High-Throughput Fluorescence Polarization Assay to Identify Ligands Using Purified G Protein-Coupled Receptor.

The development of cell-free high-throughput (HT) methods to screen and select novel lead compounds remains one of the key challenges in G protein-coupled receptor (GPCR) drug discovery. Mutational approaches have allowed the stabilization of GPCRs in a purified and ligand-free state. The increased intramolecular stability overcomes two major drawbacks for usage in in vitro screening, the low receptor density on cells and the low stability in micelles. Here, an HT fluorescence polarization (FP) assay for the neurotensin receptor type 1 (NTS1) was developed. The assay operates in a 384-well format and is tolerant to DMSO. From a library screen of 1272 compounds, 12 (~1%) were identified as primary hits. These compounds were validated in orthogonal assay formats using surface plasmon resonance (SPR), which confirmed binding of seven compounds (0.6%). One of these compounds showed a clear preference for the orthosteric binding pocket with submicromolar affinity. A second compound revealed binding at a nonorthosteric binding region and showed specific biological activity on NTS1-expressing cells. A search of analogs led to further enhancement of affinity, but at the expense of activity. The identification of GPCR ligands in a cell-free assay should allow the expansion of GPCR pharmaceuticals with antagonistic or agonistic activity.

Effectiveness of scoliosis-specific exercises for adolescent idiopathic scoliosis compared with other non-surgical interventions: a systematic review and meta-analysis.

STUDY DESIGN: Systematic review and meta-analysis. OBJECTIVE: To assess the effectiveness of scoliosis-specific exercises (SSE) on adolescent idiopathic scoliosis (AIS) compared with other non-surgical interventions. BACKGROUND: AIS is a complex deformity of the spine that develops between the age of 10years and skeletal maturity. SSE are prescribed to patients to reduce or slow curve progression, although their effectiveness is unknown. METHODS: Electronic databases were searched for relevant studies. Randomised controlled trials were eligible if they compared SSE with non-surgical interventions for individuals with AIS. Three authors independently extracted data, evaluated methodological quality and assessed the quality of evidence. Meta-analysis was performed where possible; otherwise, descriptive syntheses are reported. RESULTS: Nine randomised controlled trials were included. Four had a high risk of bias, three had an unclear risk and two had a low risk. Very-low-quality evidence indicated that SSE improved some measures of spinal deformity, function, pain and overall health-related quality of life (HRQoL). Very-low-quality evidence suggested that SSE had no effect on self-image and mental health. Very-low-quality evidence showed that bracing was more effective than SSE on measures of spinal deformity. However, SSE showed greater improvements in function, HRQoL, self-image, mental health and patient satisfaction with treatment. No differences were found for pain or trunk rotation. CONCLUSIONS: SSE may be effective for improving measures of spinal deformity for people with AIS, but the evidence is of very low quality. Future studies should evaluate relevant clinical measures and cost-effectiveness using rigorous methods and reporting standards.

An ecto-ATPase and an ecto-ATP diphosphohydrolase are expressed in rat brain.

Extracellular nucleotides acting as signaling molecules are inactivated by hydrolysis catalyzed by ecto-nucleotidases. ATP is sequentially degraded via ADP and AMP to adenosine. Enzymes that can be involved in the extracellular hydrolysis chain are ecto-ATP diphosphohydrolase (ecto-apyrase), ecto-ATPase, ecto-ADPase and 5'-nucleotidase. Mammalian ecto-ATP diphosphohydrolase is a member of a family of apyrases sharing four "apyrase conserved regions" that presumably participate in the formation of the catalytic site. We report the presence of ecto-ATP diphosphohydrolase in rat brain and the primary structure of a new mammalian member of the apyrase family. Expression in CHO cells shows that it represents an ecto-ATPase. As revealed by Northern analysis of rat tissues, the ecto-ATPase is co-expressed with ecto-ATP diphosphohydrolase in heart, kidney, spleen, thymus, lung, skeletal muscle and brain. Signals for both ecto-nucleotidases are very weak in liver. mRNAs for both proteins are present in PC12 cells, suggesting that the two nucleotidases may be co-expressed in the same neural cell. Using computer-aided sequence analysis, primary structure and membrane topography are compared with those of other members of the apyrase family.

The role of estrogen and estrogen receptor-alpha in male adipose tissue.

