The cysteine desulfurase IscS is required for synthesis of all five thiolated nucleosides present in tRNA from Salmonella enterica serovar typhimurium

Deficiency of a modified nucleoside in tRNA often mediates suppression of +1 frameshift mutations. In Salmonella enterica serovar Typhimurium strain TR970 (hisC3737), which requires histidine for growth, a potential +1 frameshifting site, CCC-CAA-UAA, exists within the frameshifting window created by insertion of a C in the hisC gene. This site may be suppressed by peptidyl-tRNAProcmo5UGG (cmo(5)U is uridine-5-oxyacetic acid), making a frameshift when decoding the near-cognate codon CCC, provided that a pause occurs by, e.g., a slow entry of the tRNAGlnmnm5s2UUG (mnm(5)s(2)U is 5-methylaminomethyl-2-thiouridine) to the CAA codon located in the A site. We selected mutants of strain TR970 that were able to grow without histidine, and one such mutant (iscS51) was shown to have an amino acid substitution in the L-cysteine desulfurase IscS. Moreover, the levels of all five thiolated nucleosides 2-thiocytidine, mnm(5)s(2)U, 5-carboxymethylaminomethyl-2-thiouridine, 4-thiouridine, and N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine present in the tRNA of S. enterica were reduced in the iscS51 mutant. In logarithmically growing cells of Escherichia coli, a deletion of the iscS gene resulted in nondetectable levels of all thiolated nucleosides in tRNA except N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine, which was present at only 1.6% of the wild-type level. After prolonged incubation of cells in stationary phase, a 20% level of 2-thiocytidine and a 2% level of N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine was observed, whereas no 4-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, or mnm(5)s(2)U was found. We attribute the frameshifting ability mediated by the iscS51 mutation to a slow decoding of CAA by the tRNAGlnmnm5s2UUG due to mnm(5)s(2)U deficiency. Since the growth rate of the iscS deletion mutant in rich medium was similar to that of a mutant (mnmA) lacking only mnm(5)s(2)U, we suggest that the major cause for the reduced growth rate of the iscS deletion mutant is the lack of mnm(5)s(2)U and 5-carboxymethylaminomethyl-2-thiouridine and not the lack of any of the other three thiolated nucleosides that are also absent in the iscS deletion mutant.     

Mycorrhizal plants of traditionally managed boreal grasslands in Norway

This paper reports on the mycorrhizal status of 82 plant species growing in traditionally managed grasslands in three different locations in the boreal and boreo-nemoral vegetation zone in the eastern part of Norway. Seventy-four species were found to have arbuscular mycorrhiza (AM). To our knowledge, we report AM for the first time in Achillea ptarmica, Ajuga pyramidalis, Alchemilla glaucescens, Carex brunnescens, Carex pallescens, Crepis praemorsa, Hieracium lactucella, Rumex longifolius, Scorzonera humilis, Trifolium aureum and Trifolium spadiceum. The rare and threatened species Arnica montana, S. humilis, C. praemorsa, Gentianella campestris, Parnassia palustris, T. aureum and T. spadiceum, all confined to grasslands, were found to possess AM fungi.     

Bioengineering of a phytoremediation plant by means of somatic hybridization

Phytoremediation is a technology that exploits a plant's ability to remove contaminants from the environment or render toxic compounds harmless. An efficient metal phytoremediating plant must combine high biomass production and established cultivation methods with high tolerance to a specific contaminant and ability for root uptake, translocation, and compartmentalization of contaminants in the above-ground biomass. Symmetric and asymmetric somatic hybridizations were used to introduce toxic metal-resistant traits from Thlaspi caerulescens into Brassica juncea. B. juncea hypocotyl protoplasts were fused with T. caerulescens mesophyll protoplasts. The hypocotyl protoplasts of B. juncea were stained with CFDA before fusion and thus fluoresced green under UV, whereas the mesophyll protoplasts of T. caerulescens had red autofluorescense. Heteroplasmic fusion products were identified and selected by flow cytometry and cell sorting. All putative hybrids grown in the greenhouse had morphological characteristics of B. juncea. A Thlaspi-specific repetitive sequence was hybridized to total DNA of plants, including the parental species. All plants from both symmetric and asymmetric fusions showed Thlaspi-specific hybridization patterns while B. juncea did not exhibit any hybridization signal. Hybrid plants, produced by asymmetric somatic hybridization between the two species, demonstrated high metal accumulation potential, tolerance to toxic metals, and good biomass production.     

