1. Biochemical characterisation of an epitope on the surface membrane antigen (Cs-gp200) of the pathogenic piscine haemoflagellate Cryptobia salmositica Katz 1951
S Feng, P T Woo Exp Parasitol. 1998 Jan;88(1):3-10. doi: 10.1006/expr.1998.4202.
A protective surface antigen (200 kDa) on C. salmositica was detected using a monoclonal antibody (mAb-001). Enzymatic studies on the epitope indicated that it was sensitive to nonspecific protease K and to site-specific trypsin and protease V8 but not to alpha-chymotrypsin. The reactivity of the epitope with mAb-001 was not affected when the antigen was denatured with 8 M urea; however, reduction of the antigen with dithiothreitol destroyed the epitope. The epitope was susceptible to sodium m-periodate oxidation and N-glycosidase F, but not to O-glycosidase or neuraminidase. It was also sensitive to mild potassium hydrochloride hydrolysis and to phospholipase C, which is specific for phosphatidylinositol. These results suggest that the epitope consists of a polypeptide, a carbohydrate, and probably a phospholipid. The asparagine-bound N-glycosidically linked hybrid-type carbohydrate chain has the minimum length of a chitobiose core unit. There is probably a phosphatidylinositol residue which anchors the polypeptide to the surface membrane. The antigen is extensively posttranslationally modified.
2. Reactions of O-acyl-L-serines with tryptophanase, tyrosine phenol-lyase, and tryptophan synthase
R S Phillips Arch Biochem Biophys. 1987 Jul;256(1):302-10. doi: 10.1016/0003-9861(87)90450-4.
The reactions of tryptophanase, tyrosine phenol-lyase, and tryptophan synthase with a new class of substrates, the O-acyl-L-serines, have been examined. A method for preparation of O-benzoyl-L-serine in high yield from tert.-butyloxycarbonyl (tBoc)-L-serine has been developed. Reaction of the cesium salt of tBoc-L-serine with benzyl bromide in dimethylformamide gives tBoc-L-serine benzyl ester in excellent yield. Acylation with benzoyl chloride and triethylamine in acetonitrile followed by hydrogenolysis with 10% palladium on carbon in trifluoroacetic acid gives O-benzoyl-L-serine, isolated as the hydrochloride salt. O-Benzoyl-L-serine is a good substrate for beta-elimination or beta-substitution reactions catalyzed by both tryptophanase and tyrosine phenol-lyase, with Vmax values 5- to 6-fold those of the physiological substrates and comparable to that of S-(o-nitrophenyl)-L-cysteine. Unexpectedly, O-acetyl-L-serine is a very poor substrate for these enzymes, with Vmax values about 5% of those of the physiological substrates. Both O-acyl-L-serines are poor substrates for tryptophan synthase, measured either by the synthesis of 5-fluoro-L-tryptophan from 5-fluoroindole and L-serine catalyzed by the intact alpha 2 beta 2 subunit or by the beta-elimination reaction catalyzed by the isolated beta 2 subunit. With all three enzymes, the elimination of benzoate appears to be irreversible. These results suggest that the binding energy from the aromatic ring of O-benzoyl-L-serine is used to lower the transition-state barrier for the elimination reactions catalyzed by tryptophanase and tyrosine phenol-lyase. Our findings support the suggestion (M. N. Kazarinoff and E. E. Snell (1980) J. Biol. Chem. 255, 6228-6233) that tryptophanase undergoes a conformational change during catalysis and suggest that tyrosine phenol-lyase also may undergo a conformational change during catalysis.
3. The role of the NMDA receptors and l-arginine-nitric oxide-cyclic guanosine monophosphate pathway in the antidepressant-like effect of duloxetine in the forced swimming test
Andréa D E Zomkowski, Daiane Engel, Mauricio P Cunha, Nelson H Gabilan, Ana Lúcia S Rodrigues Pharmacol Biochem Behav. 2012 Dec;103(2):408-17. doi: 10.1016/j.pbb.2012.09.011. Epub 2012 Sep 23.
Duloxetine is a selective serotonin and noradrenaline reuptake inhibitor used as antidepressant. However, its mechanisms of action are not fully understood. This study investigated the effect of duloxetine in the mouse forced swimming test (FST) and in the tail suspension test (TST) and the involvement of the NMDA receptors and the l-arginine-NO-cGMP pathway in its effect in the FST. Duloxetine reduced the immobility time both in the FST and in the TST (dose range of 1-30mg/kg, i.p.), without changing locomotion in an open-field. Duloxetine administered orally (1-30mg/kg) also reduced the immobility time in the FST. The effect of duloxetine (10mg/kg, p.o.) in the FST was prevented by pre-treatment with NMDA (0.1pmol/site, i.c.v.), d-serine (30μg/site, i.c.v.), (l-arginine (750mg/kg, i.p.), S-nitroso-N-acetyl-penicillamine (SNAP, 25μg/site, i.c.v) or sildenafil (5mg/kg, i.p.). The administration of MK-801 (0.001mg/kg, i.p.), 7-nitroindazole (50mg/kg, i.p.), methylene blue (20mg/kg, i.p.) or 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) (30pmol/site i.c.v.) in combination with a sub-effective dose of duloxetine (0.3mg/kg, p.o.) reduced the immobility time in the FST. Moreover, the administration of duloxetine (10mg/kg) produced a reduction in NOx levels in the hippocampus and cerebral cortex. Altogether the results suggest that the effect of duloxetine in the FST is dependent on either a blockade of NMDA receptors or an inhibition of NO. In addition, our results further reinforce the role of NMDA receptors and l-arginine-NO-cGMP pathway, besides the monoaminergic systems, in the mechanism of action of current prescribed antidepressant agents.
