorophosphate LD50, resulting in protection ratios varying from 3.1 to 9.2. The effectiveness of E2020 antidotal effect was inversely correlated to the time between pretreatment and diisopropylfluorophosphate administration, being maximal when E2020 was injected 15 min, and possibly less than 15 min, before poisoning. From these experiments it is concluded that E2020 exerts a protective action against acute diisopropylfluorophosphate-poisoning in the mouse, presumably by protecting acetylcholinesterase from irreversible inactivation by this agent.
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8039549
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Selective enhancement by cyclosporin A of collagen expression by mesangial cells 'in culture'.
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Extracellular matrix deposition in mesangial areas leading to glomerulosclerosis is the major side effect of protracted therapies with cyclosporin A. In order to define any direct correlation between a chronic therapy with the drug and glomerulosclerosis we studied the effects of cyclosporin A on extracellular matrix production by human mesangial cells in culture. By immunoprecipitation and sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) of [3H]proline-labeled mesangial cells it was found that cyclosporin A induced a dose-dependent increase in total collagen synthesis (+80%), corresponding to a net increment in collagen III (+120%) and in a component with 70 kDa molecular weight which was produced only in negligible amount by mesangial cells under standard conditions. This collagen was characterized by cyanogen bromide digestion and finger print analysis as a novel molecule, not sharing any peptide composition similarities with the already characterized collagens. These data indicate that cyclosporin A stimulates the synthesis by mesangial cells of selected collagens, mainly collagen III and a new low molecular weight component. This mechanism may be relevant in cyclosporin A induced glomerulosclerosis occurring during protracted therapies with the drug.
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8039547
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1-Methyl-4-phenylpyridinium has greater neurotoxic effect after selenium deficiency than after vitamin E deficiency in rat striatum.
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The present study was designed to assess the extent of the protective effect of antioxidative capacity of dopaminergic neurons against the possible oxidative stress produced by 1-methyl-4-phenylpyridinium. We have studied the direct effect of 1-methyl-4-phenylpyridinium on striatum slices from rats fed with selenium-deficient or vitamin E-deficient diets for 30 days. Glutathione peroxidase activity decreased significantly after selenium dietary restriction. Our results showed that the effect of 1-methyl-4-phenylpyridinium on dopamine and its metabolites 3,4-dihydroxyphenylacetic acid, homovanillic homovanillic acid and 3-methoxytyramine in animals with both restriction diets was higher than in controls. However, this effect was significantly greater in animals with low selenium diets than with vitamin E-deficient diets in terms of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid, which were all significantly more depleted by 1-methyl-4-phenylpyridinium in selenium-deficient rats than in vitamin E-deficient rats. Therefore, considering changes in the levels of dopamine and its metabolites as an index of 1-methyl-4-phenylpyridinium toxicity, our results seem to indicate that the glutathione-glutathione peroxidase system has a greater protector effect than vitamin E.
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8039546
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Sensory neuropeptide-mediated bronchoconstriction of the guinea pig lung by diamide; a comparison to hydrogen peroxide.
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The effect of the thiol oxidizing agent diamide on airway conductance, dynamic compliance and perfusion flow of isolated perfused and ventilated guinea pig lungs was investigated. When infused in the pulmonary circulation, diamide (100 microM) induced bronchoconstriction, but no effect on perfusion flow was observed. Although diamide exposure induced the formation of thromboxane A2, the thromboxane/prostaglandin endoperoxide receptor antagonist L-670,596 did not affect the decrease in conductance and compliance induced by diamide. Diamide induced the release of the sensory neuropeptide calcitonin gene-related peptide. The bronchoconstriction and the release of calcitonin gene-related peptide induced by diamide were abolished by capsaicin pretreatment of the guinea pigs. Combined pretreatment with the NK1 and NK2 receptor antagonists, CP-96,345 and SR-48968, attenuated the effect of diamide. Hydrogen peroxide-induced vaso- and bronchoconstriction was not affected by capsaicin-pretreatment, nor did hydrogen peroxide induce detectable release of calcitonin gene-related peptide. The results indicate that diamide activates sensory nerves and induces neuropeptide release and neurokinin receptor-mediated bronchoconstriction in the isolated perfused and ventilated guinea pig lung.
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8039545
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Mechanisms of hypotension in iminoctadine poisoning: pharmacological analysis in rats.
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Iminoctadine, a fungicide used widely in fruit culture, causes hypotension in human acute oral poisoning. In an attempt to elucidate this mechanism, we investigated the effects of iminoctadine on the cardiovascular system of rats. In anesthetized rats, intravenously administered iminoctadine produced hypotension and tachycardia. In isolated right atria beating spontaneously in Krebs-Ringer's solution, iminoctadine produced an increase in heart rate. It also produced a positive inotropic response in electrically driven left atria. These responses were partially diminished by atenolol, a beta 1-adrenoceptor antagonist, and also partially diminished to a similar degree in atria of reserpinized rats. Therefore, the positive inotropic and chronotropic effects of iminoctadine were partially mediated via the release of norepinephrine from sympathetic nerve terminals. In aortic ring segments, iminoctadine caused a rightward shift of the concentration-contractile response curve for phenylephrine but did not affect those for prostaglandin F2 alpha or KCl. Iminoctadine produced a potent vasodilation in aortic segments precontracted with phenylephrine. Removal of the aortic endothelium produced a rightward shift of the concentration-response curve for iminoctadine. When the aortic ring preparations were precontracted with prostaglandin F2 alpha or KCl, iminoctadine produced only slight vasodilation. Therefore, the vasodilation caused by iminoctadine is due mostly to its alpha 1-adrenoceptor antagonizing action, and partly to endothelium-dependent mechanisms our data suggest that the hypotension induced by iminoctadine is due to its vasodilator effects.
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8039544
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Lipopolysaccharide-induced pleural neutrophil accumulation depends on marrow neutrophils and platelet-activating factor.
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The involvement of platelet-activating factor (PAF) in lipopolysaccharide (LPS)-induced leukocyte accumulation in the rat pleural cavity was investigated. Intrathoracic (i.t.) injection of LPS (250 ng/cavity) induced a marked increase in the number of neutrophils at 1 h, which was maximum within 6-12 h, reducing after 24 h. In parallel, an increase in blood neutrophil counts within 1-6 h, concomitantly with a reduction in the number of these cells in the bone marrow, was observed. The number of eosinophils recovered from LPS-injected pleural cavity increased at 12 h and was maximum within 24-48 h. No change in blood or bone marrow eosinophil counts was detected. The pretreatment with WEB 2086 or PCA 4248 (20 mg/kg) significantly inhibited pleural neutrophil accumulation, blood neutrophilia and the decrease in the marrow neutrophil content, but not eosinophil accumulation. The blood neutrophilia and the decrease in marrow neutrophil counts induced by the intravenous (i.v.) injection of LPS (250 ng) were significantly lower than those observed after i.t. injection. Furthermore, WEB 2086 and PCA 4248 were ineffective against the systemic alteration induced by i.v. LPS. it was concluded that LPS-induced neutrophil, but not eosinophil, accumulation in the pleural cavity is related to the mobilization of neutrophils from the bone marrow and involves PAF dependent mechanisms.
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8039543
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Effect of tri-n-butyltin on intracellular Ca2+ concentration of mouse thymocytes under Ca(2+)-free condition.
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Effect of tri-n-butyltin at concentrations ranging from 100 nM to 1 microM on the intracellular Ca2+ concentration of mouse thymocytes was examined under Ca(2+)-free conditions in comparison with those of 50 nM A23187, 100 nM thapsigargin and 10 microM cyclopiazonic acid, using the fluorescent dye for intracellular Ca2+, fluo-3. Tri-n-butyltin persistently increased the intensity of fluo-3 fluorescence while A23187, thapsigargin and cyclopiazonic acid produced a transient augmentation of the fluorescence. Pretreatment with A23187 greatly decreased the fluorescence responses induced by 1 microM tri-n-butyltin. However, the effect of thapsigargin and cyclopiazonic acid on the tri-n-butyltin-induced response was much weaker than that of A23187. In the presence of tri-n-butyltin, the transient response produced by A23187 was greatly prolonged. Results may suggest that tri-n-butyltin increases the membrane Ca2+ permeability of the intracellular organelles (cellular calcium stores) and decreases the Ca2+ pump activity of thymocyte membrane, resulting in a sustained increase in the intracellular Ca2+ concentration under Ca(2+)-free concentration.
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8039542
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Different competition of thyroxine binding to transthyretin and thyroxine-binding globulin by hydroxy-PCBs, PCDDs and PCDFs.
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In an earlier study several hydroxylated polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) competitively displaced [125I]thyroxine (T4) from transthyretin with different potencies. Transthyretin is the major T4 transport protein in plasma of rodents. In man, however, thyroxine-binding globulin transports most of the T4 in blood. In this study, hydroxylated PCBs, PCDDs and PCDFs were tested in an in vitro competitive binding assay, using purified human thyroxine-binding globulin and [125I]T4 as the displaceable radioligand. None of the tested hydroxylated PCBs, PCDDs and PCDFs inhibited [125I]T4 binding to thyroxine-binding globulin. In addition, some T4 derived compounds, e.g., tyrosine, mono-iodotyrosine, di-iodotyrosine and tri-iodophenol were tested on both transthyretin and thyroxine-binding globulin to investigate possible differences in structural characteristics determining T4 binding to thyroxine-binding globulin and transthyretin. The T4 derived compounds also did not inhibit [125I]T4 binding to thyroxine-binding globulin as tested in the in vitro assay. However, tri-iodophenol and to a lesser extent di-iodotyrosine inhibited [125I]T4-transthyretin binding. These results indicate a marked difference in T4 binding to thyroxine-binding globulin or transthyretin. The hydroxylated PCBs, PCDDs and PCDFs can inhibit T4 binding to transthyretin, but not to thyroxine-binding globulin, and thus may cause different effects in rodents and man.
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8039540
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Grapefruit juice inhibits 7-hydroxylation of coumarin in healthy volunteers.
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The effect of grapefruit juice on the urinary excretion of 7-hydroxycoumarin after oral administration of 10 mg coumarin, as an index of cytochrome P450 dependent coumarin metabolism, has been investigated in an open, randomised cross over study in 13 healthy volunteers (7 female, 6 male). The percentage of 7-hydroxycoumarin found in urine was significantly decreased up to 8 h after simultaneous intake of 300 ml grapefruit juice. If the same volume of juice was swallowed 30 min prior to the administration of coumarin, 7-hydroxycoumarin excretion was delayed by up to 6 h. MRTexcr. of coumarin was 70% extended by coadministration of grapefruit juice. It appears that grapefruit flavonoids inhibit cytochrome P450 2A dependent metabolic pathways. The mechanism of cytochrome P450 inhibition by these flavonoids is still poorly understood.
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8039539
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Effect of theophylline on periodic breathing in congestive heart failure measured by transcutaneous oxygen monitoring.
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The effect of theophylline on periodic breathing was measured by transcutaneous oxygen monitoring in 7 patients suffering from chronic congestive heart failure. Theophylline significantly decreased the frequency of Cheyne-Stokes breathing. Transcutaneous oxygen monitoring proved to be a useful method to follow changes in the breathing pattern. The suggested mechanism of action of theophylline in reducing the incidence of periodic breathing is blockade of adenosine receptors.
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8039538
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The effect of renal function on the pharmacokinetics of ranitidine.
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This open study evaluated the influence of renal function on the pharmacokinetics of ranitidine (50 mg i.v. infusion given over 6 min). Five groups, each of 8 subjects, 1 with normal renal function and 4 with different degrees of renal impairment were studied. Renal function was assessed in each patient by 51Cr-EDTA (glomerular filtration rate, GFR), creatinine clearance (GFR) and N-methylnicotinamide clearance (reflecting glomerular and tubular function). Sixteen blood samples (5 ml) taken up to 48 h post dose from each subject were analysed for plasma ranitidine concentrations by reversed phase HPLC. Patient groups with renal impairment had significantly increased AUC infinity and t1/2 with corresponding decreases in CLp and lambda z when compared with normal subjects. There was also a significant increase in tmax but not in Cmax. There was a high linear correlation between the degree of renal impairment and ranitidine clearance. In patients with GFR < or = 20 ml min-1, the AUC infinity mean ratio (compared with normal subjects) was up to 4.6 while for patients with GFR 20-50 ml min-1, the average AUC infinity ratio was 2.6. It is recommended that the dose of ranitidine is halved in patients with GFR < or = 20 ml min-1.
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8039537
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The pharmacokinetics and pharmacodynamics of medifoxamine after oral administration in healthy elderly volunteers.
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The pharmacokinetics and psychomotor effects of medifoxamine, a 5 HT reuptake inhibitory antidepressant, were studied in healthy elderly volunteers after single and multiple dosing. The elimination half life (t1/2z) after single doses of 300 mg was 2.8 h--almost identical to that found in young volunteers. After seven days of dosing at 100 mg three times daily the mean corrected AUC after 300 mg significantly increased from 1.04 to 1.34 mg.h.l-1 and t1/2z increased to 4.0 h (NS). There were no significant changes in critical flicker fusion frequency, symbol digit substitution, continuous attention or choice reaction times.
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8039536
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Pharmacokinetics and tolerability of ascending intravenous doses of granisetron, a novel 5-HT3 antagonist, in healthy human subjects.
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The pharmacokinetics and tolerance of granisetron, a novel 5HT3-receptor antagonist which is under development as an anti-emetic agent have been studied after administration of single 30 min intravenous infusions to three groups of 8 healthy male subjects, in a series of placebo-controlled ascending dose studies (50, 80, 100 and 130 micrograms.kg-1 to group 1; 150, 180, 200 and 230 micrograms.kg-1 to group 2 and 270 and 300 micrograms.kg-1 to group 3). Plasma and urine samples were analysed for granisetron by HPLC with fluorimetric detection. Administration of granisetron was well tolerated by the volunteers and there were no serious adverse effects reported. Pharmacokinetic parameters and dose-normalised plasma levels appeared to be independent of dose in the range 50 to 300 micrograms.kg-1, although there was extensive inter-subject variability. Granisetron was extensively distributed, with mean volumes of distribution ranging from 186-264 l at the various doses. Total plasma clearance was, in general, rapid (mean values of 37.0 to 49.9 l.h-1) and predominantly non-renal, with most subjects excreting less than 20% of the dose unchanged in urine. Mean t1/2 values ranged from 4.1 to 6.3 h and MRT from 5.2 to 8.1 h.
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8039535
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Inhibition of soluble catechol-O-methyltransferase and single-dose pharmacokinetics after oral and intravenous administration of entacapone.
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The inhibition of soluble catechol-O-methyl-transferase (S-COMT) in red blood cells (RBCs) by entacapone, and the pharmacokinetics of entacapone after single oral (5-800 mg) and i.v. (25 mg) doses have been examined in an open study in 12 healthy young male volunteers. Oral entacapone dose-dependently decreased the activity of S-COMT in RBCs with a maximum inhibition of 82% after the highest dose (800 mg). The inhibition of S-COMT in RBCs was reversible and the activity recovered within 4-8 h. Entacapone showed linear pharmacokinetics over the dose range studied: Cmax and AUC were correlated with the dose of the drug. Oral absorption of entacapone was fast, with a tmax ranging from 0.4 to 0.9 h, depending on the dose. Systemic availability of entacapone varied between 30 and 46%. Entacapone was rapidly eliminated by metabolism with a half-life of 0.27-0.30 h after oral doses of 5 to 50 mg. After doses from 100 to 800 mg the disposition was best described by two phases with a t1/2 alpha of 0.27-0.37 h and t1/2 beta of 1.59-3.44 h. Over the dose range studied, the single oral and i.v. doses of entacapone were well tolerated. No haematological, biochemical or haemodynamic adverse effects were seen. The results show that entacapone is an orally effective and reversible COMT inhibitor in man and has simple, linear pharmacokinetics.
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8039534
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Influence of concomitant food intake on the oral absorption of two triazole antifungal agents, itraconazole and fluconazole.
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The influence of food on the pharmacokinetics of the triazole antimycotics fluconazole and itraconazole was investigated in a randomised, parallel group, single dose study in 24 healthy subjects. Each group took either a 100 mg capsule of fluconazole or a 100 mg capsule of itraconazole, pre-prandially or after a light meal or a full meal, in a three-way crossover design. Gastric and intestinal pH were measured with a co-administered radiotelemetric pH capsule, and gastric emptying time of the capsule (GET) was taken as the maximum gastric residence time of drug and food. The plasma AUC and Cmax of itraconazole were significantly different under the various conditions and the mean AUC was greatest after the full meal. The bioavailability (90% confidence intervals) of itraconazole relative to that after the full meal, was 54% (41-77%) on an empty stomach and 86% (65-102%) after a light meal. The criteria for bioequivalence were not attained. In contrast, the bioavailability (90% CI) of fluconazole relative to the full meal was 110% pre-prandially (100-115%) and 102% after the light meal (88-103%), and the criteria for bioequivalence were attained. Itraconazole absorption was promoted by low stomach pH, long gastric retention time and a high fat content of the coadministered meal, whereas the pharmacokinetics of fluconazole was relatively insensitive to physiological changes in the gastrointestinal tract.
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8039533
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Doxycycline carrageenate--an improved formulation providing more reliable absorption and plasma concentrations at high gastric pH than doxycycline monohydrate.
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The effect of increased gastric pH (obtained by pre-treatment with omeprazole) on the bioavailability of doxycycline monohydrate and doxycycline carrageenate has been investigated in 24 healthy volunteers, using an open, randomised, four-treatment, four-period, cross-over, 2 x 2 factorial design. Each subject received a single dose of 100 mg of each of the doxycycline formulations with and without pre-treatment with omeprazole (40 mg daily for 7 days). The two formulations were bioequivalent (rate and extent) during fasting without omeprazole pre-treatment, whereas after omeprazole, the monohydrate showed a highly significant decrease in bioavailability (38% for AUC and 45% for Cmax) compared to the carrageenate formulation, which was not affected by prior administration of omeprazole. Many of the subjects did not reach a therapeutic plasma level of doxycycline during the combination of omeprazole and doxycycline monohydrate, and most adverse events (mainly gastrointestinal) were reported after this combination. As large populations of patients have a high gastric pH due to frequent use of H2-blockers, proton pump inhibitors and antacids, as well as to physiological achlorhydria, the decreased absorption of doxycycline monohydrate may well have a clinical impact, for example when the patients are treated with tetracyclines for an infection.
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8039532
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Changes in the concentration of serum alpha 1-acid glycoprotein in epileptic patients.
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Factors leading to elevation of the serum level of alpha 1-acid glycoprotein (AAG), the principal binding protein of cationic drugs in the systemic circulation, were investigated in 142 epileptic patients receiving long-term therapy with carbamazepine (CBZ), phenytoin (DPH), phenobarbital (PB), or valproate (VPA). The mean serum activity of gamma-glutamyl transpeptidase (gamma-GTP), an indicator of enzyme induction, in the groups of patients receiving enzyme inducers such as CBZ, DPH, and/or PB was at least some 5-fold higher than in patients receiving VPA alone, which is well known to be a non-inducer. There was no significant difference in the mean serum level of AAG between the former and the latter groups, i.e. there was no correlation between AAG and gamma-GTP levels in serum, and it is unlikely that the enzyme inducing properties of CBZ, DPH and PB are associated with an increased level of AAG. The mean serum level of AAG in the patients with poorly controlled seizures was significantly higher than in those with well controlled seizures (0.81 vs. 0.56 milligram). The serum AAG level in individual patients changed in parallel with the frequency of seizures. The present results strongly suggest that elevation of serum AAG level in epileptic patients may not be associated with the enzyme-inducing activities of anticonvulsants, but with the epileptic seizures per se.
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8039531
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Rapid determination of CYP2D6 phenotype during propafenone therapy by analysing urinary excretion of propafenone glucuronides.
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Metabolism of the antiarrhythmic, propafenone, cosegregates with the sparteine/debrisoquine polymorphism. Patients devoid of CYP2D6 activity have a higher incidence of adverse effects than those with normal enzyme function. In this paper we present a method for rapid assignment of CYP2D6 phenotype using urinary excretion of intact glucuronides of propafenone (PPFG). After establishing an HPLC assay, urinary excretion of PPFG was quantified during one dosage interval and related to individual CYP2D6 activity as determined by phenotyping. We observed a close correlation of urinary excretion of PPFG with individual CYP2D6 activity (r = 0.84, P < 0.01) and conclude that this method is suitable for rapid assignment of phenotype during propafenone therapy.
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8039530
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Plasma concentrations and effect on testosterone metabolism after single doses of MK-0434, a steroid 5 alpha-reductase inhibitor, in healthy subjects.
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A four-period, two-panel, single-rising-dose study (0.1-100 mg) was conducted in healthy males to investigate the pharmacodynamics, tolerability and pharmacokinetics of MK-0434, a steroid 5 alpha-reductase inhibitor. MK-0434 was associated with a significant reduction in dihydrotestosterone, which was maximal at 24 h and maintained through 48 h post treatment. The maximum reduction was approximately 50% and occurred at all doses above 5 mg (10, 25, 50 and 100 mg). MK-0434 appeared to have no effect on serum testosterone at these single doses. Rising single doses of MK-0434 were associated with an increase in Cmax and AUC but the changes were less than proportional to dose, most likely due to nonlinear absorption. MK-0434 given in single doses up to 100 mg was without significant adverse effects in healthy male volunteers. In summary, MK-0434 is a well-tolerated, potent, orally active 5 alpha-reductase inhibitor in man.
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8039529
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Statistical evaluation of circadian blood pressure rhythm during isradipine long-term therapy.
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In this study, using the two-step truncated Fourier series with four harmonics, we analysed the diurnal blood pressure profile in 13 mild-moderate essential hypertensive patients during isradipine long-term therapy. Circadian parameters such as the amplitudes and phases of the four harmonics and the overall amplitude and phase were obtained from the model. The total duration of the study was 26 weeks. Ambulatory blood pressure was measured at 15-min, intervals using a Takeda TM 2420 device after 2 weeks of placebo and after 6 and 26 weeks of isradipine (5 mg daily) respectively. After 6 and 26 weeks therapy the blood pressure values showed a significant decrease, although the daily blood pressure curves obtained from Fourier analysis showed that the circadian rhythm was not altered by isradipine treatment. Both the night/day differences and the overall amplitude/acrophases were statistically significant at 0.6 and 26 weeks. According the nocturnal blood pressure fall, we found that long-term therapy with isradipine increased the number of patients with nocturnal blood pressure fall and reducing the early morning blood pressure rise. In conclusion, the two-step method Fourier analysis represents a novel and useful statistical approach to evaluate the presence of a significant diurnal blood pressure rhythm and to provide the circadian parameters of the 24-h blood pressure profile during pharmacological therapy.
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8039527
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Prediction of the therapeutic response to simvastatin by pretreatment lipid concentrations in 2082 subjects.