Recombinant aequorin as a reporter for receptor-mediated changes of intracellular Ca2+ -levels in Drosophila S2 cells

The bioluminescent Ca²⁺-sensitive reporter protein, aequorin, was employed to develop an insect cell-based functional assay system for monitoring receptor-mediated changes of intracellular Ca^{2 +}-concentrations. Drosophila Schneider 2 (S2) cells were genetically engineered to stably express both apoaequorin and the insect tachykinin-related peptide receptor, STKR. Lom-TK III, an STKR agonist, was shown to elicit concentration-dependent bioluminescent responses in these S2-STKR-Aeq cells. The EC₅₀ value for the calcium effect detected by means of aequorin appeared to be nearly identical to the one that was measured by means of Fura-2, a fluorescent Ca^{2 +}-indicator. In addition, this aequorin-based method was also utilised to study receptor antagonists. Experimental analysis of the effects exerted by spantide I, II and III, three potent substance P antagonists, on Lom-TK III-stimulated S2-STKR-Aeq cells showed that these compounds antagonise STKR-mediated responses in a concentration-dependent manner. The rank order of inhibitory potencies was spantide III > spantide II > spantide I. Revised version received: 12 September 2001 Electronic Publication     

Intravascular plug formation induced by poly-APS is the principal mechanism of the toxin's lethality in rats/rat tissues

Toxic water soluble polymeric 3-alkylpyridinium salts isolated from the sponge Raniera sarai strongly inhibited AChE in vitro. In vivo, experimental animals died due to plugs formed in microcirculation. The mechanism of this plug formation is unknown. In vitro, the toxin did not affect the coagulation rate, but the rate of platelet aggregation was accelerated in a dose-dependent manner. The hemolytic activity of poly-APS was diminished by the addition of serum proteins in a dose-dependent manner. These results support the conclusion that non-specific binding to proteins is the underlying mechanism of the lethality of poly APS.     

Apoptotic pathways: involvement of lysosomal proteases

Apoptosis or programmed cell death is the major mechanism used by multicellular organisms to remove infected, excessive and potentially dangerous cells. Cysteine proteases from the caspase family play a crucial role in the process. However, there is increasing evidence that lysosomal proteases are also involved in apoptosis. In this review various lysosomal proteases and their potential contribution to propagation of apoptosis are discussed.     

Intracerebroventricular interleukin-6 treatment decreases body fat in rats

Recently we found that interleukin-6 (IL-6) knockout mice develop mature-onset obesity and that a single intracerebroventricular (ICV) injection of IL-6 increases energy expenditure. In the present study we investigated if chronic ICV treatment with IL-6 can suppress body fat mass. IL-6 was injected ICV daily for two weeks to rats fed a high-fat diet. IL-6 treatment but not saline treatment decreased body weight by 8.4% and decreased the relative weights of mesenteric and retroperitoneal fat pads. Consistent with this, circulating leptin levels were decreased by 40% after IL-6 treatment but not after saline treatment. Average food intake per day was decreased in the IL-6 treated group compared to the saline treated rats. IL-6 treatment did not change hepatic expression of the acute-phase protein haptoglobin, serum levels of insulin or insulin-like growth factor-I, or the weights of the heart, liver, kidneys, adrenals, and spleen. We conclude that centrally administered IL-6 can decrease body fat in rats without causing acute-phase reaction.     

The surface exposed amino acid residues of monomeric proteins determine the partitioning in aqueous two-phase systems

It is of great interest and importance how different amino acid residues contribute to and affect the properties of a protein surface. Partitioning in aqueous two-phase systems has the potential to be used as a rapid and simple method for studying the surface properties of proteins. The influence on partitioning of the surface exposed amino acid residues of eight structurally determined monomeric proteins has been studied. The proteins were characterized in terms of surface exposed residues with a computer program, Graphical Representation and Analysis of Surface Properties (GRASP), and partitioned in two EO30PO70-dextran aqueous two-phase systems, only differing in polymer concentrations (system I: 6.8% EO30PO70, 7.1% dextran; system II: 9% EO30PO70, 9% dextran). We show for the first time that the partitioning behaviour of different monomeric proteins can be described by the differences in surface exposed amino acid residues. The contribution to the partition coefficient of the residues was found to be best characterized by peptide partitioning in the aqueous two-phase system. Compared to hydrophobicity scales available in the literature, each amino acid contribution is characterized by the slope given by the graph of log K against peptide chain length, for peptides of different length containing only one kind of residue. It was also shown that each amino acid contribution is relative to the total protein surface and the other residues on the surface. Surface hydrophobicity calculations realized for systems I and II gave respectively correlation coefficients of 0.961 and 0.949 for the linear relation between log K and calculated hydrophobicity values. To study the effect on the partition coefficient of different amino acids, they were grouped into classes according to common characteristics: the presence of an aromatic group, a long aliphatic chain or the presence of charge. Using these groups it was possible to confirm that aromatic residues have the strongest effect on the partition coefficient, giving preference to the upper EO30PO70 phase of the system; on the other hand the presence of charged amino acids on the protein surface enhances the partition of the protein to the lower dextran phase. It is also important to note that the sensitivity of the EO30PO70-dextran system for the surface exposed residues was increased by increasing the polymer concentrations. The partition coefficient of a monomeric protein can thus be predicted from its surface exposed amino acid residues and the system can also be used to characterize protein surfaces of monomeric proteins in general.