Effect of inhibitors of gamma-glutamyl transpeptidase on the uptake of glutamate and aspartate into cultured astroglial cells from the glycogen body and cerebral hemispheres of the embryonic chick

Astroglial-like cells from the glycogen body and astroglia from the cerebral hemispheres of chick embryos cultured 6 days in vitro exhibit comparable values of L-glutamate and L-aspartate uptake. The uptake is significantly reduced by inhibitors of gamma-glutamyl transpeptidase.     

Differential sensitivity to natural cell-mediated cytotoxicity of two rat colon adenocarcinoma variants differing in their tumorigenicity: identification of the effector cells as natural killer cells

Summary DHD/K12 TRb (PROb) and DHD/K12 TSb (REGb) are two cancer cell variants originating from the same rat colon adenocarcinoma. They differ in their tumorigenicity: when inoculated into syngeneic BDIX rats, PROb cells induce progressive tumors whereas REGb cells induce tumors which always regress. As previously described, there is an inverse relation between their tumorigenicity and their susceptibility to NCMC mediated by syngeneic spleen or peripheral blood lymphocytes: PROb cells are significantly less sensitive to NCMC than REGb cells. This suggests a role for NCMC in the regression of REGb tumors. In this work the BDIX NCMC effector cells active in vitro against REGb cells were identified as NK cells according to four criteria: (1) efficacy in a 4-h ⁵¹Cr release assay, (2) sensitivity to anti-asGM1 antibody plus complement, (3) LGL morphology, and (4) ability to bind with the same affinity REGb and YAC-1 cells. In spleen, these NK cells were heterogeneous with respect to their asGM1 surface density and their morphology. PROb cells were not lysed by these NK cells in a short-term cytotoxicity assay, but only in a 16-h assay. It was shown that PROb and REGb cells were bound with the same affinity by NK cells, thus they certainly differ in their ability to resist to NK lytic mechanisms. This difference could play a role in the different tumorigenicity of the two variants. Abbreviations used: NK, natural killer; NC, natural cytotoxic; NCMC, natural cell-mediated cytotoxicity; asGM1, asialo GM1; LL, large lymphocytes; LGL, large grnular lymphocytes; LAL, large agranular lymphocytes; PBMNC, peripheral blood mononuclear cells; E:T, effector to target cell ratio; C:H, cold to hot cell ratio; FBS, fetal bovine serum     

Assignment of human platelet GP2B (GPIIb) gene to chromosome 17, region q21.1-q21.3

Summary The platelet GPIIb-IIIa complex functions as a receptor for fibrinogen, fibronectin, and von Willebrand factor on activated platelets. This glycoprotein is a member of a broadly distributed family of structurally and immunologically related membrane receptors involved in cell-cell contact and cell-matrices interactions. GPIIb-IIIa is a heterodimer complex composed of GPIIb (the subunit), which consists of two disulfide-linked heavy and light chains, and GPIIIa (the subunit), which is a single polypeptide chain. Congenital absence of platelet GPIIb-IIIa in Glanzmann's thrombasthenia results in a severe bleeding disorder characterized by defective platelet aggregation and failure of fibrinogen to bind to platelets. The gene coding for GPIIb was located on 17q21.1-17q21.3 as determined by in situ hybridization with a 2650-pb GP2B (GPIIb) cDNA probe prepared from human megakaryocytes.     

An improved purification procedure and properties of casein kinase II from brain

A simple and short purification procedure applicable to casein kinase II has been developed, for fully characterizing the enzyme from calf cerebral cortex cytosol. The procedure consists of four chromatographic steps: DEAE-cellulose, phosphocellulose, phosvitin-Sepharose and ATP-agarose which yields 87% pure casein kinase II. The purified enzyme shows three major bands with apparent molecular masses of 42, 38, and 27 kDa by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and is self-autophosphorylated on its 27 kDa polypeptide. The enzyme shows all the characteristics described for casein kinase II from other sources: it is independent of cyclic nucleotides, calcium/phospholipids, and double-stranded poly(I).poly(C); it can utilize both ATP and GTP as phosphoryl donors and can phosphorylate both casein and phosvitin but not histone. The kinetic studies establish that theK _m for ATP is 12.5 M and 25.1 M when using phosvitin and casein respectively as phosphoryl acceptors. TheK _m for phosvitin is 0.91 mg/ml and for casein 1.43 mg/ml, while theV _max is 315 nmol/min/per mg protein and 479 nmol/min/per mg protein for phosvitin and casein respectively. The activity of the kinase is highly stimulated by KCl or NaCl, and almost completely inhibited by heparin concentrations of 1 g/ml (92%). This inhibition is reduced to only 33% in the presence of optimal KCl concentrations (150 mM). Spermine stimulates enzyme activity, whilst hemin produces a slight inhibition.     

Immunocytochemical investigations on the presence of neuron-specific antibodies in the CSF of Parkinson's disease cases

The CSF and sera of 7 patients with Parkinson's disease were investigated immunocytochemically, in order to see if antibodies were present which recognized DA-ergic cell bodies in glutaraldehyde fixed rat brain. In 2 patients a marked labeling of DA-ergic neurons in the substantia nigra was observed, identified by anti-DA antiserum and by 6-OHDA induced degeneration, but also other neurons in the ventral mesencephalon were recognized. The other patients were weakly positive or negative. Sera gave unspecific labelling of all neurons. In one patient the sub-classes of IgG were investigated and found to be of IgG₃ (labeling nerve terminals) and of IgG_1–2, low affinity type (recognizing perikarya). The epitopes recognized have not been identified, but are unlikely to be DA-like, since blocking experiments and ELISA-tests gave negative results. The possible clinical importance of the results are discussed.Special Issue dedicated to Prof. Holger Hydén.     

A re-examination of the Na+-independent binding of [3H]β-alanine to rat brain stem-spinal cord

The Na⁺-independent binding of [³H]-alanine to rat brain stem plus spinal cord was reinvestigated, in order to study in more detail the characteristics of previously described -alanine binding processes. Binding was absent when amino acid-free postnuclear supernatants or crude synaptic membranes were used. Experiments performed with several other Na⁺-free preparations showed a sole binding component, irrespective of the preparation used. Biochemical characterization of this Na⁺-independent binding, using frozen/thawed/washed synaptosomal-mitochodrial fractions, showed that binding reached a plateau between 7 min and 13 min, increasing thereafter. Binding was linear with fraction protein over a range of 200–415 g/ml incubation medium. Binding was completely inhibited by glycine, alanine, -aminobutyric acid, -aminoisobutyric acid, hypotaurine and strychnine, and to a lesser extent by 2,2-dimethyl--alanine, brucine and gelsemine. It was insensitive to taurine, -aminobutyric acid (GABA), 2-guanidinoethanesulfonic acid (GES), carnosine, and bicuculline methiodide. Binding was reversible, saturable (K _D 20 M), and heat sensitive.     

Antimessenger oligodeoxyribonucleotides: an alternative to antisense RNA for artificial regulation of gene expression--a review

Synthetic oligodeoxyribonucleotides (oligos) are now widely used as artificial regulators for gene expression both in cell-free media and in cultured cells. We describe the biological consequence of the various chemical modifications that have been introduced into the molecules to improve their resistance against nuclease attack, their affinity for the target mRNA and their uptake by cells. We also describe the rising generation of antimessenger oligos. Covalently linked to reactive groups these molecules direct irreversible modifications of the complementary nucleic acids. We anticipate that these oligos will be targeted to double-stranded nucleic acids to interfere with gene expression at the DNA level.