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.     

Orthopedic management of osteopetrosis: results of a survey and review of the literature

Osteopetrosis or Albers-Schonberg disease is a rare hereditary disorder of osteoclast function in which resorption of bone is diminished, resulting in abnormally dense bones. The condition is known to occur in at least four recognizable clinical patterns, each of which is variable. The optimal treatment of fractures and of bone deformity in these patients has not previously been made clear. To determine appropriate orthopedic management of the condition, we conducted a survey of the membership of the Pediatric Orthopedic Society of North America. The combined experience of 57 surgeons who treated 79 patients with osteopetrosis was compiled. Four femoral neck fractures treated by closed reduction and internal fixation had a satisfactory result, but three treated nonoperatively developed varus and required osteotomy. A total of 20 hips was treated for coxa vara by various means, none of which was free of complications. Valgus osteotomy, when used as the primary treatment for coxa vara, was the most consistently satisfactory procedure, whereas in situ pinning failed in two of three hips. Fourteen subtrochanteric fractures and 31 other fractures of the femur were treated. Good results were reported with traction or casting or both in the majority of those fractures. Twenty-nine tibia fractures were treated successfully, the majority by nonoperative means. Upper extremity fractures healed well with closed reduction and casting. Vertebral fractures, spondylolysis, and back pain were most frequently treated without surgery.     

Regulation of cell survival during B lymphopoiesis: apoptosis and Bcl-2/Bax content of precursor B cells in bone marrow of mice with altered expression of IL-7 and recombinase-activating gene-2

B cell development in mouse bone marrow depends critically upon IL-7. To examine the possible in vivo trophic role of IL-7, we have quantitated apoptosis and Bcl-2 family proteins in populations of phenotypically defined B lineage cells in IL-7-deficient and IL-7-overexpressing mice. Using immunofluorescence labeling, multiparameter flow cytometry, and a short-term culture assay, we show that the apoptotic rates of precursor B cells, but not of more mature B cells, are enhanced by IL-7 gene deletion, associated with increased intracellular content of Bax and decreased Bcl-2, while, conversely, an IL-7 transgene suppresses precursor B cell apoptosis and produces low Bax and high Bcl-2 levels. During normal B cell development, high Bax/Bcl-2 ratios characterize cells undergoing greatest apoptotic cell death. Pro-B cells in RAG-2-/- mice, all destined to abort, show elevated Bax levels and Bax/Bcl-2 ratios. By comparison with the elevated rate of pro-B cell apoptosis in RAG-2-/- mice, provisional estimates have been made for the fraction of pro-B cells undergoing apoptosis in normal mice (70%), IL-7-/- mice (85%), and IL-7 transgenic mice (35%). The results demonstrate that IL-7 strongly promotes in vivo cell survival and maintains antiapoptotic Bcl-2/Bax ratios during the development of precursor B cells in mouse bone marrow.     

The hot rectus abdominis muscle sign

We report a 2.5-year-old child who presented with an acute abdomen caused by the perforation of a sewing pin through her appendix that had started to penetrate the wall of the bladder.