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2082 hypercholesterolemic subjects were treated with simvastatin for 12 weeks. In 530 patients the dose was increased after 6 weeks from 10 to 20 mg because of persistently high cholesterol concentrations. Total cholesterol (TC) in the 1552 patients taking 10 mg fell by 1.61 mmol.l-1 (18.4%), LDL cholesterol (LDLC) by 1.53 mmol.l-1 (25.2%), and triglycerides (TG) by 0.13 mmol.l-1 (5.5%); HDL cholesterol (HDLC) significantly increased by 0.05 mmol.l-1 (3.6%). In the 530 patients taking 20 mg TC fell by 1.89 mmol.l-1 (19.9%), LDLC by 1.78 mmol.l-1 (26.0%), and TG by 0.13 mmol.l-1 (5.5%); HDLC increased by 0.05 mmol.l-1 (3.7%). The reductions in TC, LDLC, and TG were positively correlated and the increase in HDLC negatively correlated with their respective baseline values. There were independent significant correlations of the fall in LDLC with sex (MANOVA), baseline TG, and adherence to a lipid-lowering diet. The falls in TG significantly correlated with baseline fructosamine concentrations and dietary adherence. There were 571 adverse events in 16.6% of the patients but no case of myopathy. These results show that simvastatin is usually well tolerated and that its effects on TC and LDLC depend on their baseline concentrations.
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8039528
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Safety of fluconazole in the treatment of vaginal candidiasis. A prescription-event monitoring study, with special reference to the outcome of pregnancy.
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A prescription-event monitoring (PEM) study has confirmed that fluconazole, a bis-triazole oral antifungal drug, is a safe and effective treatment for vaginal candidiasis. Useful information was available on 15,015 questionnaires returned by general practitioners. The events were compared with those recorded in PEM studies of itraconazole and 31 other drugs in a total of more than 330,000 patients. The frequency of events in the study of itraconazole was almost identical. Upper respiratory tract and genito-urinary infections were reported with above-average frequency but the relationship was with the disease being treated rather than the drug itself. No serious adverse effects were recorded with an unacceptably high incidence. None of the 125 deaths was caused by fluconazole. Although contraindicated for vaginal candidiasis in pregnancy, fluconazole was taken by 289 women at some time during the months before or during pregnancy; a follow-up study by questionnaire of the outcome of pregnancy showed fluconazole to be without harmful effect. It is concluded that fluconazole is a well tolerated drug in the treatment of vaginal candidiasis and is associated with very few adverse effects.
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8039526
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Therapeutic traditions in type 2 diabetes--are they changing?
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The utilisation of antidiabetic drugs reflects both the prevalence of diabetes and the different therapeutic traditions of physicians. A questionnaire survey to study attitudes to the use of oral antidiabetic drugs amongst physicians and possible changes in treatment habits was carried out in a representative sample of Finnish physicians (n = 454) in 1992 and the results were compared with those of a similar survey carried out in 1985, and with drug utilisation statistics. The mean fasting blood glucose level at which a physician would start pharmacological treatment was 8.7 mmol.l-1, which was significantly lower than in the 1985 survey. The responses to various case histories suggested a more active approach to pharmacological treatment compared to the 1985 survey. Insulin treatment especially seems to have gained in popularity. This change in attitude was paralled by an increase in the consumption of antidiabetic drugs in Finland during the observation period. The increase in use of oral drugs was steeper in Finland than in Norway and Sweden. Whether this active approach will improve the metabolic control and prognosis of patients with Type 2 diabetes, remains to be demonstrated.
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8039525
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Suppression of electroencephalogram delta power density during non-rapid eye movement sleep as a result of a prolonged cognitive task prior to sleep onset.
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The effects of a prolonged cognitive task prior to sleep onset on subsequent sleep patterns were examined in 14 healthy subjects who were randomly assigned to two conditions. Those assigned to a working condition were asked to engage in a prolonged cognitive task until close to bedtime (0200 hours), whereas those assigned to a relaxing condition were instructed to perform the same task during the daytime and then to stay awake in a relaxed state until the same bedtime as the work group. Visual scoring of sleep stages showed no significant differences in the amounts of stage 4 and slow wave sleep (stage 3+4) between the two conditions. Power spectrum analysis of sleep electroencephalogram (EEG) revealed that the EEG delta (0.5-4.0 Hz) power density in the first non-rapid eye movement (REM)-REM sleep cycle was significantly lower following the prolonged cognitive task prior to sleep onset than following the relaxed wakefulness and that the decreased EEG delta power density in the first sleep cycle was not compensated for during the later part of the sleep. These findings would indicate that the prolonged cognitive task prior to sleep onset may suppress EEG delta power density during subsequent sleep, suggesting that such a task may interfere with the development of deep non-REM sleep.
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8039524
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Electrophysiological and psychophysical quantification of temporal summation in the human nociceptive system.
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Animal experiments have shown that the nociceptive reflex can be used as an indicator of central temporal integration in the nociceptive system. The aim of the present study on humans was to investigate whether the nociceptive reflex, evoked by repetitive strong electrical sural nerve stimuli, increased when summation was reported by the volunteers. The reflexes were recorded from the biceps femoris and rectus femoris muscles in eight volunteers following a series of stimulations at 0.1, 1, 2, and 3 Hz. Each series consisted of five consecutive stimuli. Using 0.1- and 1-Hz stimulation, the reflex was not facilitated in the course of the five consecutive stimuli. Following 2- and 3-Hz stimulation, the reflex size (root mean square amplitude) increased significantly during the course of the fifth stimulus. This reflex facilitation was followed by a significant increase (summation) in the pain magnitude when compared with 1- and 0.1-Hz stimulation. Furthermore, the threshold for psychophysical summation could be determined. This threshold (stimulus intensity) decreased when the stimulus frequency (1-5 Hz) of the five consecutive stimuli was increased. The nociceptive reflex and the psychophysical summation threshold might be used to clarify and quantify aspects of temporal summation within the human nociceptive system.
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8039523
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Effects of 12 weeks of aerobic exercise plus dietary restriction on body composition, resting energy expenditure and aerobic fitness in mildly obese middle-aged women.
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This study investigated the effects of 12 weeks of aerobic exercise plus voluntary food restriction on the body composition, resting metabolic rate (RMR) and aerobic fitness of mildly obese middle-aged women. The subjects were randomly assigned to exercise/diet (n = 17) or control (n = 15) groups. The exercise/diet group participated in an aerobic training programme, 45-60 min.day-1 at 50%-60% of maximal oxygen uptake (VO2max), 3-4 days.week-1, and also adopted a self-regulated energy deficit relative to predicted energy requirements (-1.05 MJ.day-1 to -1.14 MJ.day-1). After the regimen had been followed for 12 weeks, the body mass of the subjects had decreased by an average of 4.5 kg, due mainly to fat loss, with little change of fat free mass (mff). The absolute RMR did not change, but the experimental group showed significant increases in the RMR per unit of body mass (10%) and the RMR per unit of mff (4%). The increase in RMR/mff was not correlated with any increase in VO2max/mff. The resting heat production per unit of essential body mass increased by an average of 21%, but the resting heat production rate per unit of fat tissue mass remained unchanged. We concluded that aerobic exercise enhances the effect of moderate dietary restriction by augmenting the metabolic activity of lean tissue.
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8039522
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Exhausting handgrip exercise reduces the blood flow in the active calf muscle exercising at low intensity.
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The calf and forearm blood flows (Qcalf and Qforearm respectively), blood pressure, heart rate and oxygen uptake of six men and women were studied during combined leg and handgrip exercise to determine whether a reduction of exercise-induced hyperaemia would occur in the active leg when exhausting rhythmic handgrip exercise at 50% maximal voluntary contraction (MVC) was superimposed upon rhythmic plantar flexion lasting for 10 min at 10% MVC (P10) prior to this combined exercise. The Qcalf and Qforearm were measured by venous occlusion plethysmography during 5-s rests interposed during every minute of P10 exercise and immediately after combined exercise. The muscle sympathetic nerve activity (MSNA) changes were also recorded during leg exercise alone and combined exercise. During plantar flexion performed 60 times.min-1 with a load equal to 10% MVC (P10), Qcalf was maintained at a constant level, which was significantly higher than the resting value (P < 0.001). When rhythmic handgrip contraction at 50% MVC (H50) and P10 were performed simultaneously, the combined exercise was concluded due to forearm exhaustion after a mean of 51.2 (SEM 5.5) s. At exhaustion, Qcalf had decreased significantly from 20.6 (SEM 3.0) ml.100 ml-1.min-1 (10th min during P10 exercise) to 15.3 (SEM) ml.100 ml-1.min-1 (P = 0.001), whereas Qforearm had increased significantly (0.001 < P < 0.01) from 8.6 (SEM 1.9) ml.100 ml-1.min-1 (10th min of P10 exercise) to 26.2 (SEM 3.2) ml.100 ml-1.min-1. The mean blood pressure remained at an almost constant level during the 3rd to 10th min of P10 exercise and increased markedly when H50 was added.(ABSTRACT TRUNCATED AT 250 WORDS)
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8039521
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Correlation between percentage fiber type area and myosin heavy chain content in human skeletal muscle.
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Histochemical methods are routinely used to delineate skeletal muscle fiber types. In the present investigation, this qualitative determination of fiber type composition was compared to the electrophoretically determined myosin heavy chain (MHC) content from a large number of human muscle biopsy samples. Biopsies were taken from the vastus lateralis muscle at the beginning and every 2 weeks during 8 weeks of high-intensity resistance training from men (n = 13) and woman (n = 8). Muscle was also extracted from nontraining men (n = 7) and women (n = 5) at the same periods. Six muscle fiber types (I, IC, IIAC, IIA, IIAB, and IIB) were determined using basic myofibrillar adenosine triphosphatase histochemistry. Cross-sectional areas were determined for the three major fiber types (I, IIA, and IIB) and used to calculate the percentage area of these types. Electrophoretic techniques were used to separate and quantify the percentage MHC content in these same biopsy samples, and these data were then used to compare with the percentage fiber type area. Correlation analyses suggest a relationship between the histochemically assessed percentage fiber type area and the electrophoretically assessed MHC content in human limb musculature. However, because of possible histochemical misclassification of some fibers (especially in trained muscle) both techniques may be essential in yielding important information about fiber type composition and possible fiber type transformations.
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8039520
|
Effects of training and acclimation on heat tolerance in exercising men wearing protective clothing.
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This study examined the effectiveness of endurance training and heat acclimation in reducing the physiological strain imposed by exercising in the heat while wearing protective clothing. Seven young men underwent 8 weeks of physical training [60-80% maximal aerobic power (VO2max) for 30-45 min.day-1, 3-4 days.week-1 at < 25 degrees C] followed by 6 days of heat acclimation (45-55% VO2max for 60 min.day-1 at 40 degrees C, 30% relative humidity). Nine other young men underwent corresponding periods of control observation and heat acclimation. Before and after each treatment, subjects completed a treadmill walk (4.8 km.h-1, 2% grade) in a climatic chamber (40 degrees C, 30% relative humidity), wearing in turn normal combat clothing or clothing protecting against nuclear, biological, and chemical (NBC) agents. Criteria for halting this test were: (1) a rectal temperature (T(re)) of 39.3 degrees C; (2) a heart rate (fc) > or = 95% of the subject's observed maximum, maintained for 3 min; (3) unwillingness of the subject to continue; (4) the elapse of 120 min. The training regimen increased mean VO2max by 16% and mean plasma volume by 8%. When tested in normal combat clothing, the rates of increase in T(re) and fc were slower after training. However, when wearing NBC protective clothing, the only significant change induced by training was a higher mean skin temperature (Tsk) in the early part of the test. Heat acclimation increased the mean plasma volume of untrained subjects by 8%, but their VO2max remained unchanged. When tested in normal combat clothing, acclimation decreased their mean values of T(re), Tsk, fc, and metabolic rate. When wearing NBC protective clothing, the only significant decrease after acclimation was in overall T(re). In trained subjects, heat acclimation induced no further improvement in any physiological variable when wearing normal combat clothing, but reduced overall T(re) and Tsk when wearing NBC protective clothing. Training- or acclimation-induced increases of sweat secretion (an average increment of 0.14-0.23 kg.h-1) were not accompanied by any statistically significant increase in sweat evaporation when wearing NBC protective clothing. Moreover, tolerance times were unchanged in either normal combat (116-120 min) or NBC protective clothing (47-52 min). We conclude that neither endurance training nor heat acclimation do much to improve exercise tolerance when wearing NBC protective clothing in hot environments, because any added sweat secretion decreases blood volume and increases discomfort without augmenting body cooling.
