Characterization of (2S,2'R,3'R)-2-(2',3'-[3H]-dicarboxycyclopropyl)glycine binding in rat brain

[(2S,2'R,3'R)-2-(2',3'-[3H]Dicarboxycyclopropyl)glycine ([3H]DCG IV) binding was characterized in vitro in rat brain cortex homogenates and rat brain sections. In cortex homogenates, the binding was saturable and the saturation isotherm indicated the presence of a single binding site with a K(D) value of 180 +/- 33 nM and a Bmax of 780 +/- 70 fmol/mg of protein. The nonspecific binding, measured using 100 microM LY354740, was <30%. NMDA, AMPA, kainate, L(-)-threo-3-hydroxyaspartic acid, and (S)-3,5-dihydroxyphenylglycine were all inactive in [3H]DCG IV binding up to 1 mM. However, several compounds inhibited [3H]DCG IV binding in a concentration-dependent manner with the following rank order of potency: LY341495 = LY354740 > DCG IV = (2S,1'S,2'S)-2-(2-carboxycyclopropyl)glycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1'S,2'S)-2-methyl-2-(2-carboxycyclopropyl)glycine > L-glutamate = ibotenate > quisqualate > (RS)-alpha-methyl-4-phosphonophenylglycine = L(+)-2-amino-3-phosphonopropionic acid > (S)-alpha-methyl-4-carboxyphenylglycine > (2S)-alpha-ethylglutamic acid > L(+)-2-amino-4-phosphonobutyric acid. N-Acetyl-L-aspartyl-L-glutamic acid inhibited the binding in a biphasic manner with an IC50 of 0.2 microM for the high-affinity component. The binding was also affected by GTPgammaS, reducing agents, and CdCl2. In parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1, 3, and 4 > 2, 5, and 6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, subiculum, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for pre- and postsynaptic metabotropic glutamate mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to GTPgammaS show that [3H]DCG IV binding is probably to group II metabotropic glutamate receptors in rat brain.     

Anticonvulsant and glutamate release-inhibiting properties of the highly potent metabotropic glutamate receptor agonist (2S,2''R, 3''R)-2-(2'',3''-dicarboxycyclopropyl)glycine (DCG-IV)

IgA has an important function in the gastrointestinal immune system. We investigated IgA anti-ganglioside antibodies in Guillain-Barré syndrome (GBS) and Fisher's syndrome (FS) subsequent to Campylobacter jejuni enteritis. In previous studies, serological diagnosis of C. jejuni infection was based on the detection of IgG, IgA, and IgM anti-C. jejuni antibodies. Our study, however, showed that the detection of IgG anti-C. jejuni antibody alone was sufficient for the serological diagnosis of antecedent C. jejuni enteritis in GBS and FS, when the cut-off level was defined for results of sera from C. jejuni-isolated patients. Serological evidence of C. jejuni infection was found in 62 (31%) of 201 GBS patients and 12 (18%) of 65 FS patients. IgA anti-GMI antibody was detected in sera from 33 (16%) of the GBS patients, 1 (2%) of the FS patients, and none of the 46 normal control subjects. IgA anti-GM1 antibody titers were significantly higher in the GBS patients with positive C. jejuni serology than in those with negative serology (P < 0.0001) or the FS patients with positive C. jejuni serology (P = 0.007). IgA anti-GQ1b antibody was detected in sera from 18 (28%) of the FS patients, 9 (4%) of the GBS patients, and none of the normal control subjects. FS patients with positive C. jejuni serology had significantly higher titers of IgA anti-GQ1b antibody than those with negative serology (P = 0.01) or the GBS patients with positive C. jejuni serology (P < 0.0001). We conclude that anti-GM1 and anti-GQ1b IgA antibodies are closely associated with antecedent C. jejuni enteritis in GBS and FS, respectively.     

In vitro evaluation of 11C-labeled (S)-nicotine, (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole, and (R,S)-1-methyl-2-(3-pyridyl)azetidine as nicotinic receptor ligands for positron emission tomography studies

The binding characteristics of the novel 11C-labeled nicotinic ligands (R,S)-1-methyl-2-(3-pyridyl) azetidine (MPA) and (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418) were investigated in comparison with those of (S)-[11C]nicotine in vitro in the rat brain to be able to predict the binding properties of the new ligands for positron emission tomography studies in vivo. The data from time-resolved experiments for all ligands indicated fast binding kinetics, with the exception of a slower dissociation of [11C]MPA in comparison with (S)-[11C]nicotine and [11C]ABT-418. Saturation experiments revealed for all ligands two nicotinic receptor binding sites with affinity constants (K(D) values) of 2.4 and 560 nM and binding site densities (Bmax values) of 65.5 and 223 fmol/mg of protein for (S)-[11C]nicotine, K(D) values of 0.011 and 2.2 nM and Bmax values of 4.4 and 70.7 fmol/mg of protein for [11C]MPA, and K(D) values of 1.3 and 33.4 nM and Bmax values of 8.8 and 69.2 fmol/mg of protein for [11C]ABT-418. In competing with the 11C-ligands, epibatidine was most potent, followed by cytisine. A different rank order of potencies was found for (-)-nicotine, (+)-nicotine, MPA, and ABT-418 displacing each of the 11C-ligands. Autoradiograms displayed a similar pattern of receptor binding for all ligands, whereby [11C]MPA showed the most distinct binding pattern and the lowest nonspecific binding. We conclude that the three 11C-labeled nicotinic ligands were suitable for characterizing nicotinic receptors in vitro. The very high affinity of [11C]MPA to nicotinic acetylcholine receptors, its low nonspecific binding, and especially the slower dissociation kinetics of the [11C] MPA from the putative high-affinity nicotinic acetylcholine receptor binding site compared with (S)-[11C]nicotine and [11C]ABT-418 raise the level of interest in [11C]MPAfor application in positron emission tomography.     

