Cloning, isolation and characterization of the Thermotoga maritima KDPG aldolase

The Thermotoga maritima aldolase gene has been cloned into a T7 expression vector and overexpressed in Escherichia coli. The preparation yields 470 UL(-1) of enzyme at a specific activity of 9.4 U mg(-1). During retroaldol cleavage of KDPG, the enzyme shows a k(cat) that decreases with decreasing temperature. A more than offsetting decrease in K(m) yields an enzyme that is more efficient at 40 degrees C than at 70 degrees C. The substrate specificity of the enzyme was evaluated in the synthetic direction with a range of aldehyde substrates. Although the protein shows considerable structural homology to KDPG aldolases from mesophilic sources, significant differences in substrate specificity exist. A preparative scale reaction between 2-pyridine carboxaldehyde and pyruvate provided product of the same absolute configuration as mesophilic enzymes, but with diminished stereoselectivity.     

Selective excitation of GABAergic neurons in the substantia nigra of the rat by orexin/hypocretin in vitro

Dysfunction of the orexin/hypocretin neurotransmitter system leads to the sleep disorder narcolepsy. Narcolepsy is characterized by excessive daytime sleepiness and the occurrence of cataplexy--a sudden loss of muscle tone triggered by emotionally arousing events. Both symptoms can be treated with drugs that act on dopaminergic systems. Here we have investigated the effect of orexins on the firing of dopaminergic and GABAergic neurons of the substantia nigra (SN) in brain slices. Surprisingly, dopaminergic neurons in pars compacta were unaffected by orexins. In contrast, bath application of orexin A (100 nM) or orexin B (5-300 nM) greatly increased the firing rate of GABAergic neurons in pars reticulata. The orexin B-mediated excitation was unaffected by blocking synaptic transmission (using low-Ca2+/high-Mg2+ solution). However, the effect of orexin B was reduced significantly by thapsigargin (1 microM) and inhibitors of protein kinase A. The presence of orexinergic fibres in the SN pars reticulata was demonstrated by immunohistochemical methods with the fibre density increasing in the rostrocaudal direction. The orexin excitation of SN reticulata cells may help to maintain their high firing rate during waking. Furthermore, the absence of orexin effects in narcolepsy may predispose affected individuals to attacks of cataplexy.     

Characterization of a sialate pyruvate-lyase in the cytosol of human erythrocytes

Sialate pyruvate-lyases, also known as sialate aldolases (EC 4.1.3.3), reversibly catalyse the cleavage of free N-acetylneuraminic acids to form pyruvate and N-acetylmannosamine. These enzymes are widely distributed and are present in numerous pro- and eukaryotic cells, in which they are localized only in the cytosol. They play an important role in the regulation of sialic acid metabolism by controlling the intracellular concentration of sialic acids of biosynthetic or exogenous origin, thus preventing the accumulation of toxic levels of this sugar. Application of an original colorimetric micromethod for N-acetylmannosamine determination, as well as the use of [4,5,6,7,8,9-14C]N-acetylneuraminic acid, led us to evidence a cytosolic neuraminate aldolase activity in human red blood cells (RBCs) and then to define the main characteristics of this enzyme: Michaelis-Menten type, K(m:) 1.4 +/- 0.05 mM, optimal pH: 7.6 +/- 0.2, optimal temperature: 70 +/- 2 degrees C, inhibition by heavy metals: Ag(+) and Hg(++). These enzyme parameters are close to those of the bacterial and mammalian aldolases described up to now. At the moment, the presence of sialate pyruvate-lyase in the cytosol of red blood cells remains an enigma.     

Comparative cytotoxic and cytoprotective effects of taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA) in HepG2 cell line

This study was performed to compare the effects of two hydrophilic bile acids, taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA), on HepG2 cells. Cytotoxicity was evaluated at different times of exposure by incubating cells with increasing concentrations (50-800 micromol/l) of either bile acid, while their cytoprotective effect was tested in comparison with deoxycholic acid (DCA) (350 micromol/l and 750 micromol/l)-induced cytotoxicity. Culture media, harvested at the end of each incubation period, were analyzed to evaluate aspartate transaminase (AST), alanine transaminase and gamma-glutamyltranspeptidase release. In addition, the hemolytic effect of THDCA and TUDCA on human red blood cells was also determined. At 24 h of incubation neither THDCA nor TUDCA was cytotoxic at concentrations up to 200 and 400 micromol/l. At 800 micromol/l both THDCA and TUDCA induced a slight increase in AST release. At this concentration and with time of exposure prolonged up to 72 h, THDCA and TUDCA induced a progressive increase of AST release significantly (P<0.05) higher than that of controls being AST values for THDCA (2.97+/-0.88 time control value (tcv) at 48 h and 4.50+/-1.13 tcv at 72 h) significantly greater than those of TUDCA (1.50+/-0.20 tcv at 48 h and 1.80+/-0.43 tcv at 72 h) (P<0.01). In cytoprotection experiments, the addition of 50 micromol/l THDCA decreased only slightly (-5%) AST release induced by 350 micromol/l DCA, while the addition of 50 micromol/l TUDCA was significantly effective (-23%; P<0.05). Higher doses of THDCA or TUDCA did not reduce toxicity induced by 350 micromol/l DCA, but were much less toxic than an equimolar dose of DCA alone. At the concentration used in this experimental model neither THDCA nor TUDCA was hemolytic; however at a very high concentration (6 mmol/l) both bile acids induced 5-8% hemolysis. We conclude that bile acid molecules with a similar degree of hydrophilicity may show different cytotoxic and cytoprotective properties.     

