The contribution of a pollinating seed predator to selection on Silene latifolia females.

Interactions, antagonistic or mutualistic, can exert selection on plant traits. We explored the role of Hadena bicruris, a pollinating seed predator, as a selective agent on its host, the dioecious plant Silene latifolia. We exposed females from artificial-selection lines (many, small flowers (SF) vs. few, large flowers (LF)) to this moth. Infestation did not differ significantly between lines, but the odds of attacked fruits aborting were higher in SF females. We partitioned selection between that caused by moth attack and that resulting from all other factors. In both lines, selection via moth attack for fewer, smaller flowers contrasted with selection via other factors for more flowers. In LF females, selection via the two components was strongest and selection via moth attack also favoured increased fruit abortion. This suggests that the moths act as more of a selective force on flower size and number via their predating than their pollinating role.

Fine-scale spatial genetic structure and gene dispersal in Silene latifolia.

Plants are sessile organisms, often characterized by limited dispersal. Seeds and pollen are the critical stages for gene flow. Here we investigate spatial genetic structure, gene dispersal and the relative contribution of pollen vs seed in the movement of genes in a stable metapopulation of the white campion Silene latifolia within its native range. This short-lived perennial plant is dioecious, has gravity-dispersed seeds and moth-mediated pollination. Direct measures of pollen dispersal suggested that large populations receive more pollen than small isolated populations and that most gene flow occurs within tens of meters. However, these studies were performed in the newly colonized range (North America) where the specialist pollinator is absent. In the native range (Europe), gene dispersal could fall on a different spatial scale. We genotyped 258 individuals from large and small (15) subpopulations along a 60 km, elongated metapopulation in Europe using six highly variable microsatellite markers, two X-linked and four autosomal. We found substantial genetic differentiation among subpopulations (global F(ST)=0.11) and a general pattern of isolation by distance over the whole sampled area. Spatial autocorrelation revealed high relatedness among neighboring individuals over hundreds of meters. Estimates of gene dispersal revealed gene flow at the scale of tens of meters (5-30 m), similar to the newly colonized range. Contrary to expectations, estimates of dispersal based on X and autosomal markers showed very similar ranges, suggesting similar levels of pollen and seed dispersal. This may be explained by stochastic events of extensive seed dispersal in this area and limited pollen dispersal.

Evidence for inbreeding depression and post-pollination selection against inbreeding in the dioecious plant Silene latifolia.

In many species, inbred individuals have reduced fitness. In plants with limited pollen and seed dispersal, post-pollination selection may reduce biparental inbreeding, but knowledge on the prevalence and importance of pollen competition or post-pollination selection after non-self pollination is scarce. We tested whether post-pollination selection favours less related pollen donors and reduces inbreeding in the dioecious plant Silene latifolia. We crossed 20 plants with pollen from a sibling and an unrelated male, and with a mix of both. We found significant inbreeding depression on vegetative growth, age at first flowering and total fitness (22% in males and 14% in females). In mixed pollinations, the unrelated male sired on average 57% of the offspring. The greater the paternity share of the unrelated sire, the larger the difference in relatedness of the two males to the female. The effect of genetic similarity on paternity is consistent with predictions for post-pollination selection, although paternity, at least in some crosses, may be affected by additional factors. Our data show that in plant systems with inbreeding depression, such as S. latifolia, pollen or embryo selection after multiple-donor pollination may indeed reduce inbreeding.

Silene as a model system in ecology and evolution.

The genus Silene, studied by Darwin, Mendel and other early scientists, is re-emerging as a system for studying interrelated questions in ecology, evolution and developmental biology. These questions include sex chromosome evolution, epigenetic control of sex expression, genomic conflict and speciation. Its well-studied interactions with the pathogen Microbotryum has made Silene a model for the evolution and dynamics of disease in natural systems, and its interactions with herbivores have increased our understanding of multi-trophic ecological processes and the evolution of invasiveness. Molecular tools are now providing new approaches to many of these classical yet unresolved problems, and new progress is being made through combining phylogenetic, genomic and molecular evolutionary studies with ecological and phenotypic data.

Should food-deceptive species flower before or after rewarding species? An experimental test of pollinator visitation behaviour under contrasting phenologies.

