Polysaccharides of Soybean Seeds

Partial acid hydrolysate of the “hot-water-extract” fraction of soybean seed polysaccharides contained a homologous series of galacto-oligasaccharides as a major component group. Two of them were isolated by column chromatography. They gave, on methylation followed by acid hydrolysis, 2,3,4,6-tetra-, and 2,3,6-tri-O-methyl D-galactose, and were, therefore, 1,4-linked galacto-di- and trisaccharides, respectively. They were hydrolyzed with human saliva to liberate D-galactose but not with brewer’s yeast. The alditols derived from these oligosaccharides showed infrared absorptions at 885 and 895 cm^?1, respectively. These two results were strong evidences for the presence of β-linkages in the molecules of the oligossacharides. The optical rotation and the melting point of the disaccharide agreed with those of the β-1, 4-linked galactodisaccharide hitherto reported. Thus d-galacto-pyranosyl residues in the arabinogalactan are probably connected mainly by β-1,4-linkage.     

Design and synthesis of 1-(1-benzothiophen-7-yl)-1H-pyrazole,a novel series of G protein-coupled receptor 52 (GPR52) agonists

G-protein-coupled receptor 52 (GPR52) is classified as an orphan Gs-coupled G-protein-coupled receptor. GPR52 cancels dopamine D2 receptor signaling and activates dopamine D1/N-methyl-d-aspartate receptors via intracellular cAMP accumulation. Therefore, GPR52 agonists are expected to alleviate symptoms of psychotic disorders. A novel series of 1-(benzothiophen-7-yl)-1H-pyrazole as GPR52 agonists was designed and synthesized based on compound 1b. Compound 1b has been reported by our group as the first orally active GPR52 agonist, but high lipophilicity and poor aqueous solubility still remained as issues for candidate selection. To resolve these issues, replacement of the benzene ring at the 7-positon of compound 1b with heterocylic rings, such as pyrazole and pyridine, was greatly expected to reduