GPR40 is expressed in glucagon producing cells and affects glucagon secretion

The free fatty acid receptor, GPR40, has been coupled with insulin secretion via its expression in pancreatic beta-cells. However, the role of GPR40 in the release of glucagon has not been studied and previous attempts to identify the receptor in alpha-cells have been unfruitful. Using double-staining for glucagon and GPR40 expression, we demonstrate that the two are expressed in the same cells in the periphery of mouse islets. In-R1-G9 hamster glucagonoma cells respond dose-dependently to linoleic acid stimulation by elevated phosphatidyl inositol hydrolysis and glucagon release and the cells become increasingly responsive to fatty acid stimulation when overexpressing GPR40. Isolated mouse islets also secrete glucagon in response to linoleic acid, a response that was abolished by antisense treatment against GPR40. This study demonstrates that GPR40 is present and active in pancreatic alpha-cells and puts further emphasis on the importance of this nutrient sensing receptor in islet function.     

Contribution to the cytotaxonomy of Oryzopsis (Poaceae)

Meiotic studies were performed in twelve populations of four Oryzopsis species (O. pubiflora, O. lateralis, O. holciformis var. longiglomis and O. barbellata) to obtain data on the ploidy level and cytological evolution of the genus. The chromosome number 2n=2x=24 was revealed in all the species and populations studied. The present and other studies show the occurrence of two basic chromosome numbers in the genus, i.e. x=11 and x=12. Although Oryzopsis species and populations studied are diploid and are expected to form only bivalents in metaphase of meiosis‐I, quadrivalents were observed in O. pubiflora and O. lateralis, possibly due to the occurrence of heterozygote translocations. B‐chromosomes of 0–2 were observed in all species and populations studied. This is the first report of the occurrence of B‐chromosomes in the genus Oryzopsis. Several meiocytes showed the presence of double chromosome number in O. lateralis, and multipolar cells were observed in populations of O. barbellata, O. lateralis and O. holciformis var. longiglomis. The occurrence of large pollen grains (possibly unreduced) was observed along with smaller (normal) pollen grains in these species. Significant differences observed in chiasma frequency and distribution among studied species may be of use in species delimitation. The Kakan population differed significantly from the other populations of O. lateralis in meiotic characteristics. If such cytological differences are accompanied by morphological variation (under investigation), we may consider this population as a new variety or subspecies.     

Insulin feedback actions: complex effects involving isoforms of islet nitric oxide synthase

The present study examined the effects of exogenous insulin on C-peptide release in relation to islet activities of neural constitutive nitric oxide synthase (ncNOS) and inducible NOS (iNOS). The dose-response curves for glucose-stimulated insulin and C-peptide release from isolated islets were practically identical: 0.05-0.1 nmol/l insulin stimulated, 1-100 nmol/l had no effect, whereas concentrations >/=250 nmol/l ("high insulin"), inhibited C-peptide release. Both the stimulatory and inhibitory effects were abolished by the phosphatidylinositol 3'-kinase inhibitor wortmannin. Addition of a NOS inhibitor partially reversed the inhibitory action of high insulin, but had no effect on the stimulatory action of low insulin (0.1 nmol/l). Moreover, high insulin markedly increased islet ncNOS activity and induced a strong iNOS activity. As shown biochemically and with confocal microscopy, the stimulatory action of high insulin on NOS activities and the associated inhibition of C-peptide release were reversed by raising cyclic AMP through addition of either glucagon-like peptide 1 (GLP-1) or dibutyryl cyclic AMP (Bt(2)cAMP) to the incubated islets. We conclude that the positive feedback mechanisms of action of insulin are independent of islet NOS activities and remain unclear. The negative feedback action of insulin, however, can be explained by its ability to stimulate both islet ncNOS activity and the expression and activity of iNOS. The effects on iNOS are most likely transduced through phosphatidylinositol 3'-kinase and are counteracted by raising islet cyclic AMP levels.     

Isolation murine mesenchymal stem cells by positive selection

Isolation and purification of mesenchymal stem cells (MSCs) from mouse via plastic adherent cultures is arduous because of the unwanted growth of hematopoietic cells and non-MSCs. In this work, homogenous populations of CD34⁺ MSCs from mouse bone marrow were isolated via positive selection. For this purpose, C57Bl/6 mice were killed and bone marrow cells were aspirated before incubation with magnetic bead conjugated to anti-CD34 antibody. A sample of positively selected CD34⁺ cells were prepared for flow cytometry to examine the expression of CD34 antigen and others were subcultured in a 25-cm² culture flask. To investigate the mesenchymal nature, the plastic adherent cultivated cells were induced to differentiate along osteoblastic and adipogenic lineages. Furthermore, the expression of some surface markers was investigated by flow cytometry. According to the result, purified populations of fibroblast-like CD34⁺ cells were achieved in the first passage (1 wk after culture initiation). The cells expressed CD34, CD44, Sca-1, and Vcam-1 antigens (markers) but not CD11b and CD45. They were capable of differentiating into osteocytes and adipocytes. This study indicated that our protocol can result in the efficient isolation of homogenous populations of MSCs from C57BL/6 mouse bone marrow. We have shown that murine bone marrow-derived CD34⁺ cells with plastic adherent properties and capability of differentiating into skeletal lineages in vitro are MSCs.     

