bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular interactions with nuclear factor κB (NF‐κB) transcription factors of a PNA–DNA chimera mimicking NF‐κB binding sites
Authors: Romanelli Alessandra , Pedone Carlo , Saviano Michele , Bianchi Nicoletta , Borgatti Monica , Mischiati Carlo , Gambari Roberto
Published: March 2003

The decoy approach against nuclear factor κB (NF‐κB) is a useful tool to alter NF‐κB dependent gene expression using synthetic oligonucleotides (ODNs) carrying NF‐κB specific cis‐elements. Unfortunately, ODNs are not stable and need to be be extensively modified to be used in vivo or ex vivo. We have previously evaluated the possible use of peptide nucleic acids (PNAs) as decoy molecules. The backbone of PNAs is composed of N‐(2‐aminoethyl)glycine units, rendering these molecules resistant to both nucleases and proteases. We found that the binding of NF‐κB transcription factors to PNAs was either very low (binding to PNA–PNA hybrids) or exhibited low stability (binding to PNA–DNA hybrids). The main consideration of the present paper was to determine whether PNA–DNA chimeras mimicking NF‐κB binding sites are capable of stable interactions with proteins belonging to the NF‐κB family. Molecular modeling was employed for the design of PNA–DNA chimeras; prediction of molecular interactions between chimeras and NF‐κB nuclear proteins were investigated by molecular dynamics simulations, and interactions between PNA–DNA chimeras and NF‐κB proteins were studied by gel shifts. We found significant differences between the structure of duplex NF‐κB PNA–DNA chimera and duplex NF‐κB DNA–DNA. However, it was found that these differences do not prevent the duplex PNA–DNA chimera from binding to NF‐κB transcription factors, being able to suppress the molecular interactions between HIV‐1 LTR and p50, p52 and nuclear factors from B‐lymphoid cells. Therefore, these results demonstrate that the designed NF‐κB DNA–PNA chimeras could be used for a decoy approach in gene therapy.
Ontogeny and subcellular localization of rat liver mitochondrial branched chain amino‐acid aminotransferase
Authors: Torres Nimbe , Vargas Carolina , Hernández‐Pando Rogelio , Orozco Héctor , Hutson Susan M. , Tovar Armando R.
Published: March 2003

Branched chain amino‐acid aminotransferase (BCAT) activity is present in fetal liver but the developmental pattern of mitochondrial BCAT (BCATm) expression in rat liver has not been studied. The aim of this study was to determine the activity, protein and mRNA concentration of BCATm in fetal and postnatal rat liver, and to localize this enzyme at the cellular and subcellular levels at both developmental stages. Maximal BCAT activity and BCATm mRNA expression occurred at 17 days' gestation in fetal rat liver and then declined significantly immediately after birth. This pattern was observed only in liver; rat heart showed a different developmental pattern. Fetal liver showed intense immunostaining to BCATm in the nuclei and mitochondria of hepatic cells and blood cell precursors; in contrast, adult liver showed mild immunoreactivity located only in the mitochondria of hepatocytes. BCAT activity in isolated fetal liver nuclei was 0.64 mU·mg−1 protein whereas it was undetectable in adult liver nuclei. By Western blot analysis the BCATm antibody recognized a 41‐kDa protein in fetal liver nuclei, and proteins of 41 and 43 kDa in fetal liver supernatant. In adult rat liver supernatant, the BCATm antibody recognized only a 43‐kDa protein; however, neither protein was detected in adult rat liver nuclei. The appearance of the 41‐kDa protein was associated with the presence of the highly active form of BCATm. These results suggest the existence of active and inactive forms of BCAT in rat liver.
Modelling of simple and complex calcium oscillations
Authors: Schuster Stefan , Marhl Marko , Höfer Thomas
Published: March 2003

This review provides a comparative overview of recent developments in the modelling of cellular calcium oscillations. A large variety of mathematical models have been developed for this wide‐spread phenomenon in intra‐ and intercellular signalling. From these, a general model is extracted that involves six types of concentration variables: inositol 1,4,5‐trisphosphate (IP3), cytoplasmic, endoplasmic reticulum and mitochondrial calcium, the occupied binding sites of calcium buffers, and the fraction of active IP3 receptor calcium release channels. Using this framework, the models of calcium oscillations can be classified into ‘minimal’ models containing two variables and ‘extended’ models of three and more variables. Three types of minimal models are identified that are all based on calcium‐induced calcium release (CICR), but differ with respect to the mechanisms limiting CICR. Extended models include IP3–calcium cross‐coupling, calcium sequestration by mitochondria, the detailed gating kinetics of the IP3 receptor, and the dynamics of G‐protein activation. In addition to generating regular oscillations, such models can describe bursting and chaotic calcium dynamics. The earlier hypothesis that information in calcium oscillations is encoded mainly by their frequency is nowadays modified in that some effect is attributed to amplitude encoding or temporal encoding. This point is discussed with reference to the analysis of the local and global bifurcations by which calcium oscillations can arise. Moreover, the question of how calcium binding proteins can sense and transform oscillatory signals is addressed. Recently, potential mechanisms leading to the coordination of oscillations in coupled cells have been investigated by mathematical modelling. For this, the general modelling framework is extended to include cytoplasmic and gap‐junctional diffusion of IP3 and calcium, and specific models are compared. Various suggestions concerning the physiological significance of oscillatory behaviour in intra‐ and intercellular signalling are discussed. The article is concluded with a discussion of obstacles and prospects.
Functional analysis of a small heat shock/α‐crystallin protein from Artemia franciscana
Authors: Crack Julie A. , Mansour Marc , Sun Yu , MacRae Thomas H.
Published: March 2003