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8039518
|
Adrenaline-induced leucocytosis: recruitment of blood cells from rat spleen, bone marrow and lymphatics.
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It is well known that adrenaline causes leucocytosis, but the sources and the mechanisms of this have not been clarified. We investigated the contributions of subpopulations of white blood cells to this leucocytosis and the importance of the spleen, bone marrow and lymphatics in releasing leucocytes into the blood stream following an injection of adrenaline. We studied possible effects of adrenaline on blood flow to the spleen and bone marrow to see if any contribution to leucocytosis from these organs could be perfusion dependent. In intact awake rats, total blood leucocytes increased within 5 min to about 220% of baseline concentration, the increases of lymphocytes and neutrophilic granulocytes being about 250% and 160%, respectively. The T and B lymphocytes and natural killer cells were all mobilized, to about 230% to 250% of baseline concentrations. The leucocytosis was short-lasting, so that the cell concentrations returned to baseline within 25 min after adrenaline injection. The bone marrow, spleen, and efferent lymphatics all contributed substantially to this leucocytosis, since band-nucleated granulocytes increased upon adrenaline injection, and splenectomized or thoracic duct drained rats showed a markedly reduced leucocytosis in response to adrenaline. Supplementary data were obtained with bone marrow depleted (with 89Sr irradiation) rats. The release of leucocytes from these organs was apparently not blood-flow dependent in the control rats since organ perfusion remained unaltered after adrenaline injection. Adrenaline was found to stimulate the release of both mono- and polymorphonuclear cells in the awake rat and the release of leucocytes from the spleen, bone marrow and efferent lymphatics to contribute significantly to the leucocytosis.
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8039519
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Plasma lactate concentration increases as a parabola with delay during ramp exercise.
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This study presents an elementary model of a system which relates plasma lactate concentration ([La-]) during ramp exercise to its rate of accumulation (Rc) within its extramuscular distribution space (S). Under the parsimonious assumptions that Rc increases linearly with time (t) with a kinetic delay (delta), and that the volume of S is constant, it is shown that plasma [La-] increases as a parabola with the kinetic delay delta when t increases. This elementary system model describes changes in plasma [La-] observed in five healthy young subjects during ramp exercise on the cycle ergometer (1 W every 2 s) with great accuracy (r > 0.99) with very small residuals (average value less than 0.01 mmol.l-1), randomly distributed around the fitting curves. The delay between the beginning of exercise and the onset of increase in Rc could be due to the fact that at the corresponding work rates: (1) rate of lactate appearance (Ra), which is equal to the rate of lactate disappearance (Rd), is not modified from rest, since the exercising muscles work in fully aerobic conditions (hypothesis of the anaerobic threshold); or (2) the increase in Ra is associated with a similar increase in Rd. An alternate or complementary hypothesis is that, during ramp exercise, plasma [La-] could reflect metabolic events within the muscles, with a significant delay.
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8039517
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Saliva electrolytes as a useful tool for anaerobic threshold determination.
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The purpose of the present study was to determine the anaerobic threshold by analysis of changes in saliva composition during an incremental exercise test on a cycle ergometer. Thirteen healthy males underwent a submaximal test with an initial load of 50 W and load increases of 50 W per 3 min, until capillary blood lactate exceeded 4 mmol.l-1. A maximal test for maximum O2 uptake (VO2max) determination (initial load of 100 W and load increases of 50 W per 2 min) was also performed. Saliva and blood samples were obtained only in the submaximal test. Saliva threshold (Thsa) was defined as the point at which the first increase in either Cl- or Na+ occurred. Catecholamine threshold (Thca) was defined as the point at which a nonlinear increase occurred in either adrenaline or noradrenaline. The lactate (Thla) and ventilatory (Thve) thresholds were determined according to published criteria. No significant differences were found between Thsa values and the other methods of threshold determination. A high correlation was found between Thsa and Thla (r = 0.82, P < 0.01), and Thsa and Thca (r = 0.75, P < 0.05). These results support the validity of Thsa as a new method for noninvasive determination of the anaerobic threshold.
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8039516
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Changes in phosphorus compounds and water content in skeletal muscle due to eccentric exercise.
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The interrelationship of the time courses of soreness and oedema, and of force and phosphorus metabolites after eccentric exercise was studied. Eight male subjects performed 120 maximal eccentric contractions with their left forearm flexors. Soreness, maximal force, flexion and extension elbow angle, and creatine kinase and myoglobin efflux were followed for 96 h after exercise. For equal periods T1 and T2 relaxation times and muscle cross-sectional area were calculated from magnetic resonance images as indications of oedema, and inorganic phosphate (P(i)) and phosphocreatine (PCr) were measured with magnetic resonance spectroscopy. Soreness on extension increased at 1 h (P = 0.043), T1 and T2 (both P = 0.01) and soreness when the arm was pressed (P = 0.028) at 24 h, and muscle cross-sectional area increased at 48 h (P = 0.01) after exercise. Soreness on extension reached a maximum at 48 h, the other four parameters at 72 h. All parameters related to oedema, and soreness, showed an increasing pattern for the period after exercise as a whole, but the largest increase between two points of measurement occurred earlier for soreness than for oedema. Creatine kinase increased significantly from baseline from 24 h onwards (P = 0.017) and myoglobin from 1 h onwards (P = 0.012). The P(i):PCr ratio differed from baseline for the first time 24 h after exercise (P = 0.018), increased to 225%, and then remained on a plateau until 72 h. Maximal isotonic force decreased to 53% at 1 h (P = 0.012).(ABSTRACT TRUNCATED AT 250 WORDS)
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8039515
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Urine excretion of androgen hormones in professional racing cyclists.
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This study was performed on 16 professional racing cyclists to examine changes in urine concentrations of androgen hormones (testosterone, epitestosterone, androsterone, etiocholanolone, 11-hydroxy-androsterone and 11-hydroxy-etiocholanolone) and plasma sex hormone binding globulin (SHBG) after training and after competition. The urinary concentrations of androgen hormones decreased during the period of training and increased during competition, this being the reverse of what happened to SHBG plasma concentrations. These changes would suggest that physical activity may have an influence on the elimination of androgen hormones and on the synthesis of its transporting protein SHBG.
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8039514
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The effects of warming by active and passive means on the subsequent responses to cold water immersion.
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Two experiments were undertaken to investigate the effects of warming the body upon the responses during a subsequent cold water immersion (CWI). In both experiments the subjects, wearing swimming costumes, undertook two 45-min CWIs in water at 15 degrees C. In experiment 1, 12 subjects exercised on a cycle ergometer until their rectal temperatures (Tre) rose by an average of 0.73 degree C. They were then immediately immersed in the cold water. Before their other CWI they rested seated on a cycle ergometer (control condition). In experiment 2, 16 different subjects were immersed in a hot bath (40 degrees C) until their Tre rose by an average of 0.9 degrees; they were then immediately immersed in the cold water. Before their other CWI they were immersed in thermoneutral water (35 degrees C; control condition). Heart rate in both experiments and respiratory frequency in experiment 1 were significantly (P < 0.05) higher during the first 30 s of CWI following active warming. In experiment 1, the rate of fall of Tre during the final 15 min of CWI was significantly (P < 0.01) faster when CWI followed active warming (2.46 degrees C.h-1) compared with the control condition (1.68 degrees C.h-1). However, this rate was observed when absolute Tre was still above that seen in the control CWIs. It is possible, therefore, that if longer CWIs had been undertaken, the two temperature curves may have converged and thereafter fallen at similar rates; this was the case with the aural temperature (Tau) seen in experiment 1 and the Tau and Tre in experiment 2. It is concluded that pre-warming is neither beneficial nor detrimental to survival prospects during a subsequent CWI.
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8039513
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Telomerase activity in germline and embryonic cells of Xenopus.
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Telomerase is a ribonucleoprotein which synthesizes telomere repeats onto chromosome ends. Telomerase activity is involved in telomere length maintenance. We used Xenopus laevis as a model system to study the expression of telomerase activity in germline cells and during early development. We identified a non-processive telomerase activity in manually dissected nuclei of Xenopus stage VI oocytes. Telomerase activity was detected throughout oogenesis and embryogenesis. Telomerase was active in both S and M phase cell cycle extracts, suggesting that telomerase activity is not regulated with chromosomal DNA replication.
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8039512
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Complex recombination events at the hypermutable minisatellite CEB1 (D2S90).
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Some minisatellite structures are the site of high rates of DNA recombination in non-pathological situations, with an excess of motif insertion events and a locus-dependent sex-specific mutation bias. We previously reported the cloning of the hypermutable minisatellite locus CEB1 (D2S90), remarkable for its 13% mutation rate in the male germline (compared to approximately 0.4% in female). We have sought to analyse the mechanisms underlying the addition or deletion of motifs at this locus using the minisatellite variant repeat mapping technique. This is possible with a high precision due to the extreme sequence polymorphism seen between different motifs. No crossing-over event was observed among 38 informative neomutations. Four of the 19 informative mutant alleles with an addition of motifs are interallelic events, the others are intra-allelic. Overall, the insertion and deletion mutations are spread along the alleles, although the subset of interallelic events shows clustering towards the analysed end. The apparently complex recombination events observed can all be interpreted as a succession of elementary duplications-deletions of inter- as well as intra-chromosomal origin, suggesting a model in which sister chromatid as well as conversion-like exchanges are involved in these mutation processes.
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8039511
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Determinants for stability of the chloroplast psbD RNA are located within its short leader region in Chlamydomonas reinhardtii.
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Stability of the chloroplast psbD mRNA encoding the D2 protein of the photosystem II reaction center is drastically decreased in the nuclear photosynthetic mutant nac2-26 of Chlamydomonas reinhardtii. Using biolistic transformation and genetic crosses we have introduced chimeric genes consisting of the psbD leader fused to a reporter gene into the chloroplast in both wild-type and mutant nuclear backgrounds. The chimeric message is destabilized in the latter, but not in the former case, indicating that the 74 nt psbD leader includes one of the target sites for psbD RNA degradation in the absence of wild-type NAC2 function. Increased instability of the psbD leader in mutant versus wild-type chloroplast lysates is also demonstrated in vitro and the primary cleavage sites have been mapped. The instability of the psbD RNA in the mutant correlates with the loss of binding of a 47 kDa protein to the psbD leader RNA, suggesting that this factor acts as message stabilizer in wild-type.
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8039510
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A nuclear mutation in maize blocks the processing and translation of several chloroplast mRNAs and provides evidence for the differential translation of alternative mRNA forms.
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A mutant designated crp1 (chloroplast RNA processing 1) was identified in a screen for transposon-induced maize mutants with defects in chloroplast gene expression. crp1 is a recessive, nuclear mutation that causes the loss of the cytochrome f/b6 complex and a reduction in photosystem I. The molecular basis for these protein losses is unique relative to previously described mutants with defects in organelle gene expression; it involves defects in the metabolism of two organellar mRNAs and in the translation of two organellar proteins. Mutants lack the monocistronic forms of the petB and petD mRNAs (encoding cytochrome f/b6 subunits), but contain normal levels of their polycistronic precursors. Pulse-labeling experiments revealed normal synthesis of the petB gene product, but a large decrease in synthesis of the petD gene product. These results suggest that petD sequences are more efficiently translated in a monocistronic than in a polycistronic context, thereby providing evidence that the elaborate RNA processing typical of chloroplast transcripts can play a role in controlling gene expression. Structural predictions suggest that the petD start codon lies in a stable hairpin in the polycistronic RNA, but remains unpaired in the monocistronic transcript. Thus, processing to a monocistronic form may increase translational efficiency by releasing the translation initiation region from inhibitory interactions with upstream RNA sequences. Synthesis of a third cytochrome f/b6 subunit, encoded by the petA gene, was undetectable in crp1, although its mRNA appeared unaltered. Two mechanisms are consistent with the simultaneous loss of both petA and petD protein synthesis: the translation of the petA and petD mRNAs might be coupled via a mechanism independent of crp1, or the crp1 gene may function to coordinate the expression of the two genes, which encode subunits of the same complex.
