Ceramide inhibits the outward potassium current in rat pinealocytes

CD1 mice lacking the CB1 receptors (knockout, KO) were compared with wild-type littermates for their ability to degrade N-arachidonoylethanolamine (anandamide, AEA) through a membrane transporter (AMT) and a fatty acid amide hydrolase (FAAH). The regional distribution and age-dependence of AMT and FAAH activity were investigated. Anandamide membrane transporter and FAAH increased with age in knockout mice, whereas they showed minor changes in wild-type animals. Remarkably, they were higher in all brain areas of 6-month-old knockout versus wild-type mice, and even higher in 12-month-old animals. The molecular mass (approximately 67 kDa) and isoelectric point (approximately 7.6) of mouse brain FAAH were determined and the FAAH protein content was shown to parallel the enzyme activity. The kinetic constants of AMT and FAAH in the cortex of wild-type and knockout mice at different ages suggested that different amounts of the same proteins were expressed. The cortex and hippocampus of wild-type and knockout mice contained the following N-acylethanolamines: AEA (8% of total), 2-arachidonoylglycerol (5%), N-oleoylethanolamine (20%), N-palmitoylethanolamine (53%) and N-stearoylethanolamine (14%). These compounds were twice as abundant in the hippocampus as in the cortex. Minor differences were observed in AEA or 2-arachidonoylglycerol content in knockout versus wild-type mice, whereas the other compounds were lower in the hippocampus of knockout versus wild-type animals.     

Enantioselective hydrolysis of racemic styrene oxide by epoxide hydrolase of<Emphasis Type="Italic">Rhodosporidium kratochvilovae</Emphasis> SYU-08

Enantioselective hydrolysis for the production of chiral styrene oxide was investigated using the epoxide hydrolase activity of a newly isolatedRhodosporidium kratochvilovae SYU-08. The effects of reaction prameters—buffer type, pH, temperature, initial substrate concentrations, phenyl-1,2-ethanediol concentrations on hydrolysis rate, and enantioselectivity—were analyzed. Optically active (S)-styrene oxide with an enantiomeric excess higher than 99 % was obtained from its racemate with a yield of 38 % (theoretically 50% maximum yield) from an initial concentration of 80 mM.     

A novel family of hemicellulolytic α-glucuronidase

Investigation of the xylanolytic enzyme system of the xylose-fermenting yeast Pichia stipitis resulted in the discovery of an extracellular α-glucuronidase efficiently debranching hardwood glucuronoxylan. This activity is not exhibited by more extensively investigated α-glucuronidases of glycoside hydrolase (GH) family 67, operating on substrates in which the uronic acid is linked to the non-reducing xylopyranosyl residues of main chain fragments. The N-terminus of the purified enzyme corresponded exactly to the P. stipitis gene ABN67901 coding for a protein of unknown function. BLAST search revealed the presence of similar genes in genomes of other microorganisms. These results lead to the emergence of a new family of α-glucuronidases.     

Degradation of carbohydrate moieties of arabinogalactan-proteins by glycoside hydrolases from Neurospora crassa

Arabinogalactan-proteins (AGPs) are a family of plant proteoglycans having large carbohydrate moieties attached to core-proteins. The carbohydrate moieties of AGPs commonly have β-(1→3)(1→6)-galactan as the backbone, to which other auxiliary sugars such as l-Ara and GlcA are attached. For the present study, an α-l-arabinofuranosidase belonging to glycoside hydrolase family (GHF) 54, NcAraf1, and an endo-β-(1→6)-galactanase of GHF 5, Nc6GAL, were identified in Neurospora crassa. Recombinant NcAraf1 (rNcAraf1) expressed in Pichia pastoris hydrolyzed radish AGPs as well as arabinan and arabinoxylan, showing relatively broad substrate specificity toward polysaccharides containing α-l-arabinofuranosyl residues. Recombinant Nc6GAL (rNc6GAL) expressed in P. pastoris specifically acted on β-(1→6)-galactosyl residues. Whereas AGP from radish roots was hardly hydrolyzed by rNc6GAL alone, β-(1→6)-galactan side chains were reduced to one or two galactan residues by a combination of rNcAraf1 and rNc6GAL. These results suggest that the carbohydrate moieties of AGPs are degraded by the concerted action of NcAraf1 and Nc6GAL secreted from N. crassa.     