Oviparously developing embryos of the brine shrimp, Artemia franciscana, synthesize abundant quantities of a small heat shock/α‐crystallin protein, termed p26. Wild‐type p26 functions as a molecular chaperone in vitro and is thought to help encysted Artemia embryos survive severe physiological stress encountered during diapause and anoxia. Full‐length and truncated p26 cDNA derivatives were generated by PCR amplification of p26‐3‐6‐3, then cloned in either pET21(+) or pRSETC and expressed in Escherichia coli BL21(DE3). All constructs gave a polypeptide detectable on Western blots with either p26 specific antibody, or with antibody to the His6 epitope tag encoded by pRSETC. Full‐length p26 in cell‐free extracts of E. coli was about equal in mass to that found in Artemia embryos, but p26 lacking N‐ and C‐terminal residues remained either as monomers or small multimers. All p26 constructs conferred thermotolerance on transformed E. coli, although not all formed oligomers, and cells expressing N‐terminal truncated derivatives of p26 were more heat resistant than bacteria expressing p26 with C‐terminal deletions. The C‐terminal extension of p26 is seemingly more important for thermotolerance than is the N‐terminus, and p26 protects E. coli against heat shock when oligomer size and protein concentration are low. The findings have important implications for understanding the functional mechanisms of small heat shock/α‐crystallin proteins.
Conditionally immortalized adrenocortical cell lines at␣undifferentiated␣states exhibit inducible expression of␣glucocorticoid‐synthesizing genes
Authors: Mukai Kuniaki , Nagasawa Hideko , Agake‐Suzuki Reiko , Mitani Fumiko , Totani Keiko , Yanai Nobuaki , Obinata Masuo , Suematsu Makoto , Ishimura Yuzuru
Published: March 2003

To facilitate studies on differentiation of adrenocortical cells and regulation of steroidogenic genes, we established cell lines from adrenals of adult transgenic mice harboring a temperature‐sensitive large T‐antigen gene of simian virus 40. Adrenal glands of the mice exhibited normal cortical zonation including a functionally undifferentiated cell‐layer between the aldosterone‐synthesizing zona glomerulosa cells and the corticosterone‐synthesizing zona fasciculata cells. At a permissive temperature (33 °C), established cell lines AcA201, AcE60 and AcA101 expressed steroidogenic genes encoding steroidogenic factor‐1, cholesterol side‐chain cleavage P450scc, and steroidogenic acute regulatory protein, which are expressed throughout adrenal cortices and gonads. Genes encoding 3β‐hydroxysteroid dehydrogenase and steroid 21‐hydroxylase P450c21, which catalyze the intermediate steps for syntheses of both aldosterone and corticosterone, were inducible in the three cell lines in temperature‐ and/or dibutyryl cAMP‐dependent manners. Notably, these cell lines displayed distinct expression patterns of the steroid 11β‐hydroxylase P45011β gene responsible for the zone‐specific synthesis of corticosterone. AcA201 cells expressed the P45011β gene at 33 °C, showing the property of the zona fasciculata cells, while AcE60 cells expressed it upon a shift to a nonpermissive temperature (39 °C). On the other hand, AcA101 expressed the P45011β gene at 39 °C synergistically with exposure to dibutyryl cAMP. None of these clones express the zona glomerulosa‐specific aldosterone synthase P450aldo gene under the conditions we tested. These results show that AcE60 and AcA101 cells display a pattern of the steroidogenic gene expression similar to that of the undifferentiated cell‐layer and are capable of differentiating into the zona fasciculata‐like cells in vitro.
Proliferating cell nuclear antigen from a basidiomycete, Coprinus cinereus
Authors: Hamada Fumika , Namekawa Satoshi , Kasai Nobuyuki , Nara Takayuki , Kimura Seisuke , Sugawara Fumio , Sakaguchi Kengo
Published: March 2003

The primary purpose of the present study was to investigate whether DNA replication at meiotic prophase also requires replication factors, especially proliferating cell nuclear antigen (PCNA). We cloned PCNA cDNAs (CoPCNA) from a cDNA library made from basidia of the basidiomycete, Coprinus cinereus. Interestingly, although CoPCNA is a single‐copy gene in the genome, two different PCNA cDNA species were isolated using degenerate primers and a meiotic cDNA library, and were designated as CoPCNA‐α and CoPCNA‐β. CoPCNA‐β was made by truncating at specific sites in CoPCNA‐α mRNA, 5′‐AAGAAGGAGAAG‐3′ and 5′‐GAAGAGGAAGAA‐3′. Both of these sequences were present in exon IV in the genomic sequence, and interestingly the former was the same as the inverse sequence of the latter. CoPCNA‐α was 107 amino acids larger than human PCNA, and so the 107 amino‐acid sequence was inserted in a loop, the so‐called D2E2 loop, in human PCNA. Northern blotting analysis indicated that CoPCNA was expressed not only at premeiotic S but also at the meiotic prophase stages such as leptotene and early zygotene, just before and when karyogamy occurs and the homologous chromosomes pair. Western blotting analysis using anti‐(CoPCNA‐α) Ig revealed that at least two CoPCNA mRNAs before and after truncation were translated at the meiotic prophase as CoPCNA‐α and CoPCNA‐β.
Effects of deoxycholate on human colon cancer cells: apoptosis or proliferation
Authors: Milovic V. , Teller I. C. , Faust D. , Caspary W. F. , Stein J.
Published: March 2003

Abstract Background  Deoxycholic acid has long been attributed as a tumour promoter in the colon. It exerts its growth‐related actions in a phorbol ester‐like manner, by stimulating protein kinase C. The aim of this study was to investigate the effect of deoxycholic acid on proliferation and apoptosis in the colon, by exposing colon cancer cells to it in increasing concentrations.

