The effect of neuropeptide FF in the amygdala kindling model.

OBJECTIVE: Neuropeptide FF (NPFF) and its receptors (NPFF1 R and NPFF2 R) are differentially distributed throughout the central nervous system. NPFF reduces cortical excitability in rats when administered intracerebroventricularly (i.c.v.), and both NPFF and NPFF1 R antagonists attenuate pilocarpine-induced limbic seizures. In this study, our aim was to determine whether NPFF exerts anticonvulsant or anti-epileptogenic effects in the rat amygdala kindling model for temporal lobe seizures. METHODS: Male Wistar rats were implanted with a recording/stimulation electrode in the right amygdala and a cannula in the left lateral ventricle. In a first group of animals, the afterdischarge threshold (ADT) was determined after a single i.c.v. infusion of saline (n = 8) or NPFF (1 nmol/h for 2 h; n = 10). Subsequently, daily infusion of saline (n = 8) or NPFF (1 nmol/h for 2 h; i.c.v.; n = 9) was performed, followed by a kindling stimulus (ADT+200 µA). Afterdischarge duration and seizure severity were evaluated after every kindling stimulus. A second group of rats (n = 7) were fully kindled, and the effect of saline or a high dose of NPFF (10 nmol/h for 2 h, i.c.v.) on ADT and the generalized seizure threshold (GST) was subsequently determined. RESULTS: In naive rats, NPFF significantly increased the ADT compared to control (435 ± 72 µA vs 131 ± 23 µA [P < 0.05]). When rats underwent daily stimulations above the ADT, NPFF did not delay or prevent kindling acquisition. Furthermore, a high dose of NPFF did not alter ADT or GST in fully kindled rats. CONCLUSIONS: I.c.v. administration of NPFF reduced excitability in the amygdala in naive, but not in fully kindled rats, and had no effect on kindling acquisition.

Family quality of life in 25 Belgian families: quantitative and qualitative exploration of social and professional support domains.

BACKGROUND: This article reports on the results of a study conducted in Belgium on family quality of life situated within a larger project focusing on the development of support strategies for young and adolescent siblings of persons with intellectual disabilities. The objectives of this article are twofold: (1) to present the results of the measures contained in the nine domains of the Family Quality of Life Survey-2006 (FQOLS-2006) from the perspective of parents (quantitative analysis); and (2) to come to a more in-depth understanding of two important domains of the FQOLS-2006 by exploring and comparing the quantitative and qualitative data from open-ended interviews with parents. METHOD: The FQOLS-2006 was completed by the main caregivers of 25 families living in one typical Belgian province. Subsequently, semi-structured interviews with one or both parents were conducted within the same families. Content analysis was carried out on the transcribed interviews using the qualitative software package MaxQDA. RESULTS: A detailed analysis of the quantitative data together with data from the content analysis of the interviews revealed important issues with regard to two family quality of life domains, support from others and support from services. In general, parents were satisfied with the professional support they received, whereas they were more critical of support from others. CONCLUSIONS: The quantitative data from the FQOLS-2006 were supported and further explained by the qualitative data. These findings highlight the importance of adequate professional support, which is a flexible and capable answer to each family's individual needs. The authors warn of the dangers of 'handicapism' and plea for a family-centred support approach that takes the whole family into account. Finally, they indicate the benefits of increased practical-pedagogical support.

Methylidene-imidazolone: a novel electrophile for substrate activation.

The recent three-dimensional structure of histidine ammonia-lyase revealed that the enzyme contains a 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) ring, which forms autocatalytically from an Ala-Ser143-Gly triad. This novel prosthetic group, which is also present in phenylalanine ammonia-lyase, activates substrates by electrophilic interaction. Modern analytical methods, theoretical calculations and molecular biology tools have given further insight into the mode of action of MIO.

[Omega-(adenosin-5'-O-yl)alkyl]cobalamins mimicking the posthomolysis intermediate of coenzyme B12-dependent rearrangements: kinetic investigations on methylmalonyl-CoA mutase.