BcXTH1, a Brassica campestris homologue of Arabidopsis XTH9, is associated with cell expansion

Xyloglucan endotransglucosylase/hydrolases (XTHs) are a group of the enzymes that are responsible for reorganization of the cellulose–xyloglucan framework by catalyzing cleavage and religation of the xyloglucan chains in the plant cell wall. In this study, we report the isolation and characterization of a XTH gene from a pistil cDNA library of Brassica campestris. Sequence analysis of the gene, designated BcXTH1, revealed that it is homologous to the XTH9 gene of Arabidopsis. The highly conserved domain (DEIDFEFLG) found among all XTHs was also present in BcXTH1 but with the two amino acid substitutions (NEFDFEFLG) also found in Arabidopsis XTH9. These results suggest that BcXTH1 is the B. campestris homologue of XTH9. Expression analysis of BcXTH1 revealed that it was expressed in most of the plant organs. In situ hybridization showed that the gene is highly expressed in the floral primodia, especially in the epidermal cell layer. Southern blot analysis indicated that the BcXTH1 gene exists as a multi-copy gene in the B. campestris genome. The function of the BcXTH1 gene was deduced from using an overexpression strategy in Arabidopsis. Interestingly, the transgenic plants showed a pronounced cell expansion phenotype. Immunoelectron microscopy shows that BcXTH1 is localized almost exclusively to the cell wall, supporting our conclusion that it participates in the regulation of cell expansion in B. campestris.     

Defining natural factors that stimulate and inhibit cellulose:xyloglucan hetero-transglucosylation

Certain transglucanases can covalently graft cellulose and mixed-linkage β-glucan (MLG) as donor substrates onto xyloglucan as acceptor substrate and thus exhibit cellulose:xyloglucan endotransglucosylase (CXE) and MLG:xyloglucan endotransglucosylase (MXE) activities in vivo and in vitro. However, missing information on factors that stimulate or inhibit these hetero-transglucosylation reactions limits our insight into their biological functions. To explore factors that influence hetero-transglucosylation, we studied Equisetum fluviatile hetero-trans-β-glucanase (EfHTG), which exhibits both CXE and MXE activity, exceeding its xyloglucan:xyloglucan homo-transglucosylation (XET) activity. Enzyme assays employed radiolabelled and fluorescently labelled oligomeric acceptor substrates, and were conducted in vitro and in cell walls (in situ). With whole denatured Equisetum cell walls as donor substrate, exogenous EfHTG (extracted from Equisetum or produced in Pichia) exhibited all three activities (CXE, MXE, XET) in competition with each other. Acting on pure cellulose as donor substrate, the CXE action of Pichia-produced EfHTG was up to approximately 300% increased by addition of methanol-boiled Equisetum extracts; there was no similar effect when the same enzyme acted on soluble donors (MLG or xyloglucan). The methanol-stable factor is proposed to be expansin-like, a suggestion supported by observations of pH dependence. Screening numerous low-molecular-weight compounds for hetero-transglucanase inhibition showed that cellobiose was highly effective, inhibiting the abundant endogenous CXE and MXE (but not XET) action in Equisetum internodes. Furthermore, cellobiose retarded Equisetum stem elongation, potentially owing to its effect on hetero-transglucosylation reactions. This work provides insight and tools to further study the role of cellulose hetero-transglucosylation in planta by identifying factors that govern this reaction.     