Methods  Human colon cancer cells (Caco‐2 and HT‐29) were treated with deoxycholate or its two structural isomers, 3‐beta‐12‐alpha‐dihydroxy‐5‐beta‐cholan‐24‐oic acid and 3‐alpha‐12‐beta‐dihydroxy‐5‐beta‐cholan‐24‐oic acid. Proliferation was evaluated by cell counting, and apoptosis by estimating percentage cell survival and assessment of nuclear morphology.

Results  Within the concentration range of up to 20 µM, deoxycholate stimulated growth of both human colon cancer cell lines. Its growth‐promoting effect was abolished after inhibition of protein kinase C. At concentrations above 100 µM, deoxycholate induced apoptosis in both cell lines. Epimers of deoxycholate were significantly less potent in stimulating growth.

Conclusion  Low‐dose deoxycholate stimulates colon cancer cell proliferation while > 100 µmol L−1 of this secondary bile acid induces apoptosis in colon cancer cells. Deoxycholate might promote the likelihood of malignant transformation by increasing epithelial cell turnover in the colon.
A new insight into aspirin‐induced asthma
Authors: Jawien J.
Published: March 2003

In about 10% of adult patients with asthma, aspirin and other nonsteroidal anti‐inflammatory drugs precipitate attacks of dyspnoea. Accumulated evidence shows that these reactions are due to the interference of aspirin‐like drugs with arachidonic acid metabolism in the lungs of the sensitive patients: inhibition of cyclooxygenase is accompanied by overproduction of cysteinyl leukotrienes. The mechanisms of these reactions and the characteristic course of aspirin‐induced asthma and its management are discussed.
In vitro determination of active bile acid absorption in small biopsy specimens obtained endoscopically or surgically from the human intestine
Authors: Ung K‐A. , Olofsson G. , Fae A. , Kilander A. , Ohlsson C. , Jonsson O.
Published: March 2003

Abstract Background In the construction of a Kock reservoir for continent urinary diversion, 70 cm of the distal ileum are used. Impaired absorption of bile acids in these patients might cause diarrhoea. Data on the absorption of bile acids in different parts of the human intestine are limited.

Methods Biopsies were taken during endoscopy from the duodenum, the terminal ileum or the right colon, and during surgery 10, 50, 100 and 150 cm proximally to the ileo‐caecal valve using standard endoscopy biopsy forceps. The biopsy specimens were incubated in vitro with radio‐labelled taurocholic acid at 37 °C for 22 or 45 min The radioactivity was determined using the liquid scintillation technique.

Results A linear increase in the uptake was observed, with increased concentrations of taurocholic acid between 100 and 500 µm in all specimens tested, that represented passive uptake or unspecific binding. The active uptake could be calculated from the intercept of the line representing passive uptake with the ordinate. The active uptake in the terminal ileum was 3–4 times greater than 100 cm proximal to the valve.

Conclusions The active absorption of bile acids in humans can be determined in small biopsy specimens taken using standard biopsy forceps during endoscopy or surgery. This method is suitable for clinical studies of bile acid absorption. Active uptake of bile acids not only takes place in the very distal part of the ileum but also to a considerable degree 100 cm proximally to the ileo‐colonic valve. This should be taken into account when selecting the ileal segment for continent urinary diversion.
The effect of anti‐allergic mattress encasings on house dust mite‐induced early‐ and late‐airway reactions in asthmatic patients. A double‐blind, placebo‐controlled study
Authors: Rijssenbeek‐Nouwens L. H. M. , Oosting A. J. , De Monchy J. G. R. , Bregman I. , Postma D. S. , De Bruin‐Weller M. S.
Published: March 2003

Background Anti‐allergic mattress encasing may provide clinical benefit in asthmatic patients. However, the effect of mattress encasings on allergen‐specific parameters, such as bronchial reactions to house dust mite (HDM) challenge, is not clear.

Objective To investigate the effect of anti‐allergic mattress encasings on allergen sensitivity in patients with moderate to severe asthma.

Methods Twenty‐seven patients with asthma and HDM allergy were studied in a double‐blind, placebo‐controlled study. Concentrations of Dermatophagoides pteronyssinus (Der p 1) were measured in mattress dust before and after 1 year of treatment; bronchial histamine challenge, bronchial challenge with HDM and intradermal skin challenges with HDM were performed. The number of eosinophils in peripheral blood was assessed.

Results In the active group, but not in the placebo group, there was a significant reduction in Der p 1 concentration in the dust collected from the mattresses after 1 year of treatment compared to before. There was a significant difference between the groups with respect to HDM‐induced early‐reaction (ER) in the airways and the number of blood eosinophils, which reflected an increase in ER and eosinophils in the placebo group without significant change in the active group. No significant improvement in PC20 histamine, late‐reaction (LR) and skin tests was found in either groups.