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8039509
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The exchange of the discriminator base A73 for G is alone sufficient to convert human tRNA(Leu) into a serine-acceptor in vitro.
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Transfer RNA (tRNA) identify is maintained by the highly specific interaction of a few defined nucleotides or groups of nucleotides, called identity elements, with the cognate aminoacyl-tRNA synthetase, and by nonproductive interactions with the other 19 aminoacyl-tRNA synthetases. Most tRNAs have a set of identity elements in at least two locations, commonly in the anticodon loop or in the acceptor stem, and at the discriminator base position 73. We have used T7 RNA polymerase transcribed tRNAs to demonstrate that the sole replacement of the discriminator base A73 of human tRNA(Leu) with the tRNA(Ser)-specific G generates a complete identity switch to serine acceptance. The reverse experiment, the exchange of G73 in human tRNA(Ser) for the tRNA(Leu-specific A, causes a total loss of serine specificity without creating any leucine acceptance. These results suggest that the discriminator base A73 of human tRNA(Leu) alone protects this tRNA against serylation by seryl-tRNA synthetase. This is the first report of a complete identity switch caused by an exchange of the discriminator base alone.
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7789330
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Tissue-specific expression of novel messenger ribonucleic acids cloned from a renin-expressing kidney tumor cell line (As4.1).
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As4.1 cells are derived from a renin-expressing kidney tumor induced by tissue-specific oncogene-mediated tumorigenesis in transgenic mice. These cells express high levels of renin messenger RNA (mRNA) and synthesize prorenin and renin; they were therefore used as a model to further investigate the molecular biology of renin-producing kidney cells by cloning and characterizing novel mRNAs expressed in these cells. One clone, designated 1.5, was randomly selected from an As4.1 complementary DNA (cDNA) library, and two other cDNA clones, designated 4.9 and 6.9, were obtained by screening the cDNA library using a strategy to identify As4.1 cell-specific mRNAs. Each clone exhibited a highly restricted tissue-specific expression profile, including high level expression in As4.1 cells and low level expression in kidney. No homology was found between the sequence of the partial 1.5 and 4.9 cDNAs and sequences in Genbank. Southern blot analysis revealed that clone 4.9 is encoded by a single copy gene containing at least two separate exons. A homology search of the sequence of clone 6.9 revealed it to encode a cDNA to serum amyloid A protein; consistent with this identification, expression of 6.9 mRNA was highly induced in both kidney and liver after treatment of mice with Escherichia coli lipopolysaccharide.
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7789329
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Transient transfection of GGH3-1' cells [GH3 cells stably transfected with the gonadotropin-releasing hormone (GnRH) receptor complementary deoxyribonucleic acid] with the carboxyl-terminal of beta-adrenergic receptor kinase 1 blocks prolactin release: evidence for a role of the G protein beta gamma-subunit complex in GnRH signal transduction.
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G proteins consist of heterotrimeric alpha-, beta-, and gamma-subunits. To assess the role of the beta gamma-subunit complex in GnRH receptor-mediated signal transduction, GGH3-1' cells were transfected with plasmids PRK5-beta ARK1 (495-689) containing complementary DNA (cDNA) of the carboxyl-terminal (Gly495-Leu689) of beta-adrenergic receptor kinase 1 (beta ARK1). GGH3-1' cells are GH3 cells that have been stably transfected with rat GnRH receptor cDNA. The carboxyl region of beta ARK1 (Gly495-Leu689) binds to the beta gamma complex and thereby inhibits its action. Twenty-four hours after stimulation, PRL release, cAMP release, and inositol phosphate (IP) production were measured in these cells and in control cells transfected with vector PRK5 cDNA alone. In cells expressing the beta ARK1-(495-689) sequence there was inhibition of basal PRL release (69.3%), cAMP release (61.2%), and IP production (75.5%) compared to cells containing vector only. When cells expressing the beta ARK1 fragment were stimulated with a GnRH analog (Buserelin; 10(-7) M), maximal responses were inhibited (66.1% for PRL release, 52.3% for cAMP release, and 79.1% for IP production). Scatchard analysis of GnRH analog binding was also performed in the two groups of transfected cells. No significant differences in Kd or receptor numbers were found between beta ARK1-(495-689)-transfected cells and control cells containing the vector alone. These data suggest a role for the beta gamma complex in mediation of cAMP release, IP production, and hormone release in response to agonist occupancy of the GnRH receptor.
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7789328
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The hemodynamic basis for the cardiac effects of parathyroid hormone (PTH) and PTH-related protein.
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PTH and PTH-related protein (PTHrP) have been regarded to have positive inotropic effects on the heart as well as positive chronotropic and vasodilator effects. However, inotropy due to a direct effect of these peptides has not heretofore been distinguished from an indirect inotropic effect as a result of altered heart rate or coronary flow. The aim of this study was to determine whether PTH and PTHrP have direct inotropic effects in isolated perfused rat hearts. Three groups of hearts were studied; in all groups, hearts contracted isovolumically and were perfused with a constant coronary pressure. In the control group, heart rate, coronary flow, peak pressure (LVPmax), peak rate of rise of LV pressure (dP/dtmax), and peak intracellular calcium (measured by aequorin) all increased with PTH and PTHrP in a dose-dependent manner. When heart rate was fixed by pacing in a second group of rats, PTH and PTHrP increased coronary flow, LVPmax, and dP/dtmax significantly, indicating that inotropic actions were not mediated solely by chronotropic effects. However, when heart rate was fixed by pacing and, additionally, coronary flow was held constant (by maximal prevasodilation with nitroprusside) in a third group of rats, there was no significant effect of either PTH or PTHrP on LVPmax, dP/dtmax, or peak intracellular calcium. To demonstrate the responsiveness of this latter preparation to inotropic stimulation, the beta-adrenergic agonist, isoproterenol, increased LVPmax, dP/dtmax, and peak calcium even when heart rate was fixed and vasodilation was maximal. Thus, PTH and PTHrP are inotropic agents by virtue of their influence on coronary flow and heart rate, but not by any direct effect on contractile elements in the heart.
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7789327
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Further evidence for a novel receptor for amino-terminal parathyroid hormone-related protein on keratinocytes and squamous carcinoma cell lines.
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PTH and PTH-related peptides (PTHrPs) interact with a common PTH/PTHrP receptor (type I), which is expressed in many tissues, including bone and kidney. Amino-terminal PTH and PTHrPs also recognize receptors in several nonclassical PTH target tissues, and in some of these, the signaling mechanisms differ qualitatively from those of the classical type I receptor. In normal keratinocytes and squamous carcinoma cell lines, PTH and PTHrP stimulate a rise in intracellular calcium, but not cAMP, suggesting the existence of an alternate, type II PTH/PTHrP receptor. SqCC/Y1 squamous carcinoma cells stably expressing the type I receptor displayed sensitive intracellular cAMP responses to PTHrP and PTH, indicating that these cells express functional GS proteins and that the type I receptor is capable of signaling through adenylyl cyclase in this cell line. Therefore, the endogenous type II receptor in SqCC/Y1 cells differs from the cloned type I receptor. We next examined whether messenger RNA (mRNA) from keratinocytes and squamous cell lines could hybridize to a human type I PTH/PTHrP receptor complementary DNA [1.9 kilobases (kb)]. No type I receptor mRNA (2.3 kb) was detected in polyadenylated RNA from any of the squamous cell lines. However, squamous cell lines did express several mRNA transcripts that hybridized with the type I receptor probe, yet were smaller (1 and 1.5 kb) or larger (3.5-5 kb) than the cloned receptor mRNA. The predominant mRNA in two squamous carcinoma cell lines and normal keratinocytes was a 1-kb transcript. Northern analysis with five different region-specific probes that span the entire coding region of the human type I receptor was used to map homologous regions within each of the transcripts. Several of the transcripts identified in squamous lines are also present in polyadenylated RNA from SaOS-2 human bone cells, but a unique 1-kb transcript hybridizing to probe 2 (nucleotides 490-870) was observed only in squamous cells. The smaller 1- and 1.5-kb transcripts did not hybridize to probes corresponding to the extreme 5'- and 3'-coding regions of the type I receptor complementary DNA. Ribonuclease protection analysis employing riboprobes that correspond to the five region-specific DNA probes revealed strong RNA signals of the expected size in SaOS-2 cells, but no hybridization with squamous cell RNA. Several smaller, but minor, bands that were unique to squamous cells were observed with riboprobe 2 only, suggesting partial homology of this region with the type I receptor.(ABSTRACT TRUNCATED AT 400 WORDS)
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7789326
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Cellular localization of estradiol-induced c-fos messenger ribonucleic acid in the rat uterus: c-fos expression and uterine cell proliferation do not correlate strictly.
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Estrogens stimulate DNA synthesis and cell proliferation in the uterus. All major uterine cell types (luminal and glandular epithelium, stroma, and myometrium) respond to 17 beta-estradiol in the immature animal, whereas primarily epithelial cells of the uterine endometrium respond in the mature animal. Rapid activation of the c-fos protooncogene by estrogen precedes the uterine growth, suggesting that c-fos plays a role in amplifying the hormonal signal. The specific uterine cell types in which estrogen induces c-fos messenger RNA (mRNA) expression, however, have not been identified in either mature or immature animals. In this study, in situ hybridization was used to determine the cell type-specific location of mRNA encoding c-fos in the uterus. In both immature and mature castrated rats at 3 h after 17 beta-estradiol administration, c-fos expression was detected primarily in uterine luminal and glandular epithelia. Expression of c-fos returned to baseline levels by 24 h post 17 beta-estradiol treatment. There was no apparent difference in the uterine cell type-specific pattern of c-fos expression stimulated by estradiol in mature vs. immature animals. Nuclear run-on transcription assay in isolated luminal epithelial cell nuclei showed that c-fos gene transcription increased rapidly in the uterus after estradiol stimulation. Treatment of adult rats with a single injection of 16 alpha-estradiol, a short-acting, nonmitogenic estrogen, induced c-fos primarily in the uterine glandular epithelia. Progesterone is known to modify the action of estrogen on the uterus by redirecting the proliferative response from epithelia to stroma. To determine if progesterone modulation of estrogen action involves shifting of c-fos expression to stromal cells, rats were treated with progesterone for 48 h and then killed 0, 3, 6, or 12 h after an estradiol injection. In situ hybridization analysis revealed that c-fos mRNA remained localized in the uterine luminal and glandular epithelia, and expression was not shifted to the stroma. Although these results support the idea that c-fos plays a role in proliferation of uterine epithelial cells, they also invite reassessment of the role played by c-fos in both epithelial and nonepithelial uterine cell types.
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7789325
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Photoperiod effects on neurohypophyseal and tuberoinfundibular dopamine metabolism in the male hamster.