Males and females both express estrogen receptor (ER) in white adipose tissue (WAT), and estrogens appear to play an important role in regulating WAT in females. However, the role of ER in male WAT was unclear. In this review, we describe our work, which used wild type (WT) and ERalpha-knockout (alphaERKO) male and female mice to determine the role of ERalpha in regulating WAT and brown adipose tissue (BAT). There were progressive increases in WAT with advancing age in alphaERKO compared with WT males; weights of various WAT depots in alphaERKO males were increased by more than 100% compared with WT controls during adulthood. Conversely, BAT weight was similar in alphaERKO and WT males at all ages. Adipocyte areas and numbers were also increased in WAT from alphaERKO compared with WT males. Compared with WT controls, alphaERKO females also had increases in WAT. The alphaERKO mice also had insulin resistance and impaired glucose tolerance, similar to humans lacking ERalpha or aromatase. The obesity in alphaERKO males appeared to involve decreased energy expenditure rather than hyperphagia. In summary, ERalpha absence causes adipocyte hyperplasia and hypertrophy in WAT, but not BAT, and is accompanied by insulin resistance and glucose intolerance in both males and females. These results are the first evidence that the estrogen/ERalpha signaling system is critical in female and male WAT deposition, and may have clinical implications.

New insights into molecular structure and function of ectonucleotidases in the nervous system.

Nucleotides can be released as signaling substances in the nervous system from both neural and glial cells. Their function is terminated by ecto-nucleotidases and sequential extracellular metabolism to the nucleoside. Recently considerable progress has been made in unraveling the molecular structure of an ecto-ATPase and an ecto-ATP diphosphohydrolase, two closely related ectoenzymes. Molecular structure, tissue distribution and functional properties of the ecto-nucleotidases are discussed with particular emphasis on the nervous system.

Nitrogen dynamics and growth of seedlings of an N-fixing tree (Gliricidia sepium (Jacq.) Walp.) exposed to elevated atmospheric carbon dioxide.

Seeds of Gliricidia sepium (Jacq.) Walp., a tree native to seasonal tropical forests of Central America, were inoculated with N-fixing Rhizobium bacteria and grown in growth chambers for 71 days to investigate interactive effects of atmospheric CO2 and plant N status on early seedling growth, nodulation, and N accretion. Seedlings were grown with CO2 partial pressures of 350 and 650 µbar (current ambient and a predicted partial pressure of the mid-21st century) and with plus N or minus N nutrient solutions to control soil N status. Of particular interest was seedling response to CO2 when grown without available soil N, a condition in which seedlings initially experienced severe N deficiency because bacterial N-fixation was the sole source of N. Biomass of leaves, stems, and roots increased significantly with CO2 enrichment (by 32%, 15% and 26%, respectively) provided seedlings were supplied with N fertilizer. Leaf biomass of N-deficient seedlings was increased 50% by CO2 enrichment but there was little indication that photosynthate translocation from leaves to roots or that plant N (fixed by Rhizobium) was altered by elevated CO2. In seedlings supplied with soil N, elevated CO2 increased average nodule weight, total nodule weight per plant, and the amount of leaf nitrogen provided by N-fixation (as indicated by leaf δ15N). While CO2 enrichment reduced the N concentration of some plant tissues, whole plant N accretion increased. Results support the contention that increasing atmospheric CO2 partial pressures will enhance productivity and N-fixing activity of N-fixing tree seedlings, but that the magnitude of early seedling response to CO2 will depend greatly on plant and soil nutrient status.

The role of CagA status in gastric and extragastric complications of Helicobacter pylori.