Coenzyme-B12 analogues carrying oligomethylene chains (C3-C7) inserted between the central Co atom and the 5'-O atom of the adenosine moiety mimicking the putative posthomolysis intermediate in coenzyme B12-dependent rearrangements were synthesized and examined for their effects on methylmalonyl-CoA mutase from Propionibacterium shermanii. All analogues proved to be inhibitors of methylmalonyl-CoA mutase and in all cases competitive inhibition with respect to coenzyme B12 was found. Inhibition constants (Ki) were determined by two independent methods and showed in both cases the predicted trend: the Ki values versus chain length had minima at the C6 analogue in which the distance is about 10 A between the central Co atom and the 5'carbon of the adenosine, assuming a zig-zag chain conformation. This is the postulated distance between the Co and 5'-methylene paramagnetic centers generated in the methylmalonyl-CoA-coenzyme B12 complex after homolytic cleavage of the Co-C bond.

Sequence determination in oligosaccharides by relayed NOE experiments in the rotating frame.

An hybrid experiment, composed of 1H-NMR total correlation spectroscopy (TOCSY) and rotating frame nuclear Overhauser enhancement spectroscopy (ROESY) steps, makes it possible to determine both the partial (disaccharide) sequences, and the sequential order, of whole linear and branched oligosaccharide chains.

Kinetic investigations with inhibitors that mimic the posthomolysis intermediate in the reactions of coenzyme-B12-dependent glycerol dehydratase and diol dehydratase.

Kinetic investigations were performed on the coenzyme-B12-dependent glycerol dehydratase and diol dehydratase reactions using 1,2-propanediol as substrate and [omega-(adenosin-5'-O-yl)alkyl]cobalamins as mimics of the posthomolysis intermediate state of the coenzyme. All the coenzyme-B12 analogues with oligomethylene chains (C3-C7) inserted between the central Co atom and the 5' O of the adenosine moiety were competitive inhibitors with respect to coenzyme B12. The apparent inhibition constants (Ki) of the shorter-chain inhibitors, especially the C5 inhibitor, were smaller for both enzymes than those of the longer-chain (C6, C7) compounds. These results are in agreement with the expected (0.6-0.9 nm) distance between the Co and 5'-methylene paramagnetic centers in the posthomolysis intermediate state of coenzyme B12 in these reactions.

Ground water discharge and the related nutrient and trace metal fluxes into Quincy bay, Massachusetts.

Measurement of the rate and direction of ground water flow beneath Wollaston Beach, Quincy, Massachusetts by use of a heat-pulsing flowmeter shows a mean velocity in the bulk sediment of 40 cm d(-1). The estimated total discharge of ground water into Quincy Bay during October 1990 was 1324-2177 m(3) d(-1), a relatively low ground water discharge rate. The tides have only a moderate effect on the rate and direction of this flow. Other important controls on the rate and volume of ground water flow are the limited thickness, geographic extent, and permeability of the aquifer. Comparisons of published streamflow data and estimates of ground water discharge indicate that ground water makes up between 7.4-12.1% of the gaged freshwater input into Quincy Bay. The data from this study suggest the ground water discharge is a less important recharge component to Quincy Bay than predicted by National Urban Runoff Program (NURP) models.The high nitrate and low nitrite and ammonia concentrations in the ground water at the backshore well sites and low nitrate and high nitrite and ammonia concentrations in the water flowing from the foreshore suggests that denitrification is active in the sediments. The low ground water flow rates and low nitrate concentrations in the foreshore samples suggest that little or no nitrate is surviving the denitrification process to affect the planktonic community. Similarly, oxidizing conditions in the aquifer and low trace metal concentrations in the ground water samples suggest that the metals may be precipitating and binding to sedimentary phases before impacting the bay.

Measurement of inter-glycosidic 13C-1H coupling constants in a cyclic beta(1-->2)-glucan by 13C-filtered 2D (1H,1H)ROESY.

A method for measuring three-bond 13C-1H scalar coupling constants across glycosidic bonds in a cyclic beta(1-->2)-glucan icosamer is presented. This oligosaccharide molecule, with its high degree of symmetry, represents a particular challenge for NMR spectroscopy to distinguish inter-residue from intra-residue heteronuclear coupling effects. Chemically equivalent H2 protons in adjacent glucosyl residues are distinguished on the basis of their different through-space, dipolar interactions with the anomeric protons (H1). The strong NOE contact between anomeric (H1) and aglyconic (H2') protons permits the selective observation of the inter-residue heteronuclear couplings 3JC1H2' and 3JC2'H1 in a natural-abundance 13C-omega 1-half-filtered (1H,1H)ROESY experiment.