Conclusion Our data suggest that encasings protect against a further increase in allergen sensitivity in asthmatic patients, so their use should be recommended.
Ecological impact of large herbivores on the woody vegetation at selected watering points on the eastern basaltic soils in the Kruger National Park
Authors: Brits J. , Van Rooyen M. W. , Van Rooyen N.
Published: March 2003

A lack of quantitative data on the impact of large herbivores on woody vegetation around watering points in the Kruger National Park gave rise to an in depth study. This paper reports on trends found at two artificial watering points on the basaltic soils in the Satara Land System. As a result of the almost complete lack of woody individuals in the immediate vicinity of the watering points, this area could be described as a ‘sacrifice area’. Shrub density increased with distance from the watering point, with the impact of large herbivores on shrub density extending up to 2.8 km. The practice of providing artificial water in troughs at windmills in the Kruger National Park causes the formation of a multiple ‘piosphere’ effect, with the existence of a zone of high utilization in woody vegetation extending far beyond the sacrifice area.
An excursion in the French–Italian Alps: the ‘Atlantic’‐type ophiolite at Mont Chenaillet
Authors: Winterflood Ian
Published: March 2003

In his famous address to the Geological Society of America in 1957, H. H. Read concluded that ‘there are granites and granites’. This is equally true for ophiolites, slices of oceanic lithosphere produced by sea‐floor spreading and preserved by obduction during plate collision. Although they form in similar ways, it is clear that there are different types of ophiolite which originate under different conditions. Compared to the ‘classic’ ophiolites of Oman, many, such as those in the Alps, lack a sheeted dyke complex and were for a long time considered abnormal. Analogues for this type have now been found forming today and they occur when the rate of spreading is slow.
Wing wear, aerodynamics and flight energetics in bumblebees (Bombus terrestris): an experimental study
Authors: Hedenström A. , Ellington C. P. , Wolf T. J.
Published: March 2003

Previous work has shown that wing wear increases mortality rate in bumblebees. Two proximate explanations have been suggested to account for this: increased energy flight costs and increased predation risk due to reduced manoeuvrability.

 Wing wear was mimicked by experimentally clipping the forewing distal trailing edge, causing a 10% wing area reduction. Experimental and sham control bumblebees were induced to hover in a flight respirometry chamber for measuring metabolic rate of hovering. Simultaneous video and sound recordings were taken for wingbeat kinematic data required for an aerodynamic analysis.

 In the experimental group with reduced wing area we measured increased wingbeat frequency, lift coefficient and induced power, but a reduced profile power. The mechanical power output, assuming perfect elastic storage in the flight system, remained largely unchanged after the wing‐trimming treatment.

 Metabolic flight costs (CO2 production rate) did not increase significantly in the reduced wing area group, which is in line with the aerodynamic power output.

 Our results indicate that an increase of flight cost due to wing wear is not a likely explanation for increased mortality rate in bumblebees. Wing wear may, however, affect escape performance from predators.
Interspecific defoliation responses of trees depend on sites of winter nitrogen storage
Authors: Millard P. , Hester A. , Wendler R. , Baillie G.
Published: March 2003

To determine if the response of trees to herbivory is related to their ability to store nitrogen, saplings of Pinus sylvestris L., Betula pendula Roth. and Sorbus aucuparia L. were clipped, either when dormant or at spring bud burst, to remove half the previous year’s shoot growth. The impact of clipping on N remobilization and uptake was quantified in relation to their growth responses.

Pinus sylvestris stored N during the winter in needles grown the previous summer, whereas B. pendula and S. aucuparia used their woody roots and older stems. Therefore, N remobilization was unaffected by clipping the deciduous species, but was reduced by half in the evergreen.

For both P. sylvestris and B. pendula, root uptake contributed N for leaf growth immediately after bud burst, concurrently with remobilization. Sorbus aucuparia had remobilized half the N from storage before any N was taken up by the roots.

Pinus sylvestris, which has a fixed pattern of growth, produced a lower total needle mass when clipped, but individual needles were heavier. Nitrogen uptake during summer was reduced by 26 and 44% for winter‐ and spring‐clipped saplings, respectively. Both deciduous species showed compensatory leaf growth such that, by the end of the summer, leaf mass (and B. pendula leaf area) were unaffected by clipping. Nitrogen uptake by both deciduous species was unaffected by clipping.

We conclude that the site of N storage during winter is a crucial factor in determining the response of a sapling to herbivory, as interactions with the growth patterns of the three species can lead to different responses to damage. We suggest that for evergreen trees, the production of antiherbivory compounds serves primarily to protect N stored in their foliage, rather than the leaves themselves.
Asymmetric competition between plant species
Authors: Freckleton R. P. , Watkinson A. R.
Published: March 2003

Asymmetric competition is an unequal division of resources amongst competing plants. Thus, competition may be asymmetric in the sense that some individuals remove a disproportionately large amount of resource. Alternatively, competition may be asymmetric in that one species removes a disproportionately large amount of resource. The mechanisms determining the two forms of asymmetry may be similar, for example through initial size advantage or over‐topping.

 We explore the consequences of these two forms of asymmetry for competition models that predict mean performance as a function of the density of interacting species. We do so using neighbourhood models that explicitly consider the allocation of resources to individuals within an interacting mixture.

 Asymmetric individual competition is modelled by assuming that individuals are formed into a competitive hierarchy such that individuals at the top of the hierarchy are able to remove more resources than those at the bottom. Mean performance declines exponentially, moving from top to bottom of the hierarchy. Asymmetric species‐level competition is modelled by assuming that one species occupies all of the upper positions in the competitive hierarchy and hence dominates the resource.