Two major markers of virulence have been described in H. pylori. The first is a secreted protein (VacA) that is toxic to human cells in tissue culture. This cytotoxin causes vacuolation of epithelial cells in vitro and induces epithelial cell damage in mice. The second is a 40-Kb pathogenicity island for which the gene cagA (cytotoxin-associated gene A) is a marker. Approximately 60% of H. pylori isolates in Western countries are cagA+. The protein encoded by cagA+ has a molecular weight of 120-140 kDa and exhibits sequence heterogeneity among strains isolated from Western and Eastern countries. Although no specific function has been identified for CagA, there is increasing evidence that cagA+ strains are associated with increased intensity of gastric inflammation and increased mucosal concentration of particular cytokines including interleukin 8. Inactivation of picB (Hp 0544) or any of several other genes in the cag island ablates the enhanced IL-8 secretion of human gastric epithelial cells in tissue culture. Furthermore, persons colonized with cagA+ strains have an increased risk of developing more severe gastric diseases such as peptic ulcer and distal (non-cardia) gastric cancer than those harboring cagA- strains. We investigated the role of cagA status in both gastroduodenal and extragastroduodenal disease with H. pylori. Among the diseases limited to the antrum and body of the stomach and the duodenum, we demonstrated a correlation between CagA seropositivity and peptic ulcer disease. We also showed correlation between distal gastric cancer rated and CagA prevalence in populations in both developed and developing countries. In addition, we found that for several Asian populations, the relationship between CagA seropositivity and gastroduodenal diseases was complex. For extragastroduodenal diseases, our results confirmed previous reports that demonstrated that CagA status did not play a role in diseases such as rheumatoid arthritis and hyperemesis gravidarum. However, we found a clear negative association between the presence of a positive response to CagA and esophageal diseases. Therefore, CagA seropositivity (and thus gastric carriage) is associated with increased risks of certain diseases (involving the lower stomach and duodenum) and decreased risks of GERD and its sequelae. This apparent paradox can best be explained by differences in the interaction of cagA+ and cagA- strains with their hosts.

Pharmacokinetic interactions between lithium and fluoxetine after single and repeated fluoxetine administration in young healthy volunteers.

Pharmacokinetic interactions following coadministration of fluoxetine and lithium were investigated in 10 young healthy subjects. Both drugs were administered orally in a non-blinded design with 3 consecutive treatment periods: single oral dose of lithium (32.4 mmol lithium as acetate, Quilonum; coadministration of single oral doses of lithium (32.4 mmol) and fluoxetine (Fluctin, 60 mg); and single oral dose of lithium after 7-day pretreatment with fluoxetine (20 mg t.i.d.). Periods 1 and 2 were separated by a 1-week washout phase, while period 3 followed on immediately after period 2. Lithium serum concentrations were practically identical in periods 1 and 3 (administration of lithium alone and after chronic fluoxetine dosing). However, in period 2, when the 2 drugs were coadministered as single oral doses, the lithium concentrations were lower in the first 4 hours after medication compared with treatment periods 1 and 3. Cmax was also significantly lower in period 2. The times to peak, however, were not significantly changed by any fluoxetine comedication. The parameters AUC0 --> infinity, t1/2, total clearance (Cltot) and renal clearance (Clren) determined after administration of lithium alone did not differ statistically from values determined after single or after repeated fluoxetine dosing. Coadministration of lithium and fluoxetine did not produce any clinically relevant changes in hemodynamics, ECGs or laboratory parameters. After single doses of both drugs the most frequently reported symptoms were gastrointestinal complaints, while mild sedative symptoms were predominant when lithium was given after repeated fluoxetine medication.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic administration of nimodipine and propranolol in elderly normotensive subjects--an interaction study.

Nimodipine (30 mg t.i.d.) and propranolol (40 mg t.i.d.) were given orally to 24 healthy elderly subjects in a randomized, un-blinded, threefold crossover study. Each of the study periods lasted 8 days with a 5-day treatment phase separated by 2-week washout phases. Mean peak nimodipine plasma concentration was decreased after combined administration of the two drugs (16.1 +/- 8.1 micrograms/l vs. 12.4 +/- 9.5 micrograms/l). Nimodipine AUCss slightly decreased under propranolol co-medication from 44.9 +/- 15.1 micrograms x l/l to 38.8 +/- 22.5 micrograms x h/l, resulting in an AUC ratio of 88.8 +/- 44.5%. The relative bioavailability of propranolol was 104.1 +/- 38.3% after the combined propranolol and nimodipine medication, all other pharmacokinetic parameters remained unchanged. The pharmacological effects on the cardiovascular system were negligible after nimodipine alone. The reductions in blood pressure and pulse rate and the prolongations of typical ECG times observed after propranolol monotherapy and after the combination therapy were of similar size and were almost solely attributed to the action of the beta-blocker. The findings of this study indicate that chronic treatment with nimodipine together with propranolol should not be associated with a clinically relevant interaction.

The influence of nimodipine on hemodynamic parameters and peak and trough plasma concentrations of nifedipine chronically administered to elderly hypertensive patients.

