A modification of the Roe procedure for determination of fructose in tissues with increased specificity

A new modification of the procedure of Roe for determination of fructose concentrations in tissues is described. The modification shows no reaction with glucose or glucose phosphates. Tissues are homogenized in dilute perchloric acid, filtered, and reacted with alcoholic resorcinol and 30% HCl for 1 hr at 80°C. As little as 0.33 μg/ml of fructose may be reliably determined. Fructose phosphates react in this test but to a lesser extent than pure fructose.     

Alternatives to donor matching for control of graft-versus-host disease

Graft-versus-host disease (GvHD) after bone-marrow transplantation in dogs is controlled by many different genetic systems. In littermate combinations identical for the major histocompatibility complex (MHC) the number of systems that influence GvHD is related to the number of donor lymphocytes injected. If the number of donor lymphocytes administered is sufficiently low, minor histocompatibility systems do not influence survival after bone-marrow transplantation. With increasing numbers of donor lymphocytes the beneficial influence of MHC matching on GvH incidence and severity disappears and minor histocompatibility antigens, coded for on at least two other autosomal chromosomes as well as possibly the Y chromosome, can cause severe GvHD. In contrast, the X chromosome does not appear to carry a histocompatibility system that is of relevance to GvHD control. The severity and tissue distribution of histological signs of GvHD in recipients of bone-marrow and lymph-node cells from MHC-identical donors are similar to those in recipients of MHC-mismatched bone-marrow cells. Female donors do appear to cause severe GvHD more frequently than males. In contrast to rhesus monkey and human bone-marrow cells, dog bone-marrow cells are negative in PHA tests. This is in accordance with the generally benign course of GvHD in dogs that are treated with bone-marrow cells only from histocompatible littermate donors. The influence of the sex of the bone-marrow donor on GvHD incidence and severity is not reflected in differences between PHA tests with male and female dog lymphocytes. A better predictive test for GvH potential than the PHA test appears to be needed. Alternatives to additional donor selection for the prevention of GvHD in histocompatible recipients appear to be the use of a male donor and the removal of lymphocytes from bone-marrow-cell suspensions prior to transplantation.     

MicroRNA 218 Acts as a Tumor Suppressor by Targeting Multiple Cancer Phenotype-associated Genes in Medulloblastoma

Aberrant expression of microRNAs has been implicated in many cancers. We recently demonstrated differential expression of several microRNAs in medulloblastoma. In this study, the regulation and function of microRNA 218 (miR-218), which is significantly underexpressed in medulloblastoma, was evaluated. Re-expression of miR-218 resulted in a significant decrease in medulloblastoma cell growth, cell colony formation, cell migration, invasion, and tumor sphere size. We used C17.2 neural stem cells as a model to show that increased miR-218 expression results in increased cell differentiation and also decreased malignant transformation when transfected with the oncogene REST. These results suggest that miR-218 acts as a tumor suppressor in medulloblastoma. MicroRNAs function by down-regulating translation of target mRNAs. Targets are determined by imperfect base pairing of the microRNA to the 3′-UTR of the mRNA. To comprehensively identify actual miR-218 targets, medulloblastoma cells overexpressing miR-218 and control cells were subjected to high throughput sequencing of RNA isolated by cross-linking immunoprecipitation, a technique that identifies the mRNAs bound to the RNA-induced silencing complex component protein Argonaute 2. High throughput sequencing of mRNAs identified 618 genes as targets of miR-218 and included both previously validated targets and many targets not predicted computationally. Additional work further confirmed CDK6, RICTOR, and CTSB (cathepsin B) as targets of miR-218 and examined the functional role of one of these targets, CDK6, in medulloblastoma.     

Involvement of Na,K-pump in SEPYLRFamide-mediated reduction of cholinosensitivity in Helix neurons

SEPYLRFamide acts as an inhibitory modulator of acetylcholine (ACh) receptors in Helix lucorum neurones. Ouabain, a specific inhibitor of Na,K-pump, (0.1 mM, bath application) decreased the ACh-induced inward current (ACh-current) and increased the leak current. Ouabain decreased the modulatory SEPYLRFamide effect on the ACh-current. There was a correlation between the effects of ouabain on the amplitude of the ACh-current and on the modulatory peptide effect. Ouabain and SEPYLRFamide inhibited the activity of Helix aspersa brain Na,K-ATPase. Activation of Na,K-pump by intracellular injection of 3 M Na acetate or 3 M NaCl reduced the modulatory peptide effect on the ACh-current. An inhibitor of Na/Ca-exchange, benzamil (25 muM, bath application), and an inhibitor of Ca(2+)-pump in the endoplasmic reticulum, thapsigargin (TG, applied intracellularly), both prevented the effect of ouabain on SEPYLRFamide-mediated modulatory effect. Another inhibitor of Ca(2+)-pump in the endoplasmic reticulum, cyclopiazonic acid (applied intracellularly), did not prevent the effect of ouabain on SEPYLRFamide-mediated modulatory effect. These results indicate that Na,K-pump is responsible for the SEPYLRFamide-mediated inhibition of ACh receptors in Helix neurons. Na/Ca-exchange and intracellular Ca(2+) released from internal pools containing TG-sensitive Ca(2+)-pump are involved in the Na,K-pump pathway for the SEPYLRFamide-mediated inhibition of ACh receptors.     

Resistance of fibrogenic responses to glucocorticoid and 2-methoxyestradiol in bleomycin-induced lung fibrosis in mice

Bleomycin-induced lung fibrosis in mice reproduces some key features of pulmonary fibrosis in humans including alveolar inflammation, myofibroblast proliferation, and collagen deposition. Glucocorticoids have been used as first-line therapy for the treatment of lung fibrosis, although their clinical efficacy is equivocal. We examined the effect of the glucocorticoid, methylprednisolone (MP), and the estrogen metabolite, 2-methoxyestradiol (2MEO) on bleomycin-induced bronchoalveolar inflammation, fibrosis, and changes in lung function. The characterization of the time-course of the bleomycin-induced fibrosis indicated that lung dry mass and hydroxyproline content showed less variance than histopathological assessment of fibrosis. The bleomycin-induced increases in bronchoalveolar lavage (BAL) fluid cell number and protein levels were not significantly influenced by treatment with either MP (1 mg.(kg body mass)(-1).day(-1), i.p.) or 2MEO (50 mg.(kg body mass)(-1).day(-1), i.p.). Lung fibrosis, measured histopathologically or by hydroxyproline content, was not significantly influenced by either MP or 2MEO treatment, whereas the latter agent did reduce the increment in lung dry mass. The enlargement of alveolar airspaces and the decline in lung compliance were exacerbated by MP treatment. These data suggest that bleomycin-induced pulmonary fibrosis is resistant to inhibition by concurrent treatment with either glucocorticoids or 2MEO.