 When competition is asymmetric at the species level, yield–density responses follow an exponential decline. Otherwise, arithmetic mean performance follows a classic hyperbolic response.

 Using this approach, we explore the asymmetry of competition between wheat and three species of weeds.
Variation of fuelling rates among sites, days and individuals in migrating passerine birds
Authors: Schaub M. , Jenni L.
Published: March 2003

The seasonal migration of birds is divided into alternating phases of stopover and flight. The fuel deposition rate at stopover sites is the crucial factor determining overall speed of migration and its success. Therefore, field data about the variation in fuel deposition rates at different levels (among sites, seasons, days, individuals) are essential to explain the observed behavioural reactions to environmental variability and migration strategies.

 Fuel deposition rates of four species of passerine migrant birds captured at 14 stopover sites from northern Europe to sub‐Saharan Africa during autumn and spring were analysed. Plasma concentrations of triglycerides and β‐hydroxy‐butyrate were used to estimate relative fuel deposition rates (named fattening index).

 The largest variation in fattening indices was between sites which was only weakly explained by geographical position relative to ecological barriers and did not differ between spring and autumn. Therefore it is expected that the selection of appropriate stopover sites is of paramount importance for the success of migration.

 Variation in fattening index between days within sites was comparatively small with no obvious seasonal trend over the restricted period of mid‐migration and with no correlation between subsequent days. However, only samples during good weather days were examined and the full range of this variation could not be estimated. Air temperature and duration of rainfall could explain part of this variation, probably because invertebrate availability is higher during warm and dry weather.

 The variation of fattening rates between birds on the same day and site was low, and could partially be explained by body mass. Heavy birds had higher fattening rates than light birds.

 From this analysis, it appears to be difficult for migrating birds to predict the fattening rate of the next stopover site, but relatively easy to assess the fattening rate of the current site in a short time. This may result in larger departure fat loads than would be necessary to reach the next stopover site.
Carotenoid access and intraspecific variation in plumage pigmentation in male American Goldfinches (Carduelis tristis) and Northern Cardinals (Cardinalis cardinalis)
Authors: McGRAW K. J. , HILL G. E.
Published: March 2003

Behavioural ecologists have shown much interest recently in the factors that promote variation in carotenoid‐based colour displays. Because vertebrates cannot synthesize the pigments needed to attain this form of bright coloration, most research has focused on the effect of dietary access to pigments on carotenoid ornamentation.

 However, few studies have considered experimentally how the availability of carotenoid pigments in the diet influences variability in the expression of carotenoid‐based coloration.

 Dietary access to carotenoids was manipulated in captive groups of male American Goldfinches (Carduelis tristis L.) and Northern Cardinals (Cardinalis cardinalis L.) to assess the role of pigment acquisition in shaping intraspecific variation in carotenoid‐based plumage pigmentation.

 Substantial variation was detected in the plumage displays of captive male goldfinches and cardinals within a diet treatment, such that birds with unlimited access to the same diet type displayed carotenoid‐based plumage variation that did not differ significantly from that typical of wild males from each species. Additionally, the natural plumage brightness that male goldfinches displayed at capture predicted the degree to which they grew colourful plumage under controlled conditions in our experiments.

 These results suggest that carotenoid‐based plumage variation in male goldfinches and cardinals is less sensitive to pigment availability in the diet than colour variation observed in other studied taxa. Differences in ornamental coloration among males from these two passerine species may instead reflect their ability to utilize ingested pigments physiologically.
Environmentally induced variation in floral traits affects the mating system in Datura wrightii
Authors: Elle E. , Hare J. D.
Published: March 2003

If pollination is unpredictable, selection may favour the production of selfed seeds in the absence of pollen vectors, even in plant species with obvious adaptations for outcrossing. Pollination may be less predictable for plants growing in certain environments if environmental factors affect the floral phenotype. Through effects on flower morphology and the floral display, the environment may affect the outcrossing rate.

We manipulated two environmental factors, water availability and exposure to insect herbivores, in a common‐garden experiment using a perennial herb, Datura wrightii. We measured herkogamy (the separation of anthers and stigmas within flowers), total flower length, and flower number, and used a single‐gene trichome dimorphism as a marker to determine per‐plant outcrossing rates.

The large amount of variation in herkogamy was affected by trichome type, irrigation and herbivory. In addition, watered plants had longer corollas, and plants attacked by herbivores had fewer open flowers. Thus environmental factors affect floral phenotype.

However, irrigation and herbivory did not directly affect outcrossing rate. There were indirect effects of these treatments on outcrossing because plants with increased herkogamy and fewer open flowers had higher outcrossing rates.

  A greenhouse experiment showed that autonomous selfing is more likely when herkogamy is reduced, and can occur both as the flower opens and when the corolla is shed.

  These experiments are among the first to show that within‐population variation in the mating system can be due to environmentally induced variation in floral traits.
A population pharmacokinetic analysis of sildenafil citrate in patients with erectile dysfunction
Authors: Milligan Peter A. , Marshall Scott F. , Karlsson Mats O.
Published: March 2003

Aims  To analyse the pharmacokinetics of sildenafil citrate in patients with erectile dysfunction in order to characterize covariate relationships and assist in the development of rational dosage strategies.

Methods  A population pharmacokinetic sampling strategy was incorporated into five phase III clinical study protocols. Overall, 2077 patients, 1335 of whom received sildenafil, were asked to take an additional dose of study drug before their scheduled clinic visits on four or five occasions throughout the study duration. A single plasma sample was obtained at random times postdose (range 1–7 h), and a total of 4582 samples were assayed (average 3.4 samples per individual).

