Synthesis and Properties of the Bi-Based Superconducting Powder Prepared by the Pechini Process

Bi-based superconductors are of great interest in high-temperature superconductors. We describe what is believed to be the first synthesis of these materials using the Pechini process, a low-temperature synthetic method that often yields inorganic oxide of excellent phase purity and well-controlled stoichiometry. Using this process, it has been possible to synthesize phase-pure Bi-2223 phase by calcining the appropriate polymeric precursors in air at 800°C for several hours. The superconductivity studies show that the Pechini-synthesized materials appear to offer high-quality performance.     

N-Methyl-D-aspartate attenuates opioid receptor-mediated G protein activation and this process involves protein kinase C

The effects of N-methyl-D-aspartate (NMDA) on opioid receptor-mediated G protein activation were explored in neuroblastoma X glioma hybrid (NG108-15) cells. Treatment of the cells with NMDA resulted in a remarkable attenuation of [35S]guanosine-5'-O-(3-thio)triphosphate binding stimulated by [D-Pen2,D-Pen5]-enkephalin (DPDPE), a delta-opioid receptor agonist. The effects of NMDA were dose and time dependent with an IC50 value of 5 nM and could be blocked by NMDA receptor antagonists. After NMDA treatment, the DPDPE dose-response curve shifted to the right (EC50 value increased approximately 7-fold, from 6 to 40 nM), and the maximal response induced by DPDPE was reduced by approximately 60%. The effects of NMDA were reversible, and the DPDPE response could recover within 60 min. The functional responses of delta-, mu-, and kappa-opioid receptors in primarily cultured neurons also were attenuated significantly by NMDA treatment. The inhibitory effects of NMDA on opioid receptor-mediated G protein activation could be blocked by coadministration of the protein kinase C (PKC) inhibitors or by elimination of the extracellular Ca2+. Correspondingly, NMDA treatment of NG108 cells significantly elevated cellular PKC activity and stimulated Gialpha2 phosphorylation. Transient transfection into NG108-15 cells of the wild-type Gialpha2 and a mutated Gialpha2 (Ser144Ala) resulted in a 2-fold increase in DPDPE-stimulated G protein activation. The DPDPE responses were greatly inhibited by NMDA treatment in the wild-type Gialpha2-transfected cells but much less affected in the mutant Gialpha2-transfected cells. In summary, NMDA attenuates opioid receptor/G protein coupling, and this process requires activation of PKC.     

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.     

Aspects of oligonucleotide and peptide sequencing with MALDI and electrospray mass spectrometry

Biopolymer sequencing with mass spectrometry has become increasingly important and accessible with the development of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). Here we examine the use of sequential digestion for the rapid identification of proteolytic fragments, in turn highlighting the general utility of enzymatic MALDI ladder sequencing and ESI tandem mass spectrometry. Analyses were performed on oligonucleotides ranging in size from 2 to 50 residues, on peptides ranging in size from 7 to 44 residues and on viral coat proteins. MALDI ladder sequencing using exonuclease digestion generated a uniform distribution of ions and provided complete sequence information on the oligonucleotides 2-30 nucleic acid residues long. Only partial sequence information was obtained on the longer oligonucleotides. C-terminal peptide ladder sequencing typically provided information from 4 to 7 amino acids into the peptide. Sequential digestion, or endoprotease followed by exoprotease exposure, was also successfully applied to a trypsin digest of viral proteins. Analysis of ladder sequenced peptides by LCMS generated less information than in the MALDI-MS analysis and ESI-MS2 normally provided partial sequence information on both the small oligonucleotides and peptides. In general, MALDI ladder sequencing offered information on a broader mass range of biopolymers than ESI-MS2 and was relatively straightforward to interpret, especially for oligonucleotides.     

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.     

Osteogenesis of electrically stimulated bone cells mediated in part by calcium ions

Culture selected and expanded osteoblastic cells may be able to be reintroduced into massive skeletal defects to accelerate cell mediated regeneration of skeletal tissues, especially in bone ingrowth in total joint replacement, fracture healing, and osteoporosis. In vitro osteogenic cell culture is a useful model in studying the mechanism of bone metabolism under direct current stimulation. In this study, an osteoblastlike cell line was isolated from newborn rat calvaria. The osteogenic processes of the in vitro cultured cell line were studied by cytochemical, electron microscopic, and energy dispersive x-ray analysis techniques that resembled those observed in membrane bone ossification centers in vivo. Direct current stimulation of 100 microA/cm2 accelerated greatly the proliferation and calcification of the in vitro cultured cells. Intracellular free calcium ion metabolism was measured with an Adherent Cell Analysis and Sorting Machine. Under direct current stimulation, intracellular free calcium ion concentration increased an average of 2.3 times of the original level, which may play a key role in regulating osteogenesis and bone metabolism.     

Health-promoting behaviors of rural adolescent women

A part of a larger study of the health behaviors of adolescent women, this investigation examined health-promoting behaviors and the influence of cognitive, social, and environmental factors on these health-promoting behaviors of rural adolescent women. The sample consisted of 128 rural African-American and white adolescent women. Forty-four percent of the variance in health-promoting behavior of this sample was explained by five variables: self-image, problem solving, mother's education, employment status, and family structure. Self-image was the most salient predictor of health-promoting behavior, explaining 33% of the variance.