Identification of an opioid kappa receptor subtype-selective N-substituent for (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine

A three-component library of compounds was prepared in parallel using multiple simultaneous solution-phase synthetic methodology. The compounds were biased toward opioid receptor antagonist activity by incorporating (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (a potent, nonselective opioid pure antagonist) as one of the monomers. The other two monomers, which included N-substituted or unsubstituted Boc-protected amino acids and a range of substituted aryl carboxylic acids, were selected to add chemical diversity. Screening of these compounds in competitive binding experiments with the kappa opioid receptor selective ligand [3H]U69,593 led to the discovery of a novel kappa opioid receptor selective ligand, N-?(2'S)-[3-(4-hydroxyphenyl)propanamido]-3'-methylbutyl?-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (8, RTI-5989-29). Additional structure-activity relationship studies suggested that 8 possesses lipophilic and hydrogen-bonding sites that are important to its opioid receptor potency and selectivity. These sites appear to exist predominantly within the kappa receptor since the selectivity arises from a 530-fold loss of affinity of 8 for the mu receptor and an 18-fold increase in affinity for the kappa receptor relative to the mu-selective ligand, (+)-N-[trans-4-phenyl-2-butenyl]-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (5a). The degree of selectivity observed in the radioligand binding experiments was not observed in the functional assay. According to its ability to inhibit agonist stimulated binding of [35S]GTPgammaS at all three opioid receptors, compound 8 behaves as a mu/kappa opioid receptor pure antagonist with negligible affinity for the delta receptor.     

Sucrosemia in untreated celiac disease: a potential screening test

During studies to develop serum tests of small intestinal permeability, we detected an unidentified disaccharide in HPLC traces of sera from untreated celiacs. This present study aimed to identify the disaccharide and determine whether the presence of the disaccharide in the serum after an oral challenge had potential as a simple screening test for celiac disease. The disaccharide was identified as sucrose by incubation studies of sera with disaccharidases. Twenty untreated celiacs, 15 treated celiacs, and 20 normal or dyspeptic controls were studied for the presence of sucrose in their serum after an oral load (8 g). The results in celiacs were compared with the presence of serum IgA endomysial antibodies. The 10 normal controls were also given a larger sucrose challenge (50 g). Ten of the untreated celiacs and 10 controls had their brush border disaccharidase activities measured. Sucrose eluted in the same position as the unidentified disaccharide in the HPLC trace and the latter could be removed by incubation with sucrase. All untreated celiacs but none of the treated celiacs had sucrose in their serum after the 8-g oral challenge. None of the controls had sucrose in their serum after the 8-g or 50-g challenges. Three untreated celiacs were IgA endomysial antibody negative as were all the treated cases. Brush border sucrase activity was low in untreated celiac disease. The presence of sucrose in the serum after an oral load shows promise as a noninvasive test for celiac disease.     

Adeno-associated virus 2-mediated high efficiency gene transfer into immature and mature subsets of hematopoietic progenitor cells in human umbilical cord blood

Recombinant adeno-associated virus 2 (AAV) virions were constructed containing a gene for resistance to neomycin (neoR), under the control of either the herpesvirus thymidine kinase (TK) gene promoter (vTK-Neo), or the human parvovirus B19 p6 promoter (vB19-Neo), as well as those containing an upstream erythroid cell-specific enhancer (HS-2) from the locus control region of the human beta-globin gene cluster (vHS2-TK-Neo; vHS2-B19-Neo). These recombinant virions were used to infect either low density or highly enriched populations of CD34+ cells isolated from human umbilical cord blood. In clonogenic assays initiated with cells infected with the different recombinant AAV-Neo virions, equivalent high frequency transduction of the neoR gene into slow-cycling multipotential, erythroid, and granulocyte/macrophage (GM) progenitor cells, including those with high proliferative potential, was obtained without prestimulation with growth factors, indicating that these immature and mature hematopoietic progenitor cells were susceptible to infection by the recombinant AAV virions. Successful transduction did not require and was not enhanced by prestimulation of these cell populations with cytokines. The functional activity of the transduced neo gene was evident by the development of resistance to the drug G418, a neomycin analogue. Individual high and low proliferative colony-forming unit (CFU)-GM, burst-forming unit-erythroid, and CFU-granulocyte erythroid macrophage megakaryocyte colonies from mock-infected, or the recombinant virus-infected cultures were subjected to polymerase chain reaction analysis using a neo-specific synthetic oligonucleotide primer pair. A 276-bp DNA fragment that hybridized with a neo-specific DNA probe on Southern blots was only detected in those colonies cloned from the recombinant virus-infected cells, indicating stable integration of the transduced neo gene. These studies suggest that parvovirus-based vectors may prove to be a useful alternative to the more commonly used retroviral vectors for high efficiency gene transfer into slow or noncycling primitive hematopoietic progenitor cells, without the need for growth factor stimulation, which could potentially lead to differentiation of these cells before transplantation.     