Results  For the population average patient (age 58 years; aspartate transaminase [AST], 24 IU l−1; weight, 87 kg; not receiving CYP3A4 potential inhibitors), typical values for sildenafil (mean±SE) were 58.5±1.4 l h−1 for apparent clearance (CL/F), 310±6.92 l for volume of distribution (V/F), and 2.6±0.176 h−1 for first‐order absorption constant (ka). The value for ka is associated with meal consumption within 2 h predose, at all other times ka was equivalent to an instantaneous bolus administration. The interindividual variabilities were 29% for CL/F, 20% for V/F, and 210% for ka. Over a dose range of 25–100 mg sildenafil, the pharmacokinetics exhibited dose proportionality. There was evidence of nonproportionality (40% increase on average) in relative bioavailability with respect to the 200‐mg dose (P<0.001) relative to the other doses. Age, AST concentration, and co‐administration with CYP3A4 potential inhibitors significantly influenced CL/F of sildenafil (P<0.001, for each relationship). For age and AST, the extent of the linear relationships (extrapolated from population average values) included a 4% decrease in CL/F for every decade increase and a 6% decrease in CL/F for every 10‐unit increase, respectively. Following co‐administration of CYP3A4 potential inhibitors, a 14% decrease in CL/F was estimated. Only body weight was found to significantly (P<0.001) influence V/F (a 6% increase in V/F for every 10‐kg increase).

Conclusions  The pharmacokinetics of, and covariate influences on, sildenafil in patients with erectile dysfunction were shown to be consistent with those demonstrated in phase I volunteer studies.
The effects of sildenafil on human sperm function in healthy volunteers
Authors: Purvis Ken , Muirhead Gary J. , Harness Jane A.
Published: March 2003

Aims  This double‐blind, randomized, four‐period, two‐way crossover study was conducted to evaluate the acute effects of oral sildenafil (100‐mg single dose) on sperm motility, count, density, morphology and vitality as well as ejaculate volume and viscosity in healthy male subjects. The concentrations of sildenafil and its primary circulating metabolite UK‐103,320 were measured in ejaculate and compared with those in plasma. The study also included assessments of safety and tolerability.

Methods  A total of 17 healthy male volunteers aged 19–34 years were randomized to receive a single 100‐mg dose of sildenafil for two periods and a single dose of placebo for two periods, with each period separated by a minimum of 5–7 days. Sperm and ejaculate properties were evaluated from semen samples taken at screening and 1.5 h after dose. An additional semen sample was collected 4 h after dose, and drug and metabolite concentrations were measured in this sample and the sample taken 1.5 h after dose for comparison with plasma concentrations. Blood samples were collected before each dose and 0.25, 0.5, 1, 2, 3, 4 and 6 h after dose for measurement of sildenafil and metabolite concentrations.

Results  Sildenafil had no statistically significant effect on sperm motility, count or density; the percentage of abnormal sperm forms; or the percentage of living sperm. It also did not affect ejaculate volume or viscosity. All measures were within normal ranges. Sildenafil distributed into the semen rapidly, resulting in significant correlations between concentrations of sildenafil in the semen and total (R2=0.588) or free (R2=0.454) plasma concentrations (P<0.0001). Total semen concentrations of sildenafil were 18% of total plasma concentrations. UK‐103,320 appeared to distribute more slowly from the plasma into the semen, resulting in a lack of correlation between semen and plasma concentrations. The amount of sildenafil and UK‐103,320 in the ejaculate was small (<2×10−4% of the administered dose at 1.5 h). Sildenafil was well tolerated; no patient withdrew from the study due to adverse events attributed to sildenafil.

Conclusions  These results indicate that a single 100‐mg oral dose of sildenafil does not have an adverse effect on sperm function or ejaculate quality.
The regulation of medical devices and the role of the Medical Devices Agency
Authors: Jefferys D. B.
Published: March 2003

This article reviews the regulation of medical devices in the UK and Europe and compares the regulatory regime with that for pharmaceuticals. The regulation of devices follows the ‘New Approach’ policy of the EC Commission and involves more self‐regulation and conformity assessment. The controls are relatively recent beginning in 1993 for Active Implantable Devices and concluding with the In Vitro Diagnostic Directive implemented in June 2000. The article describes how the directives have been implemented in the UK, the role of the Notified Bodies and the role of the Medical Device Agency (MDA) as the competent authority. In particular the Agency's compliance and standards work is described along with the strategy and post marketing surveillance and adverse incident scheme. The MDA is a key international device regulatory agency and its international role is discussed. So too is its device evaluation programme for the NHS and how this complements the work of NICE. The article also considers the future direction of the MDA and changes in the device sector.
Influence of β‐adrenoceptor antagonists on the pharmacokinetics of rizatriptan, a 5‐HT1B/1D agonist: differential effects of propranolol, nadolol and metoprolol
Authors: Goldberg Michael R. , Sciberras David , De Smet Marina , Lowry Richard , Tomasko Lisa , Lee Yih , Olah Timothy V. , Zhao Jamie , Vyas Kamlesh P. , Halpin Rita , Kari Prasad H. , James Ian
Published: March 2003

Aims  Patients with migraine may receive the 5‐HT1B/1D agonist, rizatriptan (5 or 10 mg), to control acute attacks. Patients with frequent attacks may also receive propranolol or other β‐adrenoceptor antagonists for migraine prophylaxis. The present studies investigated the potential for pharmacokinetic or pharmacodynamic interaction between β‐adrenoceptor blockers and rizatriptan.