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Exposure of golden hamsters to a short photoperiod (< 12.5 h light/day) leads to suppression of gonadal function secondary to reduced gonadotropin and PRL secretion. PRL secretion is decreased despite a reduction of tuberoinfundibular dopaminergic activity. In the present study, the ability of photoperiod to affect tuberohypophyseal dopamine (DA) turnover was evaluated in long day (LD; 16 h of light, 8 h of darkness) and short day (SD; 8 h of light, 16 h of darkness) male hamsters. Exposure to SD led to decreases in testicular weight within 10 weeks and decreases in plasma PRL levels within 1 week. DA turnover in the neurointermediate lobe of the pituitary, as estimated by measuring the depletion of DA 60 min after tyrosine hydroxylase inhibition with alpha-methyl-p-tyrosine (250 mg/kg), was significantly elevated 1 and 4 weeks after transfer to SD, but returned by 10 weeks to the levels seen in LD animals. After 14 days of SD exposure an enhanced lactotroph sensitivity to DA was demonstrated and may also have contributed to suppression of PRL levels. Similarly to the findings of previous studies, DA turnover in the median eminence was depressed in animals housed in SD. The DA content of the anterior pituitary was not significantly affected by photoperiod. The data from this study suggest that decreases in PRL secretion associated with the transfer of hamsters from LD to SD conditions are at least in part caused by an increase in DA turnover by neurohypophyseal neurons. However, the involvement of other PRL-inhibiting or -stimulating factors in mediating the effects of photoperiod on PRL secretion cannot be ruled out.
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7789324
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Differential effects of dimethylsulfoxide on steroidogenesis in mouse MA-10 and rat R2C Leydig tumor cells.
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The effects of dimethylsulfoxide (DMSO) on steroidogenesis in MA-10 mouse Leydig tumor cells and R2C rat Leydig tumor cells were compared. MA-10 cells produce low basal levels of progesterone (as the major steroid synthesized), which increase several hundred-fold when stimulated with tropic hormone or the cAMP analog (Bu)2cAMP. R2C cells, on the other hand, constitutively produce high levels of progesterone in the absence of hormone stimulation of any kind. When incubated in the presence of 5% DMSO, MA-10 cells demonstrated an almost complete inhibition of progesterone production, whereas the synthesis of this steroid was virtually unaffected in R2C cells. The inhibition of steroid production in the MA-10 cell could not be attributed to an effect on protein synthesis, because this was unaffected by DMSO during the course of the incubations. The activity of the cholesterol side-chain cleavage enzyme was also unaffected by DMSO, as demonstrated by incubation of the cells with 22R-hydroxycholesterol. The production of cAMP in response to tropic hormone (hCG) and forskolin stimulation was significantly inhibited in MA-10 cells, but was much less affected in R2C cells in response to forskolin treatment. However, DMSO appeared to have no effect on the overall phosphorylation of proteins when tested either in a completely in vitro system or when MA-10 cell homogenates were used as a source of exogenous protein. Strikingly, DMSO treatment resulted in the highly specific inhibition of a series of 30-kilodalton mitochondrial proteins (named StAR for Steroidogenic Acute Regulatory protein), which we have recently shown to be indispensable for the production of steroids in MA-10 cells. The synthesis of these same proteins was much less affected in R2C cells. Although the mechanism of action by which DMSO inhibits steroidogenesis remains unknown, these results show that its action results in the complete cessation of synthesis of the StAR protein, which is required for the acute regulation of steroidogenesis in MA-10 cells.
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7789323
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Retinoic acid inhibits estrogen-induced uterine stromal and myometrial cell proliferation.
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Retinoic acid, a potent natural derivative of vitamin A, influences proliferation in many cell types. However, little is known about the role of retinoic acid in estrogen-induced proliferation in normal physiological systems. In this study we sought to determine if in vivo administration of retinoic acid influences the proliferation of a normal estrogen target tissue, the immature rat uterus. The results indicate that treatment of animals with 30 mg/kg all-trans-retinoic acid for 3 days before 17 beta-estradiol (E2) administration diminishes DNA synthesis and cell division by approximately 50% in uterine stromal and myometrial cells. Luminal epithelial cell proliferation is not inhibited, indicating that the antiproliferative effects of all-trans-retinoic acid treatment are cell type-specific. The inhibition is retinoid-specific and fully reversible 1 week after discontinuing all-trans-retinoic acid treatment. The inhibitory effect of all-trans-retinoic acid is not due to a change in E2 receptor levels assessed by ligand binding. E2 induction of c-jun, a gene expressed primarily in myometrial cells, is unaffected in retinoid-treated animals. This is the first demonstration that retinoic acid inhibits estrogen-induced proliferation of uterine stromal and myometrial cells in a physiological setting.
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7789322
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Histamine directly stimulates gonadotropin-releasing hormone secretion from GT1-1 cells via H1 receptors coupled to phosphoinositide hydrolysis.
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It is unclear whether the central stimulating effect of histamine on GnRH secretion is exerted directly on GnRH neurosecretory neurons or indirectly via multisynaptic pathways, and controversy exists about the nature of the receptors involved. The current studies were undertaken to examine whether GnRH secretion from immortalized GnRH cell lines is directly regulated by histamine and, if so, to determine the identity of the receptors and the signaling pathways coupling this action. Histamine stimulated GnRH release from GT1-1 cells in a sustained and reversible manner and in a dose-dependent fashion. This effect was blocked by the selective H1 histamine receptor antagonist, mepyramine, but not by the H2 or H3 antagonists, ranitidine or thioperamide, respectively. Saturable and specific binding sites for [3H]mepyramine were demonstrated in GT1-1 cells, showing high affinity (apparent Kd, 37.8 nM) and density (apparent binding capacity, 279 fmol/mg protein) comparable to respective values in brain tissue. Competition of [3H]mepyramine binding was achieved with mepyramine at concentrations 3 orders of magnitude lower than those of ranitidine. Histamine also increased the production of inositol phosphates in GT1-1 cells in a dose- and time-dependent manner. This response was mimicked by the selective H1 receptor agonist 2-thiazolylethylamine and blocked by the H1 antagonists mepyramine, chlorpheniramine, and triprolidine. In contrast, histamine did not alter the formation of cAMP in GT1-1 cells. The present results show a direct action of histamine on immortalized GnRH neurons, suggesting that histamine may stimulate the reproductive axis by activation of H1 receptors on the surface of GnRH neurons coupled to the formation of inositol phosphates.
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7789321
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Modulation of adenosine triphosphate-sensitive potassium channel and voltage-dependent calcium channel by activin A in HIT-T15 cells.
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The ATP-sensitive potassium channel (KATP channel) determines the membrane potential of pancreatic beta-cells and plays a critical role in the regulation of insulin secretion. The present study was conducted to investigate the effect of activin A, a member of the transforming growth factor-beta supergene family, on the KATP channel in HIT-T15 clonal hamster insulinoma cells. In an excised inside-out patch, ATP-sensitive currents with a single channel conductance of approximately 20 picosiemens were observed. In an outside-out patch, currents with identical unitary conductance were also observed. In either case, the currents were augmented by diazoxide and blocked by glibenclamide, verifying that they were KATP channel currents. When KATP channel currents were monitored in an outside-out patch, activin A added to the bath solution inhibited KATP channel currents. Upon removal of activin A, the KATP channel currents were restored, suggesting that the inhibition was not due simply to spontaneous disappearance of channel activity (run-down). The KATP channel activity was markedly reduced after the addition of activin A and was reversed by diazoxide. Besides the inhibition of KATP channel, activin A increased, in a perforated patch, the amplitude of the inward Ba2+ current in response to a depolarizing pulse from -40 to +10 mV. Under the current clamp condition, activin A induced gradual depolarization, followed by a burst of action potentials. Activin-mediated action potentials were accompanied by an elevation of the cytoplasmic free calcium concentration. These results indicate that activin A causes depolarization of the plasma membrane by inhibiting the activity of the KATP channel. In addition, activin A directly modulates the voltage-dependent calcium channel and augments calcium entry.
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7789320
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Spontaneous oscillations of intracellular calcium in single bovine parathyroid cells may be associated with the inhibition of parathyroid hormone secretion.
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PTH secretion is tightly regulated by extracellular calcium ([Ca2+]e) and in turn regulates calcium homeostasis through its action on target tissues. We investigated the mechanism and physiological significance of intracellular calcium ([Ca2+]i) levels in relation to the secretion of PTH in single bovine parathyroid cells. [Ca2+]i was recorded using digital imaging microscopy, and secretion of PTH was correlated in the same cell using the reverse hemolytic plaque assay. In individual parathyroid cells, oscillations of [Ca2+]i were present in response to specific stimuli. Like secretory activity, response to [Ca2+]e concentrations was heterogeneous. Oscillations of [Ca2+]i occurred spontaneously in 22% of cells at inhibitory concentrations of [Ca2+]e. Oscillations were present only in high [Ca2+]e (> or = 1.8 mM) and not noted at lower concentrations of [Ca2+]e. The interval and amplitude of [Ca2+]i oscillations were 42 +/- 2 sec, and 20 +/- 1 nM (mean +/- SE), respectively. Oscillations were rapidly abolished when [Ca2+]e was removed by EGTA, and this effect was reversible. Addition of Mg2+ or polycationic antibiotics such as neomycin resulted in an [Ca2+]i spike, but oscillations were absent. Lanthanum, which blocks Ca2+ influx through calcium channels in various cells, rarely caused oscillations even in the presence of high concentrations of [Ca2+]e. To test the role of cAMP in [Ca2+]i oscillations, we added the beta-agonist isoproterenol. The addition of isoproterenol, however, did not cause oscillations. The number of cells that released PTH was significantly lower in cells with oscillations compared with cells without oscillations. We suggest that spontaneous [Ca2+]i oscillations are due to the influx of [Ca2+]e through ion channels rather than release from [Ca2+]i stores and may be a specific intracellular signal associated with inhibition of PTH secretion.
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7789319
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Relaxin acts directly on rat mammary nipples to stimulate their growth.
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Previously, we demonstrated that endogenous circulating relaxin promotes growth of the mammary nipples during the second half of pregnancy in the rat. The objective of this study was to determine whether relaxin acts directly on rat nipples to promote their growth. Initially, specific relaxin-binding cells were identified to assure that relaxin binds to the same cell types in the nipples of nonpregnant rats as those we previously described in pregnant rats. To examine relaxin-induced growth of the mammary nipples, 5 days after ovariectomy, 48 nonpregnant rats were assigned (12 rats/group) to 1 of the following 4 treatment groups: ovariectomized controls, estrogen treated, relaxin treated, and estrogen plus relaxin treated. Estrogen (0.05 micrograms 17 beta-estradiol) or estradiol vehicle (0.1 ml stripped corn oil) was administered sc on the dorsal side of the neck daily for the entire 10-day treatment period. Porcine relaxin (12.5 micrograms) or relaxin vehicle (0.05 ml 5% beeswax in corn oil) was administered sc at the base of the left abdominal nipple daily for the last 5 days of the 10-day treatment period. After hormone treatments, the lengths and wet weights of the left (relaxin-treated) and right (untreated) abdominal nipples were measured. There were three findings. First, the presence of specific relaxin binding in the epithelial cells of the lactiferous duct, smooth muscle cells, and skin of the nipples in nonpregnant rats was identical to the sites of specific relaxin binding in the nipples of pregnant rats. Second, relaxin-induced increases in nipple length and wet weight were mediated at least in part by the direct effects of relaxin in the nipple. Third, estrogen was not required for relaxin-induced increases in nipple length and wet weight. We conclude that relaxin stimulates the growth of rat mammary nipples at least in part through direct actions in the nipples, and that estrogen is not required for these actions.
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7789317
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Differential pathways of insulin action upon the hepatitis B surface antigen gene expression and cell proliferation in human hepatoma cells.