Possible drug interactions between the two calcium channel blockers nimodipine and nifedipine were investigated in 12 elderly patients with stable mild to moderate hypertension. Their individually adjusted nifedipine treatment had been unchanged for at least 5 weeks. There was no evidence of significant changes in nifedipine efficacy as seen from blood pressure and heart rate after a 7-day comedication of 30 mg nimodipine t.i.d. as compared with the findings after combined administration of nifedipine and placebo. Nifedipine steady-state trough and peak concentrations were not significantly altered by concomitant nimodipine administration. On the other hand, nifedipine did not affect nimodipine trough and peak concentrations when compared with published data. Differences in clinical chemistry or tolerance between both treatment regimens did not occur or were marginal, respectively. In conclusion, a clinically relevant drug interaction between nifedipine and nimodipine which might be potentially harmful for patients with hypertension was not observed in this study.

The effect of multiple oral dosing of nimodipine on glibenclamide pharmacodynamics and pharmacokinetics in elderly patients with type-2 diabetes mellitus.

Possible interactions between the calcium channel blocker nimodipine and the hypoglycemic sulphonylurea glibenclamide were investigated in patients with type-2 diabetes mellitus. These 11 patients had taken their individually adjusted antidiabetic treatment unchanged for at least 3 months and showed a satisfactory stabilization on their disease. The concomitant administration of nimodipine 30 mg t.i.d. for 6 days did not change glibenclamide pharmacokinetics as compared with the findings after glibenclamide monotherapy. The normalized AUCss,norm were 11.6 (5.0) kg x h x 1(-1) at glibenclamide monotherapy and 12.3 (5.1) kg x h x 1(-1) after combined medication, resulting in an AUC-ratio for glibenclamide of 109 (23%). Mean elimination half-lives were determined as 2.7 (0.9) h after glibenclamide alone and 3.6 (1.9) h after nimodipine comedication. There was no evidence of significant alterations in glibenclamide efficiency as seen from glucose and insulin kinetics after the simultaneous administration of glibenclamide and nimodipine (insulin Cmax, 57.0 (30.8) mU/l after glibenclamide and 64.4 (32.1) mU/l after combined treatment). Nimodipine pharmacokinetics under nimodipine and glibenclamide steady-state conditions were similar to findings in literature: AUCss,norm 0.10 (0.04) microgram x h x 1(-1), Css,max 20.7 (8.3) microgram x 1(-1). Hemodynamics, clinical chemistry and tolerance did not differ during both treatments. Thus, a clinically relevant drug interaction between nimodipine 30 mg t.i.d. and glibenclamide during long-term treatment can be excluded.

Development and delivery of an exercise intervention for rheumatoid arthritis: strengthening and stretching for rheumatoid arthritis of the hand (SARAH) trial.

This paper describes the development and implementation of a hand exercise intervention for rheumatoid arthritis (RA) as part of a large multi-centred randomised controlled trial in a U.K. National Health Service (NHS) setting. Participants are eligible if diagnosed with RA according to American College of Rheumatology criteria, have a history of disease activity, functional deficit or impairment in the hand and/or wrist, and have been on a stable medication regime for at least 3 months. The intervention development was informed by the current evidence base, published guidelines, clinician and expert opinion, and a pilot study. The exercise programme targets known, potentially modifiable physical impairments of the hand with 5 exercise sessions and a home exercise component over a 12 week period. The intervention will be provided to 240 participants along with usual care. A further 240 will receive usual care only as part of the control arm. Specific details of the treatments delivered are described. [ISRCTN no: 89936343].

[A new metal tube for laser surgery of the laryngotracheal region]. / Ein neuer Metall-Tubus für Laser-Eingriffe im Laryngotracheal-Bereich.

Various measures have been taken to minimize laser-specific hazards during laryngotracheal microsurgery with CO2-laser equipment. Endorsing Norton's view that only a metal tube ensures complete safety during laser surgery, we tested the "Laser-Flex", a new endotracheal tube (ETT) of high-grade stainless steel. This reconstructed, flexible, gas-tight ETT was designed to avoid perforation and even ignition of the tube with consecutive inhalation of combustion products known to be very strong lung irritants. The dull surface of the spiral ETT prevents damage to the surrounding mucosa by diverging the laser beam. The flexibility and greater stability compared with PVC- or silicone armored tubes makes intubation possible without a guide even under difficult conditions. A small PVC-tube with two high-volume-low-pressure cuffs is attached to the distal end of the metal ETT. The cuffs are filled via different inlets clearly distinguished by color as well as by the inscriptions "proximal" and "distal". Both inlet lines float inside the metal tube, thus being protected from accidental laser beams. The cuffs are filled with saline solution. In case of inflammation and destruction of the proximal cuff, the discharged solution will extinguish the flame and also protect the distal cuff, ensuring normal ventilation. Thus none of the laser operations had to be interrupted during our test period if accidentally perforation of the proximal cuff occurred. The high price of the "Laser-Flex" should not be a reason to abstain from using this tube. In view of the higher margin of patient safety, this tube might be used even from an economic point of view. In our tests we reused each tube five to eight times, thus lowering the costs with each reuse. Checks following sterilization after each use did not show any dysfunction of the cuffs or valves.