Carbohydrate dynamics at a micellar surface: GD1a headgroup transformations revealed by NMR spectroscopy.

The conformational dynamics of the carbohydrate headgroup of ganglioside GD1a, NeuAc alpha 2-->3Gal beta 1-->3GalNAc beta 1-->4[NeuAc alpha 2-->3]Gal beta 1-->4Glc beta 1-->1Cer, anchored in a perdeuterated dodecylphosphocholine micelle in aqueous solution, were probed by high resolution NMR spectroscopy. The observed 1H/1H NOE interactions revealed conformational averaging of the terminal NeuAc alpha 2-->3Gal and Gal beta 1-->3GalNAc glycosidic linkages. The pronounced flexibility of this trisaccharide moiety was substantiated further by two-dimensional proton-detected 13C T1, T1 rho and 1H/13C NOE measurements. The anchoring effect of the micelle allowed the detection of conformational fluctuations of the headgroup on the time scale of a few hundred picoseconds. NMR experiments performed on the GD1a/DPC micelles in H2O at low temperatures permitted the observation of hydroxyl proton resonances, contributing valuable conformational information.

Solution conformation of Forssman antigen probed by NOE and exchange interactions.

The conformation of Forssman glycolipid, GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1ceramide, was analysed with the aid of the rotating frame NOE and Hartmann-Hahn spectroscopy. NOE contacts between C-, O-, and N-linked protons were used for distance mapping. The glycosidic bonds that are common to globotriaosylceramide and globoside showed a similar flexibility as found for these compounds [Poppe et al., (1990) Eur. J. Biochem. 189, 313-325; J. Am. Chem. Soc. 112, 7762-7771]. In contrast, the conformational mobility of the terminal GalNAc alpha 1-3GalNAc beta linkage appears to be restrained. A new approach, based on 2D exchange spectroscopy, was proposed for revealing of spatial proximities between exchangeable protons in Me2SO solution.

The behavior of substrate analogues and secondary deuterium isotope effects in the phenylalanine ammonia-lyase reaction.

Metacresol and glycine can be thought as a dissection of metatyrosine, which is an excellent substrate of phenylalanine ammonia-lyase (PAL) (B. Schuster and J. Rétey, PNAS 92, 8433, 1995). Whereas metacresol was a very weak inhibitor and glycine was inert, simultaneous addition of both compounds led to synergistic inhibition of PAL. [2H5]Phenylalanine as a substrate showed a kinetic deuterium isotope effect of 9% (kH/k2H = 1.09 +/- 0.01) while its Km value was identical to that of the unlabeled substrate. The following substrate analogues were synthesized and assayed with PAL: cyclooctatetraenyl (COT)-d,l)-alanine as well as 2-pyridyl-, 3-pyridyl-, and 4-pyridyl-(l)-alanines. While COT-(d,l)-alanine turned out to be a rather reluctant substrate, all three isomers of pyridyl-(l)-alanines were converted with a comparable or even higher Vmax than l-phenylalanine into the corresponding pyridyl acrylic acids. Their Km values were, however, an order of magnitude higher than that of the natural substrate. These results are discussed in terms of the novel mechanism which implies an electrophilic attack of the prosthetic dehydroalanine at the aromatic ring. The heats of formation of the putative sigma complexes of the electrophilic substitution at the pyridine ring have been calculated using semiempirical force-field methods. The results show the feasibility of the proposed mechanism also with the substrate analogues.

The solution conformation of sialyl-alpha (2----6)-lactose studied by modern NMR techniques and Monte Carlo simulations.