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We have shown previously that insulin at the physiological concentration suppresses hepatitis B surface antigen (HBsAg) gene expression in cultured human hepatoma Hep3B cells, and this suppression phenomenon is concomitant with the stimulation of cell proliferation. We have now examined whether these two distinct insulin actions on the Hep3B cells are mediated through the same or different signaling pathways. After prolonged treatment with high concentration of tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), the protein kinase C-alpha (PKC-alpha) level in the Hep3B cells was diminished and could not be detected by Western blot analysis. Under this condition, TPA treatment has no effect on the number or affinity of the insulin receptor on Hep3B cells. However, insulin-stimulated cell proliferation was completely abolished in the PKC-alpha depleted cells. In contrast, insulin still suppressed HBsAg gene expression with the same ED50 (approximately 0.5 nM) as the control cell. The induction of proto-oncogene egr-1 (early-growth-regulatory-1) by insulin and TPA under similar conditions were also examined. Both insulin and TPA stimulated egr-1 gene expression up to 10-fold in the control cell, but neither of these two agents showed any effect on egr-1 gene expression in the PKC-alpha down-regulated Hep3B cells. These observations indicate that the PKC-alpha may be involved in the insulin induced egr-1 expression and cell proliferation but not in the insulin suppressed HBsAg gene expression in human hepatoma cells.
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7789318
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The role of sodium in mediating adrenocorticotropin secretion by perifused rat anterior pituitary cells.
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We studied the role of sodium ions in mediating basal and stimulated ACTH release from perifused rat anterior pituitary cells by exposing the cells to the sodium channel opener veratridine or the Na+/K(+)-adenosine triphosphatase inhibitor ouabain to increase the intracellular Na+ concentration or, conversely, by omitting Na+ from the perifusion medium or blocking Na+ entry into the cell with tetrodotoxin, a voltage-dependent sodium channel blocker, to decrease the intracellular Na+ concentration. Neither tetrodotoxin nor Na(+)-free medium had a significant effect on 100 nM arginine vasopressin (AVP) or 10 nM ovine corticotropin-releasing hormone (CRH)-induced ACTH secretion. Veratridine increased basal ACTH secretion by 122% (41.3 +/- 2.9 vs. 18.6 +/- 0.4 pg/min; P < 0.001), the initial spike phase of the response to AVP by 65% (0.28 +/- 0.01 vs. 0.17 +/- 0.03 ng/3 min; P < 0.005), the subsequent sustained phase to AVP by 129% (0.16 +/- 0.01 vs. 0.07 +/- 0.01 ng/7 min; P < 0.005), and the total response to CRH by 70% (0.39 +/- 0.01 vs. 0.23 +/- 0.04 ng/10 min; P < 0.05). Ouabain increased basal ACTH secretion by 39% (45.7 +/- 2.8 vs. 32.9 +/- 2.1 pg/min; P < 0.05), the initial spike phase of the response to AVP by 88% (0.32 +/- 0.02 vs. 0.17 +/- 0.01 ng/3 min; P < 0.005), the sustained phase response to AVP by 67% (0.10 +/- 0.01 vs. 0.06 +/- 0.01 ng/7 min; P < 0.05), and the total integrated response to CRH by 49% (0.88 +/- 0.09 vs. 0.59 +/- 0.03 ng/10 min; P < 0.05). However, the effects of both veratridine and ouabain on basal ACTH secretion were significantly attenuated in Ca(2+)-free EGTA-containing medium, suggesting that this effect was indirect, due to membrane depolarization and consequent influx of extracellular Ca2+. Dexamethasone (100 nM) had no effect on the ACTH response to either veratridine or ouabain. We conclude that changes in the intracellular Na+ concentration and sodium channel activity are not directly involved in AVP- or CRH-induced ACTH secretion.
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7789316
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Growth hormone (GH) receptor and GH-binding protein messenger ribonucleic acids with alternative 5'-untranslated regions are differentially expressed in mouse liver and placenta.
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Two 5'-untranslated regions (5'UTRs) with distinctly different sequences, designated 5'UTR L1 and 5'UTR L2, were obtained by amplification of complementary DNA from mouse liver and placenta with primers complementary to sequences from the hormone-binding domain common to GH receptor (GHR) and GH-binding protein (GHBP) messenger RNAs (mRNAs). The presence of an open reading frame in the 5'UTR L2 and the high GC content of this sequence suggest that mRNAs containing this 5'UTR may be translated with a lower efficiency than those containing 5'UTR L1. Expression studies showed that 5'UTR L1 and 5'UTR L2 are present in GHR and GHBP mRNAs in both tissues. However, the relative expression of the two 5'UTRs differs between liver and placenta and in liver from different physiological states. The different expression patterns of the L1 and L2 5'UTRs predict that the corresponding 5'-noncoding exons of the GHR/GHBP gene are associated with different regulatory elements. The expression patterns of the 5'UTRs also indicate that there is a linkage between the 5'UTR present in GHR/GHBP gene transcripts and the alternative splicing of these transcripts to yield either GHR or GHBP mRNAs. The 5'-noncoding exon used for transcription of the GHR/GHBP gene, therefore, may be involved in regulating both the ratio of GHR to GHBP transcripts and the efficiency of translation of these transcripts. Transcription from the different 5'-noncoding exons of the GHR/GHBP gene thus may be a critical element in the regulation of the expression of GHR and GHBP and thereby in the control of the responses of different tissues to GH.
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7789315
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Retinoid X receptor alpha binds with the highest affinity to an imperfect direct repeat response element.
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The regulation of gene expression by retinoids is mediated by two classes of receptors, retinoic acid receptors and retinoid X receptors (RXR). RXR can bind to specific target genes as homodimers, and these homodimers can activate gene expression in the presence of the ligand 9-cis-retinoic acid. A direct repeat of AGGTCA with a 1 base pair spacer (DR1) acts as a RXR homodimer response element in the presence of 9-cis-retinoic acid. However, it is not known if this represents the highest affinity binding site for the RXR homodimer. To investigate this question, we used a nonbiased strategy to isolate from a pool of random DNA those sequences that have the highest affinity for RXR alpha homodimers. The imperfect DR1 sequence 5'-GGGGTCAAAGGTCA displayed the highest in vitro binding affinity for RXR alpha homodimers. Transient transfection studies confirmed that this sequence is a more potent response element than is a perfect DR1 of either AGGTCA or GGGGTCA. The results also indicate that for RXR alpha homodimers, the receptor bound to the 5' half-site dislays different DNA binding specificity than that bound to the 3' half-site. Thus, DNA binding specificity is determined not only by the amino acid sequence of the protein but also by its protein-protein interactions and its position on the response element (5' vs. 3').
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7789313
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Rapid protection of rat spermatogenic stem cells against procarbazine by treatment with a gonadotropin-releasing hormone antagonist (Nal-Glu) and an antiandrogen (flutamide).
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GnRH antagonist (Nal-Glu) treatment combined with the antiandrogen flutamide was used to suppress rat spermatogenesis to achieve protection of spermatogonial stem cells against the anticancer drug procarbazine. Daily injections with Nal-Glu alone suppressed spermatogenesis in a dose-responsive manner. However, it was necessary to combine Nal-Glu (600 micrograms/kg.day) with flutamide at 20 mg/kg.day to decrease testicular weight in 2 weeks to less than 0.6 g, a level previously demonstrated sufficient to protect stem cells in our model system. The Nal-Glu-flutamide pretreatment suppressed serum gonadotropin levels and intratesticular testosterone levels (6% of control) and action, resulting in a reversible decrease in the number of late spermatids to 1% of control levels. When rats were given Nal-Glu-flutamide for 2 weeks before a 250 mg/kg dose of procarbazine, recovery of spermatogenesis, as measured by testis weight, testicular sperm head counts, and repopulation indexes, was significantly better than in control rats (no hormonal pretreatment). The protection achieved with Nal-Glu-flutamide was better than that achieved with 2 weeks of testosterone and estradiol treatment. The present results show that Nal-Glu-flutamide protects spermatogonial stem cells against procarbazine and suggest a method of hormonal pretreatment to achieve rapid and efficient protection of spermatogenesis in humans.
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7789314
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Regulation of follistatin messenger ribonucleic acid in steroidogenic rat granulosa cell lines.
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Follistatin is a 35-kilodalton monomer isolated from follicular fluid that acts on pituitary gonadotropes to suppress the production of FSH. Transfection of rat granulosa cells with specific oncogenes, such as simian virus 40 (SV40) DNA and Ha-ras oncogene, leads to their immortalization concomitant with preservation of their capacity for inducible steroidogenesis. Experiments were designed to investigate the regulation of follistatin messenger RNA (mRNA) accumulation upon stimulation with forskolin, 2-O-tetradecanol-phorbol-13-acetate (TPA), FSH, and hCG in four different granulosa cell lines. Granulosa cells were transfected with SV40 DNA alone (POGS5); with SV40 DNA and Ha-ras oncogene (POGRS1); with SV40 DNA, Ha-ras oncogene, and LH/CG receptor (GLHR15); or with FSH receptor (GFSHR17) expression plasmid. Cells were cultured to reach confluence and then stimulated for 6 or 24 h with ovine FSH (oFSH; 0.004-4 nM), hCG (9 nM), forskolin (50 microM), and TPA (50 nM), alone or in combination. In the POGS5 cell line, forskolin caused a 15-fold stimulation of follistatin mRNA after 24-h incubation. The POGRS1 cell line showed a time-dependent stimulation of follistatin gene expression induced by both forskolin (5.7-fold) and TPA (9.4-fold). In the GFSHR17 cells, forskolin, oFSH, and TPA induced an increase in follistatin mRNA. When oFSH (1.6 nM) was added to cells treated with forskolin (50 microM) or TPA (50 nM), no additional stimulation was observed. The GLHR15 cell line treated with hCG showed a 2.7-fold increase in follistatin mRNA accumulation within 6 h. Our data demonstrate that 1) follistatin mRNA is detectable and induced by forskolin and TPA in transformed granulosa cell lines that do not express the FSH or LH receptors; 2) in the GFSHR17 cell line, FSH, forskolin, and TPA caused a time- and dose-dependent regulation of the gene; and 3) follistatin gene expression is up-regulated by hCG in the GLHR15 cell line. We conclude that these transformed steroidogenic cell lines can serve as a useful model to study the regulation of follistatin gene expression, a peptide known to regulate pituitary and ovarian hormone secretion and differentiation of granulosa cells by its activin-binding action. Moreover, this gene can be regulated in immortalized granulosa cells by both the protein kinase A and protein kinase C pathways, although these cells express the large T antigen and the Ha-ras oncogenic proteins.
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7789312
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N-methyl-D-aspartic acid receptor messenger ribonucleic acid levels and luteinizing hormone release in immature female rats: effects of stage of pubertal development and exposure to ethanol.
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This research was designed to determine 1) whether changes occur in the levels of N-methyl-D-aspartic acid (NMDA) receptor (NMDA-R) messenger RNA (mRNA) in the reproductive hypothalamus of female rats as they approach puberty, 2) whether NMDA-R stimulation would promote differential LH responses during the specific stages of peripubertal development, and 3) whether ethanol (ETOH), which is known to affect the NMDA-R in other brain systems, can alter NMDA-R-activated LH secretion at puberty. In the first experiment, female rats were killed at 15, 20, 25, and 34-36 days of age to determine the levels of mRNA that code for the NMDA-R, specifically NMDA-R1, in the arcuate nucleus-median eminence (AN-ME) and preoptic area (POA) during pre- and peripubertal development by a ribonuclease protection assay. Results indicate that in juvenile animals, NMDA-R mRNA levels in the AN-ME increased at 25 days (P < 0.01). In the POA, the levels increased at 20 days (P < 0.05), but were unchanged at 25 days. During the peripubertal period, NMDA-R gene expression in the AN-ME did not change; however, gene expression in the POA increased (P < 0.05) during first proestrus, then declined during first estrus. In the second experiment, NMDA-R stimulation with N-methyl-D,L-aspartic acid (NMA; 2.5 mg/kg) produced differential stimulatory effects on LH release depending upon the stage of pubertal development. In this regard, significant post-NMA percent increases in LH released over pre-NMA (basal) levels occurred during anestrus (46%; P < 0.01) and first proestrus (95%; P < 0.01), with nonsignificant increases of 18% and 28% during first estrus and diestrus, respectively. Finally, a 3 g/kg dose of ETOH given intragastrically 90 min before the NMA challenge blocked (P < 0.05) NMA-induced LH release during first proestrus. In conclusion, these findings demonstrate regional differences in the timing of NMDA-R gene expression in the reproductive hypothalamus during pubertal development, show differential responses of LH to NMDA-R activation during the peripubertal period, and continue to demonstrate the vulnerability of the hypothalamic-pituitary axis to the detrimental effects of ETOH at this critical time of development.