Many plant species reward their pollinators, whereas some species, particularly among orchids, do not. Similarity of floral cues between co-flowering species influences how rapidly pollinators learn to avoid deceptive plants. This learning process, which affects the reproductive success of deceptive plants, may additionally depend on relative timing of flowering of sympatric rewarding and deceptive species. We tested the combined effects of corolla colour similarity and flowering order of rewarding and deceptive artificial inflorescences on visitation by naïve bumblebees. When deceptive inflorescences were offered after rewarding inflorescences, bumblebees visited them four times more often if both species were similar compared with when they were dissimilar. Pollinator visitation rate to deceptive inflorescences offered before rewarding inflorescences was intermediate and independent of similarity. Thus, early-flowering deceptive species avoid the costs of dissimilarity with rewarding species. This mechanism may favour adaptive evolution of flowering phenology in deceptive species and explain why temperate deceptive orchids usually flower earlier than rewarding ones.

Determination of memantine in human plasma by liquid chromatography-electrospray tandem mass spectrometry: application to a bioequivalence study.

A simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of memantine (I) in human plasma is presented. Sample preparation consisted of the addition of amantadine (II) as internal standard (IS), liquid-liquid extraction in basic conditions using a mixture of diethyl ether-chloroform (7:3, v/v) as extracting solvent, followed by centrifugation, solvent evaporation and sample reconstitution in methanol. Both I and II (internal standard) were analyzed using a C18 column and a mobile phase composed of methanol-water-formic acid (80:20:0.1, v/v/v). Eluted compounds were monitored using positive mode electrospray (ES) tandem mass spectrometry. The analyses were carried out by selected reaction monitoring (SRM) using the parent to daughter combinations of m/z 180>163 (memantine) and m/z 152>135 (amantadine). The peak areas from the analyte and IS were used for quantification of I. The achieved limit of quantification (LOQ) was 0.1 ng/mL; the assay exhibited a linear dynamic range of 0.1-50.0 ng/mL with a determination coefficient (r2) of at least 0.98. Validation results on linearity, specificity, accuracy, precision and stability, as well as on application to the analysis of samples taken up to 320 h after oral administration of 20mg (two 10mg capsules) of I in healthy volunteers demonstrated the applicability to bioequivalence studies.

Effect of Helicobacter pylori infection and acid blockade by lansoprazole on clarithromycin bioavailability.

The effect of proton pump inhibitors and Helicobacter pylori infection on the bioavailability of antibiotics is poorly understood. We determined the effects of 5-day oral administration of 60 mg lansoprazole on the bioavailability of clarithromycin in individuals with and without H. pylori infection. Thirteen H. pylori-infected and 10 non-infected healthy volunteers were enrolled in a study with an open-randomized two-period crossover design and a 21-day washout period between phases. Plasma concentrations of clarithromycin in subjects with and without lansoprazole pre-treatment were measured by liquid chromatography coupled to a tandem mass spectrometer. Clarithromycin Cmax and AUC0-10 h were significantly reduced after lansoprazole administration. In addition, lansoprazole treatment of the H. pylori-positive group resulted in a statistically significant greater reduction in Cmax (40 vs 15%) and AUC0-10 h (30 vs 10%) compared to lansoprazole-treated H. pylori-negative subjects. Thus, treatment with lansoprazole for 5 days reduced bioavailability of clarithromycin, irrespective of H. pylori status. This reduction, however, was even more pronounced in H. pylori-infected individuals.

Validated method for determination of bromopride in human plasma by liquid chromatography--electrospray tandem mass spectrometry: application to the bioequivalence study.

A simple, sensitive and specific liquid chromatography-tandem mass spectrometry method for the quantification of bromopride I in human plasma is presented. Sample preparation consisted of the addition of procainamide II as the internal standard, liquid-liquid extraction in alkaline conditions using hexane-ethyl acetate (1 : 1, v/v) as the extracting solvent, followed by centrifugation, evaporation of the solvent and sample reconstitution in acetonitrile. Both I and II (internal standard, IS) were analyzed using a C18 column and the mobile-phase acetonitrile-water (formic acid 0.1%). The eluted compounds were monitored using electrospray tandem mass spectrometry. The analyses were carried out by multiple reaction monitoring (MRM) using the parent-to-daughter combinations of m/z 344.20 > 271.00 and m/z 236.30 > 163.10. The areas of peaks from analyte and IS were used for quantification of I. The achieved limit of quantification was 1.0 ng/ml and the assay exhibited a linear dynamic range of 1-100.0 ng/ml and gave a correlation coefficient (r) of 0.995 or better. Validation results on linearity, specificity, accuracy, precision and stability, as well as application to the analysis of samples taken up to 24 h after oral administration of 10 mg of I in healthy volunteers demonstrated the applicability to bioequivalence studies.

Validation of a selective method for determination of paroxetine in human plasma by LC-MS/MS.