SUMO-1 targets RanGAP1 to kinetochores and mitotic spindles

RanGAP1 was the first documented substrate for conjugation with the ubiquitin-like protein SUMO-1. However, the functional significance of this conjugation has not been fully clarified. We sought to examine RanGAP1 behavior during mitosis. We found that RanGAP1 associates with mitotic spindles and that it is particularly concentrated at foci near kinetochores. Association with kinetochores appeared soon after nuclear envelope breakdown and persisted until late anaphase, but it was lost coincident with nuclear envelope assembly in telophase. A mutant RanGAP1 protein lacking the capacity to be conjugated to SUMO-1 no longer associated with spindles, indicating that conjugation was essential for RanGAP1's mitotic localization. RanBP2, a nuclear pore protein that binds SUMO-1-conjugated RanGAP1 during interphase, colocalized with RanGAP1 on spindles, suggesting that a complex between these two proteins may be involved in mitotic targeting of RanGAP1. This report shows for the first time that SUMO-1 conjugation is required for mitotic localization of RanGAP1, and suggests that a major role of SUMO-1 conjugation to RanGAP1 may be the spatial regulation of the Ran pathway during mitosis.     

Isolation and characterization of a potato cDNA corresponding to a 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene differentially activated by stress

1-Aminocyclopropane-1-carboxylate (ACC) oxidase enzyme catalyses the final step in ethylene biosynthesis, converting 1-aminocyclopropane-1-carboxylic acid to ethylene. A cDNA clone encoding an ACC oxidase, ST-ACO3, was isolated from potato (Solanum tuberosum L.) by differential screening of a Fusarium eumartii infected-tuber cDNA library. The deduced amino acid sequence exhibited similarity to other ACC oxidase proteins from several plants species. Northern blot analysis revealed that the ST-ACO3 mRNA level increased in potato tubers upon inoculation with F. eumartii, as well as after treatment with salicylic acid and indole-3-acetic acid, suggesting a cross-talk between different signalling pathways involved in the defence response of potato tubers against F. eumartii attack.     

Cyanobacterial leader peptides for protein secretion

The leader peptide of the major secreted protein PilA1 of the cyanobacterium Synechocystis sp. strain PCC 6803 and several artificial leader peptides have been used to study secretion of the reporter protein lichenase to the culture medium. The strains of Synechocystis carrying lichenase with the leader sequences of PilA and with the leader sequence of Slr2016 efficiently secreted the reporter protein. The artificial leader sequence that was characterized by the overall positive charge (as PilA1 and Slr2016 leaders) also allowed secretion. The artificial leader with negative charge, however, did not allow secretion of the reporter protein. Moreover, no secreted proteins have been isolated from this strain using conventional techniques for preparation of secreted proteins. These data suggest that the general secretion pathway in cyanobacteria, at least for pilins, recognizes the overall charge of the leader sequences, and operates in a sequence-non-specific manner.     

Transformation of tobacco with a gene for the thermophilic acyl-lipid desaturase enhances the chilling tolerance of plants

The desC gene for the acyl-lipid Delta9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus was introduced into Nicotiana tabacum under control of the 35S promoter. Expression of the desaturase was confirmed by Western blotting. Lipid analysis revealed that lipid content and the extent of fatty acid unsaturation significantly increased in leaves of transgenic plants. Chilling tolerance of those plants also increased, as estimated by the electrolyte leakage from the tissues damaged by cold treatments. Seeds of plants that expressed the desC gene imbibed at low temperatures demonstrated higher chilling tolerance than those of the control plants. The results demonstrate that the cyanobacterial thermophilic acyl-lipid desaturase was efficiently expressed in tobacco at ambient temperatures, and its expression resulted in the enhanced chilling tolerance of the transgenic plants.     

The Drosophila orphan nuclear receptor DHR38 mediates an atypical ecdysteroid signaling pathway

Ecdysteroid pulses trigger the major developmental transitions during the Drosophila life cycle. These hormonal responses are thought to be mediated by the ecdysteroid receptor (EcR) and its heterodimeric partner Ultraspiracle (USP). We provide evidence for a second ecdysteroid signaling pathway mediated by DHR38, the Drosophila ortholog of the mammalian NGFI-B subfamily of orphan nuclear receptors. DHR38 also heterodimerizes with USP, and this complex responds to a distinct class of ecdysteroids in a manner that is independent of EcR. This response is unusual in that it does not involve direct binding of ecdysteroids to either DHR38 or USP. X-ray crystallographic analysis of DHR38 reveals the absence of both a classic ligand binding pocket and coactivator binding site, features that seem to be common to all NGFI-B subfamily members. Taken together, these data reveal the existence of a separate structural class of nuclear receptors that is conserved from fly to humans.