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7789311
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Characterization of newly established testicular peritubular and prostatic stromal cell lines: potential use in the study of mesenchymal-epithelial interactions.
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Testicular peritubular and prostatic stromal cells produce extracellular matrix elements and paracrine factors that modulate the cytodifferentiation and function of the corresponding epithelial cells. The present paper describes the establishment and characterization of five rat testicular cell lines with peritubular characteristics and one prostatic stromal cell line. Four peritubular cell lines were isolated after transfection of a mixed peritubular-Sertoli cell culture with a v-myc-containing plasmid. The same immortalization procedure applied to prostatic stromal cells yielded one cell line. An additional testicular cell line arose by spontaneous immortalization during serial subculture. Except for one testicular cell line (RTC-8T1), the morphology of all of the immortalized cell lines strongly resembled that of primary cultures of peritubular and stromal cells. Flow cytometric analysis demonstrated that all cell lines scored positive for alpha-smooth muscle isoactin and negative for cytokeratins, confirming their myofibroblast-like nature. None of the cell lines, however, stained positive for alkaline phosphatase, and androgen receptor expression was also lost. Typical Leydig cell characteristics, such as steroidogenesis, and Sertoli cell markers, such as transferrin secretion, were absent. Coculture of the cell lines with Sertoli cells resulted in the formation of tubular structures. A cell attachment assay and an enzyme-linked immunosorbent assay for fibronectin confirmed the production of extracellular matrix elements by all of the established cell lines. Media conditioned by the cell lines stimulated Sertoli cell transferrin production. The active principle was partially purified and resembled the P-MOD-S-like factors produced by primary cultures of peritubular and stromal cells. It is concluded that the immortalized cell lines have retained several of the characteristics of primary cultures of peritubular and stromal cells and may be useful for further studies on mesenchymal-epithelial interactions in testis and prostate.
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7789310
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1,25-Dihydroxyvitamin D3 evokes oscillations of intracellular calcium in a pancreatic beta-cell line.
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The steroid hormone 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] can elicit biological responses via a nongenomic pathway that involves rapid opening of the plasma membrane Ca2+ channels. There is also evidence that 1,25-(OH)2D3 influences insulin secretion in the pancreatic beta-cell, which is primarily mediated by a rapid rise in the concentration of intracellular free Ca2+ ([Ca2+]i). We employed fluorescent digital ratiometric video imaging at the single cell level to study the effects of 1,25-(OH)2D3 on [Ca2+]i in a pancreatic beta-cell line, RINr1046-38. In RIN cells equilibrated at a steady state glucose concentration (5.5 mM), 1,25-(OH)2D3 (2-20 nM) rapidly, within 5-10 sec, increased [Ca2+]i and evoked sinusoidal [Ca2+]i oscillations with a frequency of 1.87 +/- 0.13 min-1 and an amplitude of 236 +/- 3 nM (from the initial basal level of 110 +/- 2 nM). The [Ca2+]i oscillations were acutely dependent on extracellular Ca2+, but not on extracellular glucose. Further, we investigated the mechanisms of activation by 1,25-(OH)2D3 of the Ca2+ entry pathway in the plasma membrane and analyzed the relationship between 1,25-(OH)2D3-stimulated Ca2+ entry and Ca2+ release from intracellular stores. The 1,25-(OH)2D3-evoked [Ca2+]i oscillations were mediated by nonselective Ca2+ channels, which are permeable to Mn2+ and suppressed by extracellular La3+. Blockage of voltage-dependent Ca2+ channels by nifedipine significantly decreased the amplitude of the oscillations. Depletion of intracellular Ca2+ stores with thapsigargin did not affect the 1,25-(OH)2D3-stimulated Ca2+ entry estimated by the Mn2+ entry and fura-2 fluorescence quench, which implies that the hormone directly activates nonselective Ca2+ channels. The 1,25-(OH)2D3-evoked increase in the background Ca2+ influx appears to generate [Ca2+]i oscillations by triggering Ca2+ release through the ryanodine receptor/Ca2+ release channel, but not through activation of the inositol 1,4,5-triphosphate receptor. Our findings are consistent with a role of the plasmalemmal vitamin D receptor coupled to the plasma membrane Ca2+ channels in mediating rapid effects of the hormone. We propose that the 1,25-(OH)2D3-mediated Ca2+ signaling pathway may be involved in the regulation of insulin secretion from the pancreatic beta-cell.
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7789309
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Studies on the repression of basal transcription (silencing) by artificial and natural human thyroid hormone receptor-beta mutants.
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Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that regulate target gene transcription. Interestingly, in the absence of ligand, TRs also can repress basal transcription of positively regulated target genes, suggesting that unliganded TR may have a distinct role in gene regulation. In this paper, DNA binding, truncation, and natural human TR beta mutants were used in cotransfection and electrophoretic mobility shift assays to study various aspects of TR-mediated basal repression. Presently, little is known about the role(s) of natural human TR beta mutants on basal repression. These results show that: 1) TR binding to DNA likely is required for basal repression; 2) the amino-terminal region of TR is not required for basal repression; 3) TR homodimer binding is not absolutely required for basal repression, as TR mutants that selectively form TR-retinoid X receptor heterodimers can mediate basal repression; and 4) TR mutants with poor T3-binding affinity likely have constitutive basal repression, even in the presence of ligand. These findings provide new insight on the mechanism of basal repression by unliganded TRs.
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7789308
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Regulation of polymeric immunoglobulin A receptor messenger ribonucleic acid expression in rodent uteri: effect of sex hormones.
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Previously we have shown that estradiol stimulates the production of secretory component, the external domain of the polymeric immunoglobulin A (IgA) receptor (pIgR) responsible for transporting IgA from tissues into secretions. In the present study, levels of pIgR messenger RNA (mRNA) in uterine tissues of rats were correlated with pIgR expression in epithelial cells and secretory component in uterine secretions. Analysis of uterine pIgR mRNA and pIgR expression in epithelial cells during the estrous cycle indicated that levels were high at proestrus and estrus and low at diestrus. When ovariectomized rats were treated with estradiol for 3 days, and pIgR mRNA was measured 4 and 12 h after the last injection, levels of uterine pIgR mRNA were significantly greater than those in saline-treated controls. High levels of pIgR were also detected in uterine epithelial cells and uterine secretions. When estradiol and progesterone were given in combination, progesterone partially reversed the effect of estradiol on pIgR mRNA levels and expression of pIgR in epithelial cells. These studies demonstrate that changes in uterine pIgR mRNA levels correlate with pIgR expression during the estrous cycle and in response to estradiol and progesterone. These findings suggest that mucosal immune responses in the reproductive tract are regulated in part by the actions of estradiol and progesterone on pIgR mRNA expression.
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7789307
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Electron microscopic visualization of insulin translocation into the cytoplasm and nuclei of intact H35 hepatoma cells using covalently linked Nanogold-insulin.
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Insulin affects numerous metabolic processes as well as nuclear events such as gene transcription. Our previous ultrastructural and biochemical studies demonstrated insulin accumulation in nuclei of cultured and rapidly proliferating cells, and biochemical evidence suggested that insulin entered the cell cytoplasm before accumulating in the nucleus. The present study was undertaken to develop a covalently linked electron-dense insulin complex that could be used to visualize the intracellular translocation of insulin and confirm that insulin enters the cytoplasm of cells. Insulin was cross-linked to 1.4-nm diameter Nanogold particles. The complex binds to the plasma membrane insulin receptor, is biologically active, and is degraded by cellular insulin-degradative enzymes. Ultrastructural analysis after silver intensification of the gold particles confirmed that insulin internalization culminates in the translocation of some internalized insulin to the cytoplasm and nuclei. When cytoplasmic insulin-degrading enzyme (IDE) activity was inhibited with 1,10-phenanthroline, an increase in the number of cytoplasmic and nuclear Nanogold-insulin particles was observed. The results of this and previous studies suggest that 1) the translocation of insulin to the cytoplasm, 2) the regulation of insulin degradation in the cytoplasm by IDE, 3) the possible interaction of insulin with cytoplasmic proteins other than IDE, and 4) the subsequent accumulation of intact insulin or insulin complexed with cytoplasmic proteins in nuclei may play a role in insulin's regulation of gene transcription and cell proliferation.
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7789306
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Regulation of glutamine:fructose-6-phosphate amidotransferase gene transcription by epidermal growth factor and glucose.
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In preparation for the cellular proliferation stimulated by growth factors, the rate of macromolecular synthesis must be increased to allow for the enlargement of the cell that proceeds mitosis. The increased glycoprotein synthesis that follows growth factor stimulation would consume the hexosamines required for protein modification. Glutamine:fructose-6-phosphate amidotransferase (GFAT) is the rate-limiting enzyme controlling the synthesis of the hexosamines used in these biosynthetic pathways. We tested the idea that growth factors might activate the transcription of the GFAT gene to increase the cellular content of this rate-limiting enzyme in hexosamine synthesis. We employed a human breast cancer cell line, MDA468 cells, which express high numbers of epidermal growth factor (EGF) receptors, to determine whether EGF could stimulate transcription of the GFAT gene. Our experiments showed that EGF stimulated the accumulation of GFAT messenger RNA (mRNA) to a level 4-fold higher than that in unstimulated cells. This accumulation could be largely accounted for by an increase in transcription, as assessed by nuclear run-on experiments. Furthermore, the GFAT mRNA was highly stable and not further stabilized by EGF. This effect of EGF on GFAT gene transcription required stimulation for 12-16 h with EGF. Interestingly, when cells were exposed to 25 mM glucose instead of 5 mM glucose, this effect of EGF was blocked. Glucose had no effect on the stability of the GFAT mRNA, implying that the effect of glucose was to antagonize the transcriptional effect of EGF on the GFAT gene. Glucosamine had an effect opposite that of glucose, in that it stimulated GFAT mRNA accumulation and had an additive effect with EGF on the accumulation of this mRNA. These results demonstrate that the GFAT gene undergoes a late transcriptional response to EGF and that the provision of high glucose concentrations to the cells blocks this EGF activation. This effect of glucose does not appear to result from its metabolism through GFAT to glucosamine.
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7789291
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Ceftazidime. An update of its antibacterial activity, pharmacokinetic properties and therapeutic efficacy.
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Ceftazidime is a third generation cephalosporin antibacterial agent which, since its introduction in the early 1980s, has retained a broad spectrum of in vitro antimicrobial activity and clinical utility in serious infections. However, increasing resistance to ceftazidime and other third generation cephalosporins, particularly among Enterobacteriaceae, due to the emergence of plasmid-mediated extended spectrum beta-lactamases and the class I chromosomally mediated beta-lactamases, is of concern. There is now a wealth of information on the pharmacokinetics of the drug. enabling ceftazidime to be used predictably, and with a low potential for adverse effects, in a diversity of patient populations. Overall, ceftazidime remains an effective agent for the treatment of serious infection, particularly those due to major nosocomial pathogens, and respiratory infections in patients with cystic fibrosis. Ceftazidime-containing regimens also remain an important option for the empirical therapy of febrile episodes in neutropenic patients. The tolerability profile of ceftazidime makes the drug a useful option in seriously ill patients who are at risk of developing adverse events with other antibacterial agents. Although patterns of bacterial resistance have changed in the ensuing years since its introduction, judicious use of this important agent will help maintain its present clinical utility.
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