PURPOSE: A sensitive, robust, and selective liquid chromatographic-tandem mass spectrometric method (LC-MS/MS) was developed and validated for paroxetine quantification in human EDTA plasma. METHODS: Sample preparation was based on liquid-liquid extraction using a mixture of ethyl acetate/hexane (50/50; v/v) to extract the drug and internal standard from plasma. Chromatography was performed on a C-18 analytical column and the retention times were 1.6 and 1.7 for paroxetine and fluoxetine (IS), respectively. The ionization was optimized using ESI(+) and selectivity was achieved by tandem mass spectrometric analysis using MRM functions, 330.0 --> 70.0 and 310 --> 43.9 for paroxetine and fluoxetine. RESULTS: Analytical curve ranged from 0.2 to 20.0 ng/mL. Inter-day precision and accuracy of the quality control (QC) samples were < 15% relative standard deviation (RSD). Analyte stability during sampling processing and storage were established. CONCLUSION: Validation results on linearity, specificity, accuracy, precision as well as application to the analysis of samples taken up to 120 h after oral administration of 20 mg of paroxetine in 28 healthy volunteers were found to be of good performance in bioequivalence study.

Plasma hydroxy-metronidazole/metronidazole ratio in hepatitis C virus-induced liver disease.

It has been suggested that the measurement of metronidazole clearance is a sensitive method for evaluating liver function. The aim of this study was to evaluate the usefulness of plasma hydroxy-metronidazole/metronidazole ratios as indicators of dynamic liver function to detect changes resulting from the various forms of chronic hepatitis C virus (HCV) infection. A total of 139 individuals were studied: 14 healthy volunteers, 22 healthy, asymptomatic, consecutive anti-HCV-positive HCV-RNA negative subjects, 81 patients with chronic hepatitis C (49 with moderate/severe chronic hepatitis and 34 with mild hepatitis), and 20 patients with cirrhosis of the liver. HCV status was determined by the polymerase chain reaction. Plasma concentrations of metronidazole and its hydroxy-metabolite were measured by reverse-phase high-performance liquid chromatography with ultraviolet detection in a blood sample collected 10 min after the end of a metronidazole infusion. Anti-HCV-positive HCV-RNA-negative individuals demonstrated a significantly reduced capacity to metabolize intravenously infused metronidazole compared to healthy individuals (0.0478 +/- 0.0044 vs 0.0742 +/- 0.0232). Liver cirrhosis patients also had a reduced plasma hydroxy-metronidazole/metronidazole ratio when compared to the other groups of anti-HCV-positive individuals (0.0300 +/- 0.0032 vs 0.0438 +/- 0.0027 (moderate/severe chronic hepatitis) vs 0.0455 +/- 0.0026 (mild chronic hepatitis) and vs 0.0478 +/- 0.0044 (anti-HCV-positive, HCV-RNA-negative individuals)). These results suggest an impairment of the metronidazole metabolizing system induced by HCV infection that lasts after viral clearance. In those patients with chronic hepatitis C, this impairment is paralleled by progression of the disease to liver cirrhosis.

DSM-III diagnoses associated with dyspepsia of unknown cause.

The frequencies of DSM-III diagnoses in 30 patients with dyspepsia of unknown origin and 20 patients with organic dyspepsia were compared. Dyspepsia of unknown origin was associated with a higher prevalence of psychiatric diagnoses (86.7% versus 25.0%), particularly anxiety disturbances (66.7%).

Amoxicillin and ampicillin are not transferred to gastric juice irrespective of Helicobacter pylori status or acid blockade by omeprazole.

BACKGROUND: The effects of proton pump inhibitors and Helicobacter pylori infection on the distribution of drugs used for the eradication of the bacteria are poorly understood. AIM: The aim of this study was to investigate the effects of a 7-day administration of 20 mg of omeprazole on the pharmacokinetics of amoxicillin and ampicillin in the plasma, saliva and gastric juice of individuals with and without H. pylori infection. METHODS: Fifty-four healthy volunteers without endoscopic lesions were enrolled. Twenty-six volunteers were included in the amoxicillin study and 28 individuals in the ampicillin study. Each study had an open randomized two-period crossover design and a 21-day washout period between phases. Plasma, saliva and gastric juice concentrations of amoxicillin and ampicillin in subjects with and without omeprazole pre-treatment were measured by reversed-phase HPLC using UV detection. RESULTS: Neither pre-treatment with omeprazole nor H. pylori infection interfered with the plasma bioavailability of amoxicillin or ampicillin, as assessed by the AUC0-2 h. Neither ampicillin nor amoxicillin were detected in saliva or gastric juice in any study phase. CONCLUSION: Short-term treatment with omeprazole does not interfere with the pharmacokinetics of amoxicillin or ampicillin. Our results also exclude the presence of a transfer mechanism for amoxicillin or ampicillin from the plasma to the gastric lumen.

