The existence of glutathione and cysteine disulfide-linked to erythrocyte carbonic anhydrase from tiger shark

In this study it is shown that the higher molecular weight previously reported for tiger shark carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) compared to other carbonic anhydrases is decreased to a normal value around 30 000 after disulfide reduction of the enzyme. This difference in molecular weight is at least partly due to the existence of disulfide-linked glutathione and cysteine residues. Approx. 3 mol glutathione and a similar amount of cysteine are shown to be bound per mol enzyme. The presence of these factors also has effects on the enzyme activity.     

Phase memory relaxation times of spin labels in human carbonic anhydrase II: pulsed EPR to determine spin label location.

Phase memory relaxation times (T(M) or T(2)) of spin labels in human carbonic anhydrase II (HCA II) are reported. Spin labels (N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide, IPSL) were introduced at cysteines, by site-directed mutagenesis at seven different positions in the protein. By two pulse electron paramagnetic resonance (EPR), electron spin echo decays at 45 K are measured and fitted by stretched exponentials, resulting in relaxation parameters T(M) and x. T(M) values of seven positions are between 1.6 micros for the most buried residue (L79C) and 4.7 micros for a residue at the protein surface (W245C). In deuteriated buffer, longer T(M) are found for all but the most buried residues (L79C and W97C), and electron spin echo envelop modulation (ESEEM) of deuterium nuclei is observed. Different deuterium ESEEM patterns for W95C and W16C (surface residue) indicate differences in the local water concentration, or accessibility, of the spin label by deuterium. We propose T(M) as a parameter to determine the spin label location in proteins. Furthermore, these systems are interesting for studying the pertaining relaxation mechanism.     

Effects of the 5-lipoxygenase inhibitors AA-863 and U-60,257 on human glioma cell lines

The effects of two specific 5-lipoxygenase inhibitors AA-863 and U-60,257 (piriprost) on the growth of two human glioma cell lines, U-343 MGa and U-251 MG were investigated. Both monolayer cultured cells and spheroids were studied. The results of the monolayer studies showed potent and dose dependent inhibitory effects on the proliferation of glioma cells (IC50/one week treatment/of AA-863: 9.0 microM, IC50 of U-60,257: 40.0 microM). The experiments made on the tumor spheroids suggested an inhibitory effect on proliferation and volume growth already at lower doses (AA-863: 0.4-2.0 microM, U-60,257: 1.0-5.0 microM), a dose range where effects were not found in monolayers. At higher doses (AA-863: 10.0-30.0 microM, U-60,257: 30.0-90.0 microM) the experiments with spheroids failed to demonstrate a further inhibitory effect on spheroid volume, probably attributed to phenomena such as swelling of cells, dissociation of spheroid structure and development of necrosis. The clearly dose dependent inhibitory effect on the proliferation of human glioma cells in monolayer culture and the inhibitory effects on spheroid growth with these specific inhibitors indicate a role for lipoxygenase products in the growth of gliomas.     

Interactions between central glutamatergic,catecholaminergic and cholinergic systems with regard to locomotor activity in monoamine-depleted mice

Summary Following injection of 5µg of the competitive NMDA receptor antagonist AP-5 into the nucleus accumbens, but not following injection of the same dose into the dorsal striatum, a pronounced locomotor stimulation in monoamine-depleted mice was produced; the-adrenoceptor agonist clonidine (1 mg/kg) administered ip caused a marked potentiation of an intraaccumbens AP-5 (2.5µg) injection.On the other hand, 10µg of AP-5 combined with an ip injection of clonidine (1 mg/kg) caused a marked locomotor stimulation following local application into the dorsal striatum but not following application into the prefrontal cortex. Likewise, in combination with systemically administered clonidine, a substantial locomotor stimulation was observed after application of the muscarine receptor antagonist methscopolamine (62µg) into the dorsal striatum but not into the prefrontal cortex.This study suggests that NMDA receptors in the nucleus accumbens exert an inhibitory influence on locomotor activity. The dorsal striatum may also be involved in such control via NMDA and muscarinic receptors.     