Dynamic polarizabilities and van der Waals coefficients of the 2 (1)S and 2 (3)S metastable states of helium

Gliosis is the most frequent and therefore important neurocellular reaction to brain insult occurring in diseases ranging from AIDS to infarction. Neuropathological diagnosis of gliosis is based on morphological changes of brain glial cells. Changes commonly agreed to reflect gliosis are qualitative increases in size, number and glial fibrillary acidic protein (GFAP) immunoreactivity of astrocytes. These parameters were morphometrically quantified in brain tissues of 22 individuals who died with 7 diseases and statistically compared to the extent of gliosis independently determined by 3 qualified observers. The data indicate that the extent of gliosis correlated with the increase in size of astrocytes in white matter (p = 0.67) and this relationship was statistically significant (P = 0.0006). In contrast, the extent of gliosis was not correlated with the density of astrocytes nor the intensity of GFAP staining.     

Excitatory amino acid receptor antagonists: resolution, absolute stereochemistry, and pharmacology of (S)- and (R)-2-amino-2-(5-tert-butyl-3-hydroxyisoxazol-4-yl)acetic acid (ATAA)

We have previously shown that (RS)-2-amino-2-(5-tert-butyl-3-hydroxyisoxazol-4-yl)acetic acid (ATAA) is an antagonist at N-methyl-D-aspartic acid (NMDA) and (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors. We have now resolved ATAA via diastereomeric salt formation using N-BOC protected ATAA and (R)- and (S)-phenylethylamine. Enantiomeric purities (ee > 98%) of (R)- and (S)-ATAA were determined using the Crownpak CR(-) and CR(+) columns, respectively. The absolute configuration of (R)-ATAA was established by an X-ray crystallographic analysis of the (R)-phenylethylamine salt of N-BOC-(R)-ATAA. Like ATAA, neither (R)- nor (S)-ATAA significantly affected (IC50 > 100 microM) the receptor binding of tritiated AMPA, kainic acid, or (RS)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid, the latter being a competitive NMDA antagonist. Electrophysiological experiments, using the rat cortical wedge preparation, showed the NMDA antagonist effect as well as the AMPA antagonist effect of ATAA to reside exclusively in the (R)-enantiomer (Ki = 75 +/- 5 microM and 57 +/- 1 microM, respectively). Neither (R)- nor (S)-ATAA significantly reduced kainic acid-induced excitation (Ki > 1,000 microM).     

Solubilities of Pb+2, Bi+3, Sb+3, and Cu+1 sulfides in (NaCl+KCl+Na2S) melts

Solubilities of common metal sulfides have been determined in the (NaCl+KCl) eutectic melt with and without Na₂S. A novel gas-phase equilibrium technique has been used for PbS, Bi₂S₃, and So₂S₃, and an improved liquid phase equilibrium technique for Cu₂S, which eliminates the errors due to physical entrapment of the sulfide phase and segregation on quenching, enabling precise measurements to be made. Solubilities in the (NaCl+KCl) eutectic melt were determined as a function of temperature in the rante 700° to 950°C, and were found to be small. The partial molar heats of mixing of the sulfides in the eutectic melt have been calculated from the solubility measurements, to be 13.3, 31.4, 37.1, and 49.0 kcal for PbSs), Sb₂S₂(l), and Cu₂S(s), respectively. Sodium sulfide addition was observed to enhance these solubilities, the effect being largest for Cu₂S followed by Sb₂S₃, Bi₂S₃, and PbS. This effect is explained qualitatively. It was observed that PbS and Sb₂S₃ obey Henry's law up to saturation in (NaCl+KCl+Na₂S) melts. O. P. MOHAPATRA, formerly Graduate Student, University of Toronto     

The Absorption Spectrum of H2S Between 2150 and 4260 cm-1: Analysis of the Positions and Intensities in the First (2nu2, nu1, and nu3) and Second (3nu2, nu1 + nu2, and nu2 + nu3) Triad Regions