[Anesthesia and laser surgery of the laryngo-nasal-ear region]. / Anaesthesie und Laserchirurgie im Hals-Nasen-Ohrenbereich.

The applications of lasers in surgery is nowadays a widely used and well appreciated technique. Besides convincing advantages (precision in surgical manipulations, excellent hemostasis, minimal postoperative swelling, rapid healing) laser-unique hazards require particular safety considerations to avoid accidental injuries to patients and medical personnel. For damage of the eyes is the greatest risk in laser surgery goggles designed to the respective wavelength of the specific laser must be worn by everyone in the operation theatre. The patient's eyes are best protected by covering them with water moistened gauze pads. To avoid reflection of the laser beam only instruments with dull surfaces should be used. The proper function of the laser itself has been tested by a well trained surgeon before operation. Especially in microlaryngeal surgery carried out under general anaesthesia, severe complications, e.g. pneumothorax, penetration of large vessels or destruction of the surrounding tissues, may be the result of beam reflection or unskilled use. However, the most serious hazard for the patient-and even that one with the highest incidence (0.4 to 1.5%)-arises from the close neighbourhood of endotracheal tube and laser beam while operating in the larynx or the upper airways. In tests it has been demonstrated that the beams of different lasers penetrate tubes of various materials (silicone, semi- or transparent pvc) to a different extent, often igniting the material. High oxygen and nitrous oxide contents support tube ignition. The inhalation of smoke and toxic pyrolysis products either of tube-material or, possibly, of volatile anaesthetics are followed by serious damage of trachea and lung tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

[Presence of high concentrations of aluminum in neurons of a patient dying from a rapidly developing dementia]. / Sur la présence d'aluminium en forte concentration dans les neurones d'un malade décédé d'une démence rapidement évolutive.

Ultrastructural observation of brain cells of a patient dead from senile dementia associated with myoclonies, has shown a number of intracytoplasmic dense structures. High concentration of Aluminium has been detected in these structures by electron probe X ray analysis. The clinical symptomatology, the evolution and the cell pathology observed in this patients are very similar to the classical features observed in dialysis encephalopathy the origin of which is an aluminium intoxication. In this patient, the possible origin of the intoxication is the absorption in the last 10 years of aluminium gels.

Trunk proprioception: enhancement through lumbar bracing.

OBJECTIVES: To examine the effects of a neoprene lumbar brace on the proprioceptive ability of subjects without pathology and to determine whether those with poorer proprioception were more affected by the brace. STUDY DESIGN: Randomized 2 x 2 Latin square cross-over design. SETTING: Laboratory in an educational institution. SUBJECTS: Forty asymptomatic subjects matched by gender, age, weight, and height. METHOD: Blindfolded subjects performed a position matching task in which they flexed the trunk in the sagittal plane. Six positions across the range of motion were examined. Angular data were recorded by a lumbar motion monitor secured by straps to the subject's trunk. MAIN OUTCOME MEASURE: Absolute, constant, and variable errors in braced and unbraced conditions. RESULTS: The mean absolute, constant, and variable errors were 3.6 degrees, .45 degrees, and 4.4 degrees, respectively. Absolute, constant, and variable errors were significantly decreased when wearing a brace. However, the magnitude of the decreases were small (<1.0 degrees) for all errors when wearing a brace. The high error group's absolute error improved significantly (p < .05), from 6.0 degrees when unbraced to 4.0 degrees when braced. In contrast, absolute error measurements for subjects in the low error group were unchanged, with an error of 2.0 degrees when unbraced and 2.3 degrees when braced. CONCLUSIONS: The findings suggest that a neoprene lumbar brace improves the somatosensory information received by the central nervous system and results in less error in trunk positioning. Persons with less ability to match trunk position will benefit more from wearing a neoprene brace than will those who are already adept at the task.