Methods  Four double‐blind, placebo‐controlled, randomized crossover investigations were performed in a total of 51 healthy subjects. A single 10 mg dose of rizatriptan was administered after 7 days' administration of propranolol (60 and 120 mg twice daily), nadolol (80 mg twice daily), metoprolol (100 mg twice daily) or placebo. Rizatriptan pharmacokinetics were assessed. In vitro incubations of rizatriptan and sumatriptan with various β‐adrenoceptor blockers were performed in human S9 fraction. Production of the indole‐acetic acid‐MAO‐A metabolite of each triptan was measured.

Results  Administration of rizatriptan during propranolol treatment (120 mg twice daily for 7.5 days) increased the AUC(0,∞) for rizatriptan by approximately 67% and the Cmax by approximately 75%. A reduction in the dose of propranolol (60 mg twice daily) and/or the incorporation of a delay (1 or 2 h) between propranolol and rizatriptan administration did not produce a statistically significant change in the effect of propranolol on rizatriptan pharmacokinetics. Administration of rizatriptan together with nadolol (80 mg twice daily) or metoprolol (100 mg twice daily) for 7 days did not significantly alter the pharmacokinetics of rizatriptan. No untoward adverse experiences attributable to the pharmacokinetic interaction between propranolol and rizatriptan were observed, and no subjects developed serious clinical, laboratory, or other significant adverse experiences during coadministration of rizatriptan with any of the β‐adrenoceptor blockers. In vitro incubations showed that propranolol, but not other β‐adrenoceptor blockers significantly inhibited the production of the indole‐acetic acid metabolite of rizatriptan and sumatriptan.

Conclusions  These results suggest that propranolol increases plasma concentrations of rizatriptan by inhibiting monoamine oxidase‐A. When prescribing rizatriptan to migraine patients receiving propranolol for prophylaxis, the 5 mg dose of rizatriptan is recommended. Administration with other β‐adrenoceptor blockers does not require consideration of a dose adjustment.
Use of selective serotonin reuptake inhibitors and risk of developing first‐time acute myocardial infarction
Authors: Meier Christoph R. , Schlienger Raymond G. , Jick Hershel
Published: March 2003

Aims  Selective serotonin reuptake inhibitors (SSRIs) have been associated with serotonin depletion in platelets, potentially leading to abnormal aggregation and prolonged bleeding time. In view of the importance of serotonin in coronary thrombosis, and decreased platelet serotonin concentrations associated with SSRIs, the present study was performed to test the hypothesis of a decreased risk of acute myocardial infarction (AMI) associated with SSRIs.

Methods  We conducted a population‐based case‐control analysis using the UK General Practice Research Database (GPRD). A total of 3319 patients aged 75 years or younger free of clinical conditions predisposing to ischaemic heart disease, with a first‐time diagnosis of AMI between 1992 and 1997, and 13 139 controls without AMI matched to cases for age, sex, general practice attended, and calendar time were included. Conditional logistic regression was used to estimate relative risks.

Results  Adjusted odds ratios (with 95% CI) for current use of SSRIs, non‐SSRIs, or other antidepressants, compared to the group of nonusers of antidepressants were 0.9 (95% CI 0.5,1.8), 0.9 (95% CI 0.7,1.2), and 1.3 (95% CI 0.6,2.8), respectively. As compared with nonuse of SSRIs, current use (regardless of any other antidepressants used) resulted in an adjusted OR of 1.1 (95% CI 0.7,1.6).

Conclusions  The current analysis provides evidence that SSRI exposure does not substantially decrease the risk of developing first‐time AMI in patients free of factors predisposing to ischaemic heart disease. However, due to relatively small numbers of exposed subjects and the resulting wide confidence intervals, further studies may be needed to document a lack of effect of SSRIs in subjects without pre‐existing diseases predisposing to AMI.
Effects of adrenomedullin on angiotensin II stimulated atrial natriuretic peptide and arginine vasopressin secretion in healthy humans
Authors: Petrie Mark C. , McDonald John E. , Hillier Chris , Morton James J. , McMurray John J. V.
Published: March 2003

Aims  Adrenomedullin is a newly described peptide that has widespread tissue distribution. Its presence in cardiovascular (including vascular endothelial cells, smooth muscle cells, and cardiac atria and ventricles) and renal tissues, together with its vasodilatory and natriuretic properties, suggest a role in blood pressure regulation and fluid and electrolyte balance.

Methods
Nine normal volunteers were studied to determine whether or not adrenomedullin influenced plasma atrial natriuretic peptide and arginine vasopressin concentrations during systemic angiotensin II infusion.

Results
A significant (P = 0.02) augmentation of atrial natriuretic peptide concentrations, but no suppression of arginine vasopressin concentrations, was found with coinfusion of adrenomedullin and angiotensin II when compared with vehicle and angiotensin II.

Conclusions
Despite its vasodilator and natriuretic action, adrenomedullin significantly augmented angiotensin II‐stimulated plasma atrial natriuretic peptide concentrations in healthy humans. This provides further evidence of a synergistic interaction between adrenomedullin and atrial natriuretic peptide and suggests that adrenomedullin may have a role in fluid and electrolyte balance and blood pressure regulation.
Recovery of platelet function after discontinuation of clopidogrel treatment in healthy volunteers
Authors: Weber Artur‐Aron , Braun Marina , Hohlfeld Thomas , Schwippert Barbara , Tschöpe Diethelm , Schrör Karsten
Published: March 2003

Aims  To study the recovery of platelet function after discontinuation of clopidogrel treatment in healthy volunteers.