First-principles vdW-DF investigation on the interaction between the oxazepam molecule and C60 fullerene

The interaction between oxazepam and C₆₀ fullerene was explored using first-principles vdW-DF calculations. It was found that oxazepam binds weakly to the fullerene cage via its carbonyl group. The binding of oxazepam to C₆₀ is affected drastically by nonlocal dispersion interactions, while vdW forces affect the corresponding geometries only a little. Furthermore, aqueous solution affects the geometries of the oxazepam approaching to fullerene slightly, while oxazepam binds slightly farther away from the nanocage. The results presented provide evidence for the applicability of the vdW-DF method and serve as a practical benchmark for the investigation of host–guest interactions in biological systems.

Synthesis, characterization and inhibitory potency of two oxono and thiono analogues of phosphoramidate compounds on acetylcholinesterase

Two novel structurally related phosphoramidate compounds, 1 and 2, with likely beta-diketone system were synthesized and characterized by 1H, 13C, 31P NMR, IR spectroscopy and elemental analysis. Compound 2 exhibited a 31P NMR signal which was significantly shielded (8 ppm) relative to compound 1. Determination of human erythrocyte acetylcholinesterase (hAChE) inhibitory activity was carried out according to Ellman's modified kinetic method and the IC50 values of compounds 1 and 2 were 1.567 and 2.986 mM, respectively. The k(i) values of 1 and 2 were 1.39 to 2.65 min(-1) respectively. A comparison of the bimolecular rate constant (k(i)) and IC50 values for the irreversible inhibitors 1 and 2 revealed that the oxono analogue has greater affinity for hAChE than the thiono compound. Furthermore effects of two conventional oximes paralidoxime (A) and obidoxime (B) on reactivation of the inhibited hAChE were studied but low reactivity was shown by both the oximes.     

High transmission of paternal plastid DNA in alfalfa plants demonstrated by restriction fragment polymorphic analysis

Summary A high frequency of paternal plastid transmission occurred in progeny from crosses among normal green alfalfa plants. Plastid transmission was analyzed by hybridization of radiolabeled alfalfa plastid DNA (cpDNA) probes to Southern blots of restriction digests of the progeny DNA. Each probe revealed a specific polymorphism differentiating the parental plastid genomes. Of 212 progeny, 34 were heteroplastidic, with their cpDNAs ranging from predominantly paternal to predominantly maternal. Regrowth of shoots from heteroplasmic plants following removal of top growth revealed the persistence of mixed plastids in a given plant. However, different shoots within a green heteroplasmic plant exhibited paternal, maternal, or mixed cpDNAs. Evidence of maternal nuclear genomic influence on the frequency of paternal plastid transmission was observed in some reciprocal crosses. A few tetraploid F₁ progeny were obtained from tetraploid (2n=4x=32) Medicago sativa ssp. sativa x diploid (2n=2x=16) M. sativa ssp. falcata crosses, and resulted from unreduced gametes. Here more than the maternal genome alone apparently functioned in controlling plastid transmission. Considering all crosses, only 5 of 212 progeny cpDNAs lacked evidence of a definitive paternal plastid fragment.Contribution No. 89-524-J from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan     

Hsp110 over-expression increases the immunogenicity of the murine CT26 colon tumor

Several studies have suggested a positive correlation between heat shock protein (hsp) expression and tumor immunogenicity. Independently, many studies have shown that hsp purified from tumors can be used as a tumor-specific vaccine. In this study, we have explored the connection between hsp expression and anti-tumor immunity by transducing murine CT26 colon carcinoma cells with the cDNA of a major hsp, i.e. hsp110. We have shown that over-expression of hsp110 has no effect on CT26 tumor cell growth in vitro, and does not inhibit their anchorage-independent growth capacity. However, in situ, hsp110 over-expressing CT26 tumor (CT26-hsp110) grew at a significantly reduced rate as compared to the wild-type CT26 tumor in immunocompetent mice. Moreover, immunization of mice with inactivated CT26-hsp110 cells significantly inhibited the growth of wild-type CT26 tumor. This immunity was associated with an increased frequency of tumor-specific T cells after vaccination. An in vivo antibody depletion assay demonstrated that inactivated CT26-hsp110 cells elicited anti-tumor responses involving CD8(+) T cells and natural killer (NK) cells, but not CD4(+) T cells. Lastly, the effect of the addition of granulocyte-macrophage colony stimulating factor (GM-CSF) to these vaccine formulations was determined. Mice immunized with irradiated CT26-hsp110 cells combined with GM-CSF-producing bystander cells revealed a complete inhibition of CT26 tumor growth, indicating a synergy between inactivated CT26-hsp110 vaccine activity and GM-CSF. These observations demonstrate that manipulation of hsp110 expression in tumors, specifically when combined with GM-CSF, represents a potentially powerful approach to cancer vaccine formulation.     

Effect of cadmium and zinc ethanolamine complexes on rat brain monoamine oxidase-B activity in vitro

Monoamine oxidase-B (MAO-B) from rat brain was inhibited strongly by the prepared cadmium and zinc ethanolamine complexes obtained from their sulphate and chloride salts. The inhibition of MAO-B by these complexes was time-dependent and fully reversible after dilution and sedimentation. In vitro, the cadmium ethanolamine complexes were more potent at inhibiting MAO-B than the zinc complexes. The inhibitory effect of these complexes follow the order: TEA>DEA>MEA, due to the alkyl residues and steric effect properties. The inhibition of MAO-B by cadmium and zinc ethanolamine complexes was a noncompetitive type. The K(i) values were calculated. The influence of the complexes on the activity of MAO-B was rather evaluated. It decreased the MAO-B activity. The IC(50) values of the two potent cadmium and zinc triethanolamine complexes on MAO-B were evaluated indicating that the complexes were tightly binding, but reversible inhibitors for MAO-B. In general, these systems may be used for preventing some neurodegenerative diseases.