We present a comprehensive strategy for detailed characterization of the solution conformations of oligosaccharides by NMR spectroscopy and force-field calculations. Our experimental strategy generates a number of interglycosidic spatial constraints that is sufficiently large to allow us to determine glycosidic linkage conformations with a precision heretofore unachievable. In addition to the commonly used [1H,1H] NOE contacts between aliphatic protons, our constraints are: (a) homonuclear NOEs of hydroxyl protons in H2O to other protons in the oligosaccharide, (b) heteronuclear [1H,13C] NOEs, (c) isotope effects of O1H/O2H hydroxyl groups on 13C chemical shifts, and (d) long-range heteronuclear scalar couplings across glycosidic bonds. We have used this approach to study the trisaccharide sialyl-alpha (2----6)-lactose in aqueous solution. The experimentally determined geometrical constraints were compared to results obtained from force-field calculations based on Metropolis Monte Carlo simulations. The molecule was found to exist in 2 families of conformers. The preferred conformations of the alpha (2----6)-linkage of the trisaccharide are best described by an equilibrium of 2 conformers with phi angles at -60 degrees or 180 degrees and of the 3 staggered rotamers of the omega angle with a predominant gt conformer. Three intramolecular hydrogen bonds, involving the hydroxyl protons on C8 and C7 of the sialic acid residue and on C3 of the reducing-end glucose residue, contribute significantly to the conformational stability of the trisaccharide in aqueous solution.

Phenylalanine ammonia-lyase: the use of its broad substrate specificity for mechanistic investigations and biocatalysis--synthesis of L-arylalanines.

Several fluoro- and chlorophenylalanines were found to be good substrates of phenylalanine ammonia-lyase (PAL/EC 4.3.1.5) from parsley. The enantiomerically pure L-amino acids were obtained in good yields by reaction of the corresponding cinnamic acids with 5M ammonia solution (buffered to pH 10) in the presence of PAL. The kinetic constants for nine different fluoro- and chlorophenylalanines do not provide a rigorous proof for but are consistent with the previously proposed mechanism comprising an electrophilic attack of the methylidene-imidazolone cofactor of PAL at the aromatic nucleus as a first chemical step. In the resulting Friedel-Crafts-type sigma complex the beta-protons are activated for abstraction and consequently the pro-S is abstracted by an enzymic base. Results from semi-empirical calculations combined with a proposed partial active site model showed a correlation between the experimental kinetic constants and the change in polarization of the pro-S Cbeta-H bond and heat of formation of the sigma complexes, thus making the electrophilic attack at the neutral aromatic ring plausible. Furthermore, while 5-pyrimidinylalanine was found to be a moderately good substrate of PAL, 2-pyrimidinylalanine was an inhibitor.

Effects of indomethacin on intestinal secretion, prostaglandin E and cyclic AMP: evidence against a role for prostaglandins in cholera toxin-induced secretion.

The effects of indomethacin on intestine mucosal cAMP, intestinal fluid secretion, and mucosal and fluid PGE were studied in rabbits in vivo following challenge with cholera toxin. Indomethacin had no effect on cholera toxin-induced fluid secretion or cAMP accumulation. Inhibition of PGE synthesis was achieved by the administration of two but not one injection of indomethacin. These studies provide evidence against a role for PGE in mediating cholera toxin-induced secretion and point out the need to measure prostaglandin levels when using prostaglandin synthetase inhibitors in vivo.

Prostaglandin E in cholera toxin-induced intestinal secretion. Lack of an intermediary role.

Prostaglandin E1 (PGE1) and cholera enterotoxin stimulate small-intestine mucosal adenylate cyclase and intestinal secretion of water and electrolytes. The previous suggestion that PGE may mediate cholera-toxin effects was explored in these studies. Closed rabbit jejunal loops were injected in vivo with cholera toxin and compared to similar loops in the same animal injected with buffer. Loop mucosal homogenates and intestinal secretions were analyzed by radioimmunoassay for cAMP and PGE concentrations. Cholera toxin produced significant increases in mucosal and intestinal fluid cAMP; however, there were no significant increases in PGE in the toxin-treated loops when compared to the control loops. In addition, there was no correlation between cAMP and PGE in the same samples. These studies indicate that cholera toxin stimulates intestinal cAMP anc secretion independent of PGE synthesis and provide evidence against a specific role for PGE in mediating cholera-toxin effects.

Three-dimensional structure of the oligosaccharide terminus of globotriaosylceramide and isoglobotriaosylceramide in solution. A rotating-frame NOE study using hydroxyl groups as long-range sensors in conformational analysis by 1H-NMR spectroscopy.