Changes in glycan branching and sialylation of the Thy-1 antigen during normal differentiation of mouse T-lymphocytes.

The glycans of the Thy-1 antigen present on thymocytes and lymph-node T-lymphocytes were investigated after external labelling of the cells. Neuraminidase, endoglycosidase H and endoglycosidase F were used in combination with sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing in order to characterize the nature of the glycans on 125I-labelled and immunoprecipitated Thy-1. Glycopeptides were prepared from Thy-1 obtained from cells labelled by periodate/boro[3H]hydride treatment. The glycopeptides were separated by affinity chromatography on concanavalin A-Sepharose and analysed by gel filtration. The results show that both types of cells possess Thy-1 molecules with three N-linked carbohydrate chains, of which one is of 'high-mannose' type and the other two of triantennary and biantennary 'complex' type. The ratio of triantennary/biantennary chains was decreased on Thy-1 of mature cells compared with that of immature cells, but instead more sialic acid was present on these chains. Deglycosylated Thy-1 appeared to be of the same size regardless of origin, indicating that only the carbohydrate moiety differs between Thy-1 molecules of the two cell types.     

Infection of DBA/2 or C3H/HeJ mice by intraperitoneal injection of vaccinia virus elicits activated macrophages,cytolytic and cytostatic for S91-melanoma tumor cells

Summary Murine peritoneal macrophages harvested 3–4 days after IP injection of vaccinia virus lysed S91-melanoma tumor cells in vitro; enhanced tumoricidal activity was measured with effector macrophages prepared 5–6 days after vaccinia virus infection. Treatment of virus-elicited macrophages prepared from DBA/2 mice with anti-asialo-GM1 antiserum, anti-Thy 1.2 antiserum or anti-Ia^d antiserum in the presence of complement so that cells sensitized with antibodies were lysed, did not reduce the measured level of tumoricidal activity indicating that macrophages [Ia(–); asialo GM1(–)] and not natural killer cells [asialo GM1(+); Thy 1.2(±)] or T-cells [Thy 1.2(+)] were responsible for mediating the lysis of S91-melanoma tumor cells. When incubated with virus-elicited macrophages but not thioglycollate-elicited macrophages, the ability of S91-melanoma tumor cells, to synthesize DNA was completely blocked. The results of these experiments support the view that one aspect of antitumor immunity enhanced during immunotherapy with vaccinia virus is the activation of macrophages which have cytolytic as well as cytostatic effects on melanoma tumor cells.     

Homogeneous penetration but heterogeneous binding of antibodies to carcinoembryonic antigen in human colon carcinoma HT-29 spheroids

Summary The monoclonal antibodies 38S1, directed against the carcinoembryonic antigen (CEA), were tested for penetration and binding in human colon carcinoma HT-29 spheroids. Penetration was studied with a method which has not previously been used in immunological investigations. The method, which allows unbound substances to be visualized, is based on freeze drying, vapour fixation, dry sectioning and dry autoradiography. The antibodies penetrated easily and all parts of the HT-29 spheroids seemed to be reached within 15 min. The penetration was even faster than in control glioma U-118MG spheroids that did not express CEA. Binding of the 38S1 antibodies was demonstrated after processing with conventional histology and autoradiography. The binding in the HT-29 spheroids was, after a 1-h incubation period, extremely heterogeneous and occurred mainly in the peripheral parts. More cells were binding the antibodies after 8-h and 32-h incubations and these cells were arranged in peripheral clusters. No binding at all was seen in the CEA-negative glioma spheroids. The distribution of CEA antigens in monolayers and in frozen sections of spheroids of HT-29 cells was analysed with immunohistochemical staining using polyclonal CEA antibodies. The CEA antigens were heterogeneously distributed in both spheroids and monolayers and were as heterogeneous as the binding of the monoclonal antibodies in the living spheroids. Thus, the heterogeneous binding in the living spheroids was not due to penetration barriers, but instead to the heterogeneity in the CEA antigen expression.