Octreotide 24-h prophylaxis in patients at high risk for post-ERCP pancreatitis: results of a multicenter, randomized, controlled trial.

BACKGROUND: Pharmacological prophylaxis of post-ERCP pancreatitis is costly and not useful in most non-selected patients, in whom the incidence of pancreatitis is 5% or less. However, it could be useful and probably cost-effective, in patients at high risk for this complication, where the post-procedure pancreatitis rate is 10% and more. AIM: To assess the efficacy of octreotide in reducing the incidence and severity of post-ERCP pancreatitis and procedure-related hospital stay, in subjects with known patient-related risk factors. METHODS: A total of 120 patients were randomly allocated to receive octreotide or not, in a multicentre, randomized, controlled trial. The drug was given subcutaneously, 200 microg t.d.s., starting 24 h before the ERCP procedure, in patients with either sphincter of Oddi dysfunction, or a history of relapsing pancreatitis or post-ERCP pancreatitis, or who were aged under 35 years, or who had a small common bile duct diameter (< 8 mm). RESULTS: A total of 114 patients (58 in the octreotide group and 56 in the control group) completed the trial. Post-procedure pancreatitis occurred in seven octreotide-treated patients (12.0%) and eight controls (14.3%). The two groups showed no significant differences in the incidence or severity of pancreatitis. Twenty-four hours after the procedure, severe hyperamylasemia (more than five times the upper normal limit) without pancreatic-like pain was recorded in three octreotide-treated patients (5.2%) and six controls (10.7%), the difference being not significant. CONCLUSION: Twenty-four-hour prophylaxis with octreotide proved ineffective in preventing post-ERCP pancreatitis and in avoiding 24-h severe hyperamylasemia in high-risk patients.

Female polyandry affects their sons' reproductive success in the red flour beetle Tribolium castaneum.

A potential benefit to females mating with multiple males is the increased probability that their sons will inherit traits enhancing their pre- or post-mating ability to obtain fertilizations. We allowed red flour beetle (Tribolium castaneum) females to mate on three consecutive days either repeatedly to the same male or to three different males. This procedure was carried out in 20 replicate lines, 10 established with wild-type, and 10 with the Chicago black morph, a partially dominant phenotypic marker. The paternity achieved by the sons of females from polyandrous vs. monandrous lines of contrasting morph was assessed in the F1, F2 and F3 generation by mating wild-type stock females to two experimental males and assigning the progeny to either sire based on phenotype. The sons of polyandrous wild-type females achieved significantly higher paternity when mating in the second male role than the sons of monandrous wild-type females. By contrast, when mating in the first male role, males produced by females from polyandrous lines tended to have lower paternity than males from monandrous lines. Both effects were independent of the number of mates of the black competitor's mother, and interacted significantly with the number of progeny laid by the female. These results provide the first evidence that manipulating the number of mates of a female can influence her sons' mating success and suggest a potential trade-off between offence and defence in this species.

Radial artery catheterization by surgical exposure in infants.

56 radial arteries were cannulated by cutdown technique in 54 critically ill infants (age (mean +/- SD) 12.2 +/- 25.9 days, weight 2710 +/- 910 g) for continuous pressure monitoring and repeated blood sampling. 30 patients survived their illness, 24 died. The mean catheterization time in the survivors was 8.1 +/- 4.1 in the non-survivors 6.9 +/- 5.6 days. Most cannulae were removed electively. 27 of the surviving infants were followed up after 10.5 +/- 5.1 months. 25 (86%) vessels were patent, 4 (14%) occluded. No trophic changes of hands or fingers were noted. We concluded that radial artery cannulation by cutdown in infants is a quick and safe technique and is suitable in patients with severe peripheral vasoconstriction.

Presence of calcified tissue in the human temporomandibular joint disc.

Large calcified areas were observed in the articular discs of the temporomandibular joint from five patients suffering from articular dysfunctions. The calcified regions were always located inside the fibrous tissue of the discs. They had a woven bone-like morphological pattern and consisted of a compact mineralized tissue containing cells in irregular lacunae. In all the samples the calcified tissue was completely surrounded by a mineralizing border rich in cells and variously arranged collagen fibrils. Energy-dispersive spectrometry showed that mineralized regions contained large amounts of Ca and P. X-ray powder diffraction identified the crystals in these areas as hydroxyapatite.

A microscopic and immunocytochemical study of structural changes in dysfunctional human temporomandibular joint discs.

