Vinculin is an essential component for normal myofibrillar arrangement in fetal mouse cardiac myocytes

Vinculin is a cytoskeletal protein that is believed to be an essential component in the linkage of cytoskeletal actin filaments to the plasma membrane. To investigate the precise function of vinculin in the development of cardiac myofibrils, antisense oligodeoxynucleotides complementary to vinculin mRNA were used to perturb the expression of the protein during myofibril assembly and arrangement in mouse cardiac myocytes. Fetal (day 18-20 post-conception) mouse cardiac myocytes were isolated by collagenase digestion, separated by Percoll density gradient centrifugation, and plated on aligned collagen gels. By 72 h of culture, mouse myocytes displayed an elongated in vivo-like phenotype in parallel with the aligned fibrils of the collagen gels with polarized arrays of myofibrils. Two different antisense oligonucleotides (20-mer) altered the formation of the tissue-like phenotype of myocytes. These antisense oligonucleotides suppressed vinculin protein expression at 43.5+/-26.8% and 48.7+/-20.9% when compared to myocytes that were not treated. Examination of these myocytes by confocal scanning laser and transmission electron microscopy revealed a disruption of the aligned in vivo-like phenotype, assembly of thick and thin filaments, and formulation of Z-bands. Random sequence 20-mer oligonucleotides used as controls had little detectable effect on vinculin protein expression (94.2+/-14.8%), cell shape, normal alignment or assembly of myofibrils. These results indicate that vinculin is a critical cytoskeletal component, that functions in the determination of cell shape and the arrangement and organization of developing myofibrils.     

Validation of lucigenin as a chemiluminescent probe to monitor vascular superoxide as well as basal vascular nitric oxide production

Lucigenin has been widely used as a chemiluminescent substrate to monitor vascular superoxide (O*-2) formation. The validity of lucigenin for detection of O*-2 has been questioned because O*-2 is generated by lucigenin itself. It has been shown that the concentration of lucigenin is a critical parameter affecting the validity of this assay. In the present studies we evaluated a reduced concentration of lucigenin (5 microM) as a tool to quantify O*-2 production in vascular tissue. Lucigenin-induced effects on endothelial function were assessed by isometric tension recording of isolated aortic rings suspended in organ baths. The effects of lucigenin on O*-2 production were studied using spin trapping and electron spin resonance spectroscopy. Lucigenin at 250 microM but not at 5 microM caused a significant attenuation of endothelium-dependent relaxations to acetylcholine, which was prevented by pretreatment with superoxide dismutase. Spin-trapping studies revealed that lucigenin at 250 microM increased vascular O*-2 production several fold while 5 microM lucigenin did not stimulate O*-2 production. Inhibition of NO synthase by NG-momomethyl-l-arginine as well as the removal of the endothelium almost doubled lucigenin-derived chemiluminescence (LDCL), indicating that basal production of endothelium-derived NO depresses the baseline chemiluminescence signal. Thus, lucigenin at a concentration of 5 microM seems to be a sensitive and valid probe for assessing O*-2 in vascular tissue. It can also be used as an indirect probe to estimate basal vascular NO release.     

Edible mushroom (Agaricus bisporus) lectin, which reversibly inhibits epithelial cell proliferation, blocks nuclear localization sequence-dependent nuclear protein import

The Galbeta1-3GalNAcalpha (TF antigen)-binding lectin (ABL) from the common edible mushroom (Agaricus bisporus) has a potent anti-proliferative effect without any apparent cytotoxicity. This unusual combination of properties prompted investigation of its mechanism of action. In contrast to soluble lectin, agarose-immobilized, and hence noninternalizable ABL had no effect on proliferation of HT29 colon cancer cells. Electron microscopy of HT29 cells incubated with fluorescein- and gold-conjugated ABL showed internalization of the lectin into endocytotic vesicles and multivesicular bodies. Confocal microscopy showed perinuclear accumulation of fluorescein isothiocyanate-conjugated lectin, which also inhibits HT29 cell proliferation, raising the possibility that the lectin might interfere with nuclear pore function. Transport of heat shock protein 70 into the nucleus in response to heat shock was blocked by preincubation of HT29 cells for 6 h with 40 micrograms/ml ABL. In digitonin-permeabilized cells, nuclear uptake of bovine albumin conjugated to a nuclear localization sequence (NLS)-containing peptide was also inhibited by a 15-min preincubation with 40-100 micrograms/ml ABL. In contrast, serum-stimulated nuclear translocation of mitogen-activated protein kinase, which is NLS-independent, was not affected by pretreatment of cells with the lectin. These results suggest that the anti-proliferative effect of ABL is likely to be a consequence of the lectin trafficking to the nuclear periphery, where it blocks NLS-dependent protein uptake into the nucleus.     

Evaluation of a multicomponent, behaviorally oriented, problem-based "summer school" program for adolescents with diabetes

A 2-week summer school program, combining problem-based learning with behavior therapy, was developed to help adolescents with insulin-dependent diabetes improve their ability to cope with obstacles to dietary management. Ten students participated in a first session, and 9 participated in a second session, serving as a waiting list control group. Outcomes were evaluated pre- and postsession and at a 4-month follow-up using 3-day food diaries, blood glucose data, and paper-and-pencil tests of diabetes-related knowledge, self-efficacy, coping strategies, and general problem solving. Improvements were observed in self-efficacy, problem-solving skills, and self-reported coping strategies. No significant changes were observed in daily intake of fat, cholesterol, calories, mean blood glucose levels or blood glucose variability, and diabetes knowledge. Comparisons between the first group and the waiting list control group do not allow the significant pre-post changes to be clearly attributed to the summer school program.     

A conserved LIM protein that affects muscular adherens junction integrity and mechanosensory function in Caenorhabditis elegans

The glycosylation of the equine interhaemal barrier and areola was studied throughout the period of gestation. Placentae of 35, 37, 50, 119, 152, 200, 280 and 300 days gestation were investigated, using semithin plastic embedded sections and a panel of 15 biotinylated lectins with an avidin-peroxidase revealing system. Glycosylation of the trophoblast and maternal epithelium showed the most change during the first 50 days of gestation, being associated with the initial stages of adhesion and attachment. In the trophoblast, non-bisected tri/tetraantennary complex N-glycan was only evident after day 37 and terminal N-acetyl galactosamine, alpha2,3- and alpha2,6-linked sialic acids disappeared at the same time. The areolar trophoblast exhibited some differences from microcotyledonary areas, especially with respect to 2-deoxy, 2-acetamido alpha-galactose and tri/tetraantennary, non-bisected complex N-glycan, suggesting that the differences in function between microcotyledonary and areolar trophoblast are reflected at both the morphological and the biochemical level. Granules of the maternal uterine epithelium bound many lectins, particularly those with specificity for bisected and non-bisected bi/triantennary N-linked glycan, 2-deoxy, 2-acetamido alpha-galactosyl, beta-galactosyl and some fucosylated termini. Binding to sialic acids in alpha2,3- and alpha2,6-linkage was sparse. Maternal and fetal capillaries showed little change in glycan expression over the period studied, being rich in bisected and non-bisected bi/triantennary N-linked glycan and sialic acids, with some terminal N-acetyl galactosamine and no detectable terminal fucosyl residues.