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.     

Characteristic chromosome abnormalities,including rearrangements of 6p,del(7q), +12, and t(12;14), in 44 uterine leiomyomas

Summary The cytogenetic analysis of 224 leiomyomas from 138 patients is presented. An insufficient number of mitoses was found in 35 tumors, normal karyotypes in 145, and clonal chromosome aberrations were detected in 44. The three previously identified cytogenetic subgroups were all represented in this series: del(7) (q21.2q31.2) was found in 11, trisomy 12 in five, and t(12;14)(q14-15;q23-24) in one leiomyoma. Rearrangements of 6p, including deletions, inversions, and various translocations, were found in eight tumors, thus delineating a new cytogenetic subgroup of uterine leiomyoma. The remaining 21 karyotypically abnormal tumors had nonrecurrent changes. One leiomyoma had two cytogenetically unrelated clones characterized by del(7)(q21.2 q31.2) and +12. Karyotypic changes in two separate leiomyomas from the same uterus were identified in five patients; in three of them, different anomalies were found in the two tumors, whereas cytogenetically identical aberrations – del(7q) and dic(21;22) – were detected in two macroscopically discrete tumors. These findings suggest that whereas some multiple leiomyomas originate independently, others may be derived from the same neoplastic clone.     

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.     

Covalent immobilization of the multienzyme enniatin synthetase

Summary The multienzyme enniatin synthetase was covalently immobilized to N-hydroxysuccinimide activated agarose. The stability of the immobilized enzyme at 25°C was enhanced compared to the soluble enzyme. Immobilization experiments also indicated that the enniatins are synthesized by a single molecule and thus do not require interactions of several enzyme molecules.     

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.     

Production and characterization of KDO-specific monoclonal antibodies recognizing lipopolysaccharides from heptoseless mutants of Salmonella

Abstract Hybrid cell lines producing monoclonal antibodies with specificity for the lipopolysaccharide (LPS) from the deep rough mutant Salmonella minnesota R595 have been established. Spleen cells from BALB/c mice immunized with live R595 bacteria were fused with Sp 2/0 myeloma cells and three hybridomas producing antibodies specific for heptoseless LPS from Salmonella were selected. All three monoclonal antibodies were shown to bind only to heptoseless, but 3-deoxy- d -manno-octulosonic acid (KDO) containing LPS when tested in enzyme-linked immunosorbent assay (ELISA) against a set of structurally defined LPS and lipid A from Salmonella, Shigella and Escherichia coli . Synthetic KDO was an efficient inhibitor of the antibody-R595 LPS interaction defining that KDO is in an immunodeterminant position interacting with the monoclonal antibodies.