Production of epoxide hydrolases in batch fermentations of <Emphasis Type="Italic">Botryosphaeria rhodina</Emphasis>

The filamentous fungus Botryosphaeria rhodina (ATCC 9055) was investigated related to its ability for epoxide hydrolase (EH) production. Epoxide hydrolase activity is located at two different sites of the cells. The larger part is present in the cytosol (70%), while the smaller part is associated to membranes (30%). In media optimization experiments, an activity of 3.5 U/gDW for aromatic epoxide hydrolysis of para-nitro-styrene oxide (pNSO) could be obtained. Activity increased by 30% when pNSO was added to the culture during exponential growth. An increase of enzyme activity up to 6 U/gDW was achieved during batch-fermentations in a bioreactor with 2.7 l working volume. Evaluation of fermentations with 30 l working volume revealed a relation of oxygen uptake rate to EH expression. Oxygen limitation resulted in a decreased EH activity. Parameter estimation by the linearization method of Hanes yielded Km values of 2.54 and 1.00 mM for the substrates S-pNSO and R-pNSO, respectively. vmax was 3.4 times higher when using R-pNSO. A protein purification strategy leading to a 47-fold increase in specific activity (940 U/mgProtein) was developed as a first step to investigate molecular and structural characteristics of the EH.     

An albumin–butyrylcholinesterase for cocaine toxicity and addiction: Catalytic and pharmacokinetic properties

Butyrylcholinesterase (BChE, EC 3.1.1.8) is important in human cocaine metabolism despite its limited ability to hydrolyze this drug. Efforts to improve the catalytic efficiency of this enzyme have led to a quadruple mutant cocaine hydrolase, “CocH”, that in animal models of addiction appears promising for treatment of overdose and relapse. We incorporated the CocH mutations into a BChE–albumin fusion protein, “Albu-CocH”, and evaluated the pharmacokinetics of the enzyme after i.v. injection in rats. As assessed from the time course of cocaine hydrolyzing activity in plasma, Albu-CocH redistributed into extracellular fluid (16% of estimated total body water) with a t_1/2 of 0.66 h and it underwent elimination with a t_1/2 of 8 h. These results indicate that the enzyme has ample stability for short-term applications and may be suitable for longer-term treatment as well. Present data also confirm the markedly enhanced power of Albu-CocH for cocaine hydrolysis and they support the view that Albu-CocH might prove valuable in treating phenomena associated with cocaine abuse.     

Expression of xyloglucan endotransglucosylase/hydrolase (XTH) genes and XET activity in ethylene treated apple and tomato fruits

Xyloglucan endotransglucosylase/hydrolase (XTHs: EC 2.4.1.207 and/or EC 3.2.1.151), a xyloglucan modifying enzyme, has been proposed to have a role during tomato and apple fruit ripening by loosening the cell wall. Since the ripening of climacteric fruits is controlled by endogenous ethylene biosynthesis, we wanted to study whether XET activity was ethylene-regulated, and if so, which specific genes encoding ripening-regulated XTH genes were indeed ethylene-regulated. XET specific activity in tomato and apple fruits was significantly increased by the ethylene treatment, as compared with the control fruits, suggesting an increase in the XTH gene expression induced by ethylene. The 25 SlXTH protein sequences of tomato and the 11 sequences MdXTH of apple were phylogenetically analyzed and grouped into three major clades. The SlXTHs genes with highest expression during ripening were SlXTH5 and SlXTH8 from Group III-B, and in apple MdXTH2, from Group II, and MdXTH10, and MdXTH11 from Group III-B. Ethylene was involved in the regulation of the expression of different SlXTH and MdXTH genes during ripening. In tomato fruit fifteen different SlXTH genes showed an increase in expression after ethylene treatment, and the SlXTHs that were ripening associated were also ethylene dependent, and belong to Group III-B (SlXTH5 and SlXTH8). In apple fruit, three MdXTH showed an increase in expression after the ethylene treatment and the only MdXTH that was ripening associated and ethylene dependent was MdXTH10 from Group III-B. The results indicate that XTH may play an important role in fruit ripening and a possible relationship between XTHs from Group III-B and fruit ripening, and ethylene regulation is suggested.