Something Old,Something New: Integrating Engineering Practice into the Teaching of Engineering Mechanics

This paper presents a multifaceted method for teaching engineering mechanics designed to satisfy the following set of desiderata: (1) integrate engineering practice into the teaching of engineering science; (2) address a wide set of learning styles; (3) provide opportunities for practice in group work and learning; (4) promote communication and synthesizing skills; (5) engage students in the teaching and learning process; (6) maintain traditional achievement levels with respect to traditional measures; (7) motivate students to continue their engineering studies; and (8) maintain reasonable resource demands. Consistent with this variety of objectives, the approach presented here is composed of a variety of ingredients. In addition to the standard homework problems and exams, these ingredients include design projects, group work, basic competency exams, computational environments for simulating and visualizing phenomena, multimedia instructional tools, hands-on experiences, and student presentations. This educational model is intended to be suitable for teaching engineering mechanics in general, but this paper focuses on a particular sophomore-level mechanics of materials course in which the method has been implemented, refined over several years of classroom use, and tested relative to traditional approaches. The paper describes the and techniques that together make up the method, followed by summaries of the results of evaluations that have been applied to the course.     

Hydrolysis studies on oleamide in simulated gastrointestinal fluids

Hydrolysis studies using simulated gastrointestinal fluids have been performed on oleamide, an important fatty acid amide slip additive used in plastics food packaging. Experiments have been conducted using the simulated gastrointestinal fluids, specified by the Scientific Committee for Food for hydrolysis studies, and with slightly modified preparations believed to be more representative of the in vivo situation. The degree of hydrolysis in simulated gastric fluids was found to be negligible after incubation for 4 h at 37 degrees C. Addition of 'bile salts' to simulated intestinal fluid was found to significantly increase the degree of hydrolysis to about 95% loss of oleamide after incubation for 4 h at 37 degrees C. Stoichiometric formation of oleic acid, an innocuous substance, was also demonstrated. It has therefore been concluded that ingestion of the very low levels of oleamide expected to migrate into most foods from food packaging is unlikely to pose a threat to the health of the consumer.     

The effect of increasing application rate of granular calcium ammonium nitrate on net nitrification in a laboratory study of grassland soils

Soil incubation studies were undertaken in controlled environment cabinets at 15°C to investigate the effect of increasing application rates of calcium ammonium nitrate (CAN) on net nitrification in two grassland soils. Granular CAN was applied to the surface of freshly collected, moist soil, at a rate equivalent to 0, 100, 200, 400, 800 and 1600µg NH ₄ ⁺ -N and NO ₃ ^- -N per gram of oven dry soil. In half the treatments finely ground CaCO₃ was incorporated into the moist soil to raise the starting pH. Changes in soil mineral N and pH were measured at weekly intervals up to six-weeks. The most probable number (MPN) technique was used to enumerate the NH ₄ ⁺ -N and NO ₂ ^- -N oxidizers at the beginning and end of the incubation.At low rates of CAN application there was considerable NH ₄ ⁺ -N oxidation to NO ₃ ^- -N during the incubation of both soils. Lime stimulated this N transformation. At high application rates (i.e. 800 and 1600 ppm) there was little change in NH ₄ ⁺ -N or NO ₃ ^- -N on either soil during the 6 week incubation, in the presence or absence of lime. The rate of NO ₃ ^- -N produced peaked at 5.6 and 3.8 mg NO ₃ ^- -N kg^–1 d^–1 on soil 1 and 2 respectively, in the presence of lime. Above a level of 400 ppm CAN (equivalent to 38 kg N ha^–1) the rate of NO ₃ ^- -N produced decreased. The higher rate of net nitrification in soil 1 compared with soil 2 was probably due to a higher number of nitrifying bacteria. Although high rates of CAN decreased the nitrifying activity of both soils there was little difference between treatments in the actual numbers of NH ₄ ⁺ -N and NO ₂ ^- -N oxidizers determined by the MPN technique.The results showed that the rate of granular CAN applied to the soil surface can influence the local activity of nitrifying bacteria and subsequent N transformations. At application rates of CAN generally used agriculturally for grass production, it is likely that net nitrification of the NH ₄ ⁺ -N in the fertilizer granule will be inhibited.     

Abrasion Resistance of Field-Assisted Ion-Exchanged Glass

The fracture strength of HF-etched samples was measured as a function of field-assisted K-Na ion exchange depth, as well as the severity of abrasion. As expected, the more severe the abrasion, the greater the depth required to make the strength insensitive to abrasion. An abrasion-independent weakening mechanism that increased with exchange depth was also found to be operative.