Methods  Ten healthy volunteers were treated with clopidogrel (75 mg day−1) for 7 days. CD62P expression and PAC‐1 binding were measured by flow cytometry.

Results  Adenosine diphosphate (ADP, 30 µM)‐induced platelet responses were almost completely inhibited by clopidogrel. After discontinuation of the drug, platelet function gradually increased and complete recovery was seen 7 days after the last clopidogrel dose. The mean difference (95% CI) for ADP‐induced PAC‐1 binding (fluorescence intensity) between baseline and 7 days after the last dose was 0.01 (0.61, −0.59). Single cell analysis provides direct evidence for an irreversible mode of action of clopidogrel.

Conclusions  This is the first report to directly demonstrate irreversibility of clopidogrel action in humans.
Genetic polymorphism of cytochrome P450 2C9 in a Caucasian and a black African population
Authors: Scordo Maria Gabriella , Aklillu Eleni , Yasar, Umit , Dahl Marja‐Liisa , Spina Edoardo , Ingelman‐Sundberg Magnus
Published: March 2003

Aims  CYP2C9 is a major enzyme in human drug metabolism and the polymorphism observed in the corresponding gene may affect the therapeutic outcome during treatment with several drugs. The distribution of variant CYP2C9 alleles was therefore investigated in an Italian and an Ethiopian population.

Methods  Allele‐specific PCR analysis was carried out in order to determine the frequencies of the two most common variant alleles, CYP2C9*2 and CYP2C9*3 in genomic DNA isolated from 157 Italians and 150 Ethiopians.

Results  The frequencies of CYP2C9*1 (80%), CYP2C9*2 (11%) and CYP2C9*3 (9%) found in the Italian population were similar to other Caucasian groups. However in the Ethiopian population CYP2C9*1, CYP2C9*2 and CYP2C9*3 were present at a frequency of 94, 4 and 2% respectively. The 95% confidence intervals in CYP2C9*1, CYP2C9*2 and CYP2C9*3 between Italians and Ethiopians were 0.098, 0.176, 0.040, 0.098 and 0.040, 0.098, respectively.

Conclusions  Our results indicate that the Ethiopian population has a unique relative distribution of the CYP2C9 alleles, which is not similar to any other ethnic group hitherto described.
Candidate gene case‐control association studies: advantages and potential pitfalls
Authors: Daly Ann K. , Day Christopher P.
Published: March 2003

There is increasing information on the importance of genetic polymorphisms in human genes. Polymorphisms occur on average once every 500–1000 base pairs in the human genome and are useful in the identification of genes involved in human disease. Some genetic polymorphisms have functionally significant effects on the gene product and are the most useful type of polymorphism in disease association studies while others are simply useful markers. There are two main approaches using polymorphisms in the identification of genes involved in polygenic diseases. The first involves examining inheritance patterns for genetic polymorphisms in family studies and the second case‐control studies which compare genotype frequencies for candidate disease genes in unrelated individuals with the disease and healthy controls. Use of family studies is generally the preferred approach but this is only feasible if the genetic component of the disease is relatively strong, DNA samples are available from other family members and the disease is relatively easy to diagnose and is not stigmatized. Population case‐control studies are useful both as an alternative and an adjunct to family studies. When performing case‐control studies factors such as study design, methods for recruitment of cases and controls, functional significance of polymorphisms chosen for study and statistical analysis of data require close attention to ensure that only genuine associations are detected. To illustrate some potential problems in the design and interpretation of association studies, some specific examples of association studies on drug response and on disease susceptibility involving receptor genes, cytochrome P450 and other xenobiotic metabolizing enzyme genes and immune system genes including TNF‐α, IL‐10 and the IL‐4 receptor are discussed.
The effects of rifampicin on the pharmacokinetics and pharmacodynamics of orally administered nilvadipine to healthy subjects
Authors: Saima S. , Furuie K. , Yoshimoto H. , Fukuda J. , Hayashi T. , Echizen H.
Published: March 2003

Aims  To study the effects of rifampicin on the pharmacokinetics and pharmacodynamics of nilvadipine.

Methods  Five healthy adult volunteers received nilvadipine (4 mg) orally before and after a 6 day treatment with rifampicin. Blood and urine were collected and assayed for plasma nilvadipine and urinary 6β‐hydroxycortisol and cortisol.

Results  The treatment with rifampicin reduced the mean (± s.d.) AUC of nilvadipine from 17.4 ± 8.4 to 0.6 ± 0.4 µg l−1 h (mean difference −16.8 µg l−1 h, 95% CI −9.4, 24.2 µg l−1 h). While the administration of nilvadipine alone elicited a significant (P < 0.05) hypotensive (mean difference for diastolic blood pressure –8 mmHg, 95% CI −4, −12 mmHg) and reflex tachycardia (mean difference 5 beats min−1, 95% CI 1, 9 beats min−1), the treatment with rifampicin abolished these responses. The urinary 6β‐hydroxycortisol/cortisol ratio showed a significant (P < 0.05) increase from 10.3 ± 4.0 to 50.3 ± 24.6 by rifampicin: mean difference 40.1, 95% CI 20.4, 59.8.

Conclusions  Because rifampicin may greatly decrease the oral bioavailability of nilvadipine, caution is needed when these two drugs are to be coadministered.