Spatial structures of the oligosaccharide parts of globotriaosylceramide, Gal(alpha 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer (Cer = ceramide) and isoglobotriaosylceramide, Gal(alpha 1-3)Gal(beta 1-4)Glc(beta 1-1)Cer were investigated in (C2H3)2SO solution by means of laboratory and rotating frame NOE, hydroxyl protons being used as long-range sensors defining the distance constraints. Both oligosaccharides were found to exist in more than one conformation interconverting rapidly on the NMR time scale. The conformation of the Gal(alpha 1-4)Gal(beta 1-4)Glc beta trisaccharide dissolved in 2H2O appeared to be the same as that of the corresponding part of the glycosphingolipid in (C2H3)2SO solution.

Solution conformation of sialosylcerebroside (GM4) and its NeuAc(alpha 2----3)Gal beta sugar component.

The solution conformations of GM4 ganglioside [NeuAc(alpha 2----3)Gal (beta 1----1)Cer] in (2H3C)2SO and its component disaccharide in 2H2O were investigated with the aid of 1H-nuclear magnetic resonance spectroscopy (nuclear Overhauser effect, analysis of coupling constants) and by energy-minimum calculations. The existence of three low-energy conformers obtained by theoretical calculations was supported by experimental findings in the case of GM4, whereas the disaccharide appears to exist as a mixture of two conformers.

A base-off analogue of coenzyme-B12 with a modified nucleotide loop--1H-NMR structure analysis and kinetic studies with (R)-methylmalonyl-CoA mutase, glycerol dehydratase, and diol dehydratase.

(Co beta-5'-Deoxyadenosin-5'-yl)-(p-cresolyl)cobamide (Ado-PCC), an analogue of the base-off form of coenzyme-B12 (CoB12), was prepared by alkylation of (Co alpha/beta-cyano/aqua)-(p-cresolyl)cobamide (PCC) with 5'-chloro-5'-deoxyadenosine. The 500 MHz 1H-NMR spectrum of Ado-PCC in D2O at pH 7.4 was completely analyzed using COSY and NOESY two-dimensional experiments. The coenzyme and inhibitory activities of Ado-PCC were tested with three coenzyme-B12-dependent enzymes: (R)-methylmalonyl-CoA mutase, glycerol dehydratase, and diol dehydratase. Ado-PCC showed strong coenzyme activity with methylmalonyl-CoA mutase, which is known to bind the base-off form of CoB12. In contrast, Ado-PCC had no coenzyme activity but acted instead as a competitive inhibitor with glycerol dehydratase and diol dehydratase, which are likely to prefer the base-on form of CoB12. These results indicate that Ado-PCC, whose structure is analogous to the base-off form of CoB12, can be used for probing the mode of coenzyme binding by coenzyme-B12-dependent enzymes.

Characterization of the active site of histidine ammonia-lyase from Pseudomonas putida.

Elucidation of the 3D structure of histidine ammonia-lyase (HAL, EC 4.3.1.3) from Pseudomonas putida by X-ray crystallography revealed that the electrophilic prosthetic group at the active site is 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) [Schwede, T.F., Rétey, J., Schulz, G.E. (1999) Biochemistry, 38, 5355-5361]. To evaluate the importance of several amino-acid residues at the active site for substrate binding and catalysis, we mutated the following amino-acid codons in the HAL gene: R283, Y53, Y280, E414, Q277, F329, N195 and H83. Kinetic measurements with the overexpressed mutants showed that all mutations resulted in a decrease of catalytic activity. The mutants R283I, R283K and N195A were approximately 1640, 20 and 1000 times less active, respectively, compared to the single mutant C273A, into which all mutations were introduced. Mutants Y280F, F329A and Q277A exhibited approximately 55, 100 and 125 times lower activity, respectively. The greatest loss of activity shown was in the HAL mutants Y53F, E414Q, H83L and E414A, the last being more than 20 900-fold less active than the single mutant C273A, while H83L was 18 000-fold less active than mutant C273A. We propose that the carboxylate group of E414 plays an important role as a base in catalysis. To investigate a possible participation of active site amino acids in the formation of MIO, we used the chromophore formation upon treatment of HAL with l-cysteine and dioxygen at pH 10.5 as an indicator. All mutants, except F329A showed the formation of a 338-nm chromophore arising from a modified MIO group. The UV difference spectra of HAL mutant F329A with the MIO-free mutant S143A provide evidence for the presence of a MIO group in HAL mutant F329A also. For modelling of the substrate arrangement within the active site and protonation state of MIO, theoretical calculations were performed.