The two triad systems of hydrogen sulfide (2nu2, nu1, and nu3 near 4 μm and 3nu2, nu1 + nu2, and nu2 + nu3 near 2.7 μm) were analyzed using 14 spectra recorded at 0.0056 and 0.011 cm-1 resolution with the McMath Fourier transform spectrometer located at Kitt Peak National Observatory. Experimental upper state levels of H232S, H234S, and H233S were obtained from assigned positions (as high as J = 20 and Ka = 15 for the main isotope). These were fitted to the A-reduced Watson Hamiltonian to determine precise sets of rotational constants through J10 and up to nine Fermi and Coriolis coupling parameters. Intensities of the two H232S triads were modeled with rms values of 2.5%, using the transformed transition moment expansion with 19 terms for 568 intensities of the first triad and 11 terms for the 526 intensities of the second triad. The second derivatives of the dipole moment with respect to normal coordinates were estimated in Debye to be: 22μx = -0.004873 (90); 12μx = 0.01372 (30); and 23μz = 0.01578 (30). This confirmed that for hydrogen sulfide some of the second derivatives are larger than the first derivatives. The calculated line intensities were summed yielding integrated band strengths (in cm-2/atm at 296 K) as follows: 0.3315 for 2nu2, 0.4522 for nu1, 0.1201 for nu3, 0.0303 for 3nu2, 1.820 for nu1 + nu2, and 2.869 for nu2 + nu3. In addition, the hot band transitions were identified in both regions. Finally, a composite database of hydrogen sulfide line parameters was predicted for the 5- to 2.5-μm region. Copyright 1998 Academic Press.     

Inhibitory effect of tumor metastasis in mice by saponins, ginsenoside-Rb2, 20(R)- and 20(S)-ginsenoside-Rg3, of red ginseng

We examined the inhibitory effect of two saponin preparations from Red ginseng, 20(R)- and 20(S)-ginsenoside-Rg3, in comparison with that of ginsenoside-Rb2, on lung metastasis produced by two highly metastatic tumor cells, B16-BL6 melanoma and colon 26-M3.1 carcinoma, in syngeneic mice. In an in vitro analysis, both saponin preparations showed a significant inhibition of adhesion to fibronectin (FN) and laminin (LM) by B16-BL6 melanoma. Similarly, they significantly inhibited the invasion of B16-BL6 cells into the reconstituted basement membrane (Matrigel)/FN in a dose-dependent manner. In an experimental metastasis model using B16-BL6 melanoma, consecutive intravenous (i.v.) administrations of 100 micrograms/mouse of 20(R)- or 20(S)-ginsenoside-Rg3 1, 2, 3 and 4 d after tumor inoculation led to a significant decrease in lung metastasis. The inhibitory effect of i.v. administration of both ginseng saponins on the tumor metastasis of B16-BL6 melanoma was also recognized in a low dose of 10 micrograms/mouse. The oral administration (p.o.) of both saponins (100-1000 micrograms/mouse) induced a significant decrease in lung metastasis of B16-BL6 melanoma. Moreover, both ginseng saponins were effective in inhibiting of lung metastasis produced by colon 26-M3.1 carcinoma. When 20(R)- or 20(S)-ginsenoside-Rg3 was orally administered consecutively after tumor inoculation in a spontaneous metastasis model using B16-BL6 melanoma, both of them significantly inhibited lung metastasis. In the experiment involving neovasculization by tumor cells in vivo, both mice groups given each saponin preparation after tumor inoculation exhibited a significant decrease in the number of blood vessels oriented toward the tumor mass, with no repression of tumor size. These findings suggest that both ginseng saponins, 20(R)- and 20(S)-ginsenoside-Rg3, possess an ability to inhibit the lung metastasis of tumor cells, and the mechanism of their antimetastatic effect is related to inhibition of the adhesion and invasion of tumor cells, and also to anti-angiogenesis activity.     

(3R,4S)-3-[4-(4-fluorophenyl)-4-hydroxypiperidin-1-yl]chroman-4,7-diol: a conformationally restricted analogue of the NR2B subtype-selective NMDA antagonist (1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)- 1-propanol

(1S,2S)-1-(4-Hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP-101,606, 1) is a recently described antagonist of N-methyl-D-aspartate (NMDA) receptors containing the NR2B subunit. In the present study, the optimal orientation of compounds of this structural type for their receptor was explored. Tethering of the pendent methyl group of 1 to the phenolic aromatic ring via an oxygen atom prevents rotation about the central portion of the molecule. Several of the new chromanol compounds have high affinity for the racemic [3H]CP-101,606 binding site on the NMDA receptor and protect against glutamate toxicity in cultured hippocampal neurons. The new ring caused a change in the stereochemical preference of the receptor-cis (erythro) compounds had better affinity for the receptor than the trans isomers. Computational studies suggest that steric interactions between the pendent methyl group and the phenol ring in the acyclic series determine which structures can best fit the receptor. The chromanol analogue, (3R,4S)-3-[4-(4-fluorophenyl)-4-hydroxypiperidin-1- yl]chroman-4,7-diol (12a, CP-283,097), was found to possess potency and selectivity comparable to CP-101,606. Thus 12a is a new tool to explore the function of the NR2B-containing NMDA receptors.