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.     

Selenite-induced variation in glutathione peroxidase activity of three mammalian cell lines: no effect on radiation-induced cell killing or DNA strand breakage

The selenium-dependent glutathione peroxidase activities of three mammalian cell lines, HT29, P31, and N-18, cultured in medium with low serum content, increased about 2-, 5-, and 40-fold, respectively, after supplementation with 100 nM selenite. Catalase, CuZn superoxide dismutase, and Mn superoxide dismutase activities were not generally influenced by selenite supplementation, and there was only a minor nonselenium-dependent glutathione peroxidase activity in the investigated cell lines. Gamma-irradiated control and selenite-supplemented cells showed no changes in the surviving fractions, as estimated by clonogenic survival or [3H]-thymidine uptake, nor were there any significant differences between the two groups in the induction of DNA strand breaks after gamma irradiation under repairing (37 degrees C) or nonrepairing (0 degrees C) conditions. The results suggest that selenium-dependent glutathione peroxidase does not contribute significantly to the radiation resistance of cultured mammalian cells.     

Construction of an artificial bifunctional enzyme, beta-galactosidase/galactose dehydrogenase, exhibiting efficient galactose channeling

The in-frame fusion between two oligomeric enzymes, beta-galactosidase and galactose dehydrogenase, is described. The lacZ gene was fused to the 3' end of the galdh gene with a linker encoding only three amino acids. The purified artificial bifunctional enzyme displayed the enzymic activity of both gene products. The hybrid protein was found in two major forms, consisting of four and six subunits, but other forms could also be identified. The molecular weight of each subunit was determined to be 145,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bifunctional enzyme shows kinetic advantages over the identical native system in conversion of lactose to galactonolactone. A higher steady-state rate and a reduction of the transient time are observed. This phenomenon is especially pronounced at low initial substrate concentrations and when the pH is adjusted to a level at which the galactose dehydrogenase activity is much higher than that of the beta-galactosidase.     

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.     

Isolation and characterization of phage F9-11 from a lysogenicDeleya halophila strain

Thirty-eight strains ofDeleya halophila species were examined for production of phage after mitomycin C induction. Thirty-two of them were able to inhibit growth of some other strains. Phage F9-11, isolated fromD. halophila strain F9-11, showed an isometric head and a noncontractile tail. The effects of salt concentrations variation on the stability and replication of this phage were established. Its replication was possible at a wide range of marine salt concentrations, from 2.5% to 15% (wt/vol). Stability seems to be influenced by osmolarity of medium rather than by NaCl level. The euryhaline character showed by F9-11 phage is evoked as an important factor for the survival of this phage in its environment.     

Cloning of cDNAs encoding human lysosomal membrane glycoproteins, h-lamp-1 and h-lamp-2. Comparison of their deduced amino acid sequences

We have isolated and sequenced cDNA clones corresponding to the entire coding sequences of the human lysosomal membrane glycoproteins, lamp-1 and lamp-2 (h-lamp-1 and h-lamp-2). The deduced amino acid sequences indicate that h-lamp-1 and h-lamp-2 consist of 416 and 408 amino acid residues, respectively, and suggest that 27 and 28 NH2-terminal residues are cleavable signal peptides. The major portions of both h-lamp-1 and h-lamp-2 reside on the luminal side of the lysosome and are heavily glycosylated by N-glycans: h-lamp-1 and h-lamp-2 were found to contain 19 and 16 potential N-glycosylation sites, respectively. The findings are consistent with the results obtained by endo-beta-N-acetylglucosaminidase F treatment of h-lamp-1 and h-lamp-2 precursors, described in the preceding paper (Carlsson, S. R., Roth, J., Piller, F., and Fukuda, M. (1988) J. Biol. Chem. 263, 18911-18919). These N-glycosylation sites are clustered into two domains separated by a hinge-like structure enriched with proline and serine in h-lamp-1 or proline and threonine in h-lamp-2. The two domains of h-lamp-1 on each side of the hinge region are homologous to each other, whereas no such homology was detected between the two domains of h-lamp-2. Both proteins have one putative transmembrane domain consisting of 24 hydrophobic amino acids near the COOH terminus, and contain a short cytoplasmic segment composed of 11 amino acid residues at the COOH-terminal end. Comparison of h-lamp-1 and h-lamp-2 sequences reveal strong homology between the two molecules, particularly in the proximity to the COOH-terminal end. It is possible that this portion is important for targeting the molecules to lysosomes. These results also suggest that lamp-1 and lamp-2 are evolutionarily related. Comparison of known lamp-1 sequences among different species, on the other hand, show that human lamp-1 has more similarity to lamp-1 from other species than to human lamp-2. This fact, taken together with the finding that h-lamp-2 lacks repeating domains, suggests that lamp-1 and lamp-2 diverged from a putative ancestor gene in early stages of evolution. These results also suggest that lamp-1 and lamp-2 probably have distinctly separate functions despite the fact that they share many structural features.     

Successional pathways of Mediterranean evergreen vegetation on Sicily

The Mediterranean evergreen vegetation of Sicily, comprised in the belt of the Quercetea ilicis, occupies a large part of the island. Human intervention (cutting, fire, pasture) has brought about a degradation of the natural vegetation. This study is based on our phytosociological research of the Quercetea ilicis belt on Sicily.

With the ‘habitat comparison’ method, the dynamical relations between the different vegetation units have been defined.

We distinguish the following stages, with reference to their vegetation structure:

• a herbaceous stage formed by steppic vegetation, preceded by various types of nitrophilous-ruderal vegetation on abandoned fields;

• a garrigue stage dominated by half-shrubs;

• a macquis stage with various distinct plant communities, four communities being important in regressive successions, and three in progressive ones;

• a woodland and shrub-woodland stage with three different substages: pre-existent forests, present woodlands, and woodlands which tend towards the final, stable stage of vegetation (potential natural vegetation).

The dynamic relationships both in progressive and regressive successions have been synthesized in a scheme. In this scheme we have shown the main stages of the vegetation in their dynamics and we have constructed different series of vegetation types in two altitudinal belts, which are determined by varying environmental conditions of today.

The results also show that in some cases the progressive series follow different pathways than the regressive series, and the final stage of the progressive series is different from the original vegetation.