The central part of 12 articular discs from patients with serious alterations in function of the temporomandibular joint were investigated. The control discs were removed at autopsy from individuals who did not have any such functional defects. The anomalous discs had an increased cellular component; fibrocytes, fibroblasts, numerous myofibroblasts and, less frequently, smooth muscle cells and mast cells were present. The myofibroblasts, which had numerous bundles of thin filaments in the cytoplasm, were positive to the immunocytochemical reaction with antibody to alpha-smooth muscle actin. In one disc only a large part had changed into a mostly fatty tissue. In the majority of the altered discs, the part examined, which usually is neither vascularized nor innervated, was characterized by the presence of numerous blood vessels. Besides the capillary network several larger vessels were present. In one disc, several myelinated and unmyelinated fibres, isolated or in nerve bundles, were also seen. These observations show that the disc fibrous tissue may undergo deep structural modifications that appear to indicate not only a capacity for repair but also an ability to adapt to new functional conditions.

Expression of vascular endothelial growth factor in human dysfunctional temporomandibular joint discs.

A high density of blood vessels is found in specimens of temporomandibular joint (TMJ) disc at any stage of internal derangement of the joint, but the factors responsible for angiogenesis in the disc have not been described. The purpose here was to investigate, in human TMJ discs, the expression of vascular endothelial growth factor (VEGF), a multifunctional cytokine that contributes to angiogenesis. Specimens, free of significant morphological alterations and with varying degrees of disc tissue degeneration/regeneration, were studied by immunohistochemistry for VEGF in order to correlate immunohistochemical with histopathological findings. In normal discs and discs with minor pathological changes, fibroblast-like cells, fibrochondrocytes and chondrocyte-like cells were either not or only weakly immunostained by VEGF antibody. In disc specimens from internal derangement of the TMJ with significant tissue degeneration/regeneration, VEGF was consistently expressed. In these specimens, immunoreaction products for VEGF were observed both in the disc and in the endothelial cells of newly formed vessels. This VEGF immunolocalization is consistent with the stimulation of angiogenesis and the morphogenesis and differentiation of chondrocytes. Therefore VEGF expression by disc chondrocyte-like cells might reflect the action of the cytokine as an inducer of angiogenesis and as an autocrine signal for cells of the chondrogenic lineage.

Cellular S-100 protein immunostaining in human dysfunctional temporomandibular joint discs.

S-100 protein was detected immunohistochemically in diseased human temporomandibular joint discs with different degrees of pathology, and the findings compared with those of normal discs. In normal discs, large nerve trunks in the posterior ligament were strongly stained by anti-S-100 antiserum; the very few chondrocyte-like cells sometimes showed faint staining, while no staining was observed in any fibrochondrocyte-like or fibroblast-like cell. In dysfunctional discs, S-100 protein immunostaining seemed to correlate with structural pathological findings. The discs showing an abnormal collagen arrangement or fragmentation of collagen fibres presented overall the same immunolabelling pattern as normal discs. In discs with fibrocartilaginous metaplasia and dystrophic cartilage formation, fibrochondrocyte cells showed a very strong immunoreaction for S-100 protein and fibroblast-like cells in some instances were also positive. These findings suggest that S-100 upregulation in disc cells can be considered an attempt at tissue repair by chondroid metaplasia following an injury in that it enables fibroblast-like cells and fibrochondrocytes to acquire a chondrogenic phenotype.

Immunolocalization of gelatinase-A (matrix metalloproteinase-2) in damaged human temporomandibular joint discs.

The fibrous tissue of the articular disc of the dysfunctional temporomandibular joint undergoes deep and variable structural modifications. Here the concurrence of morphological changes and the expression of matrix metalloproteinase-2 (MMP-2) in damaged discs from individuals suffering joint dysfunction was investigated. Microscopic, ultrastructural and immunocytochemical investigations were made on variously damaged articular discs and on one control sample. Disaggregation of collagen fibres, an increase in cellular components and calcification of large areas of tissue were observed in the damaged discs. These modifications were accompanied by a positive immunoreaction pattern for MMP-2. Fibroblast-, chondroblast- and osteoblast-like cells displayed a positive cytoplasmic reaction. In samples displaying evidence of synovial hyperplasia, some cells of the synovial protrusions were MMP-2 immunoreactive. No MMP-2 staining was observed in the control sample. These findings demonstrate that structural modifications of the articular disc could be specific responses to changes in the function of the temporomandibular joint. Variations in extrinsic stimuli may activate intrinsic factors, such as MMPs, that induce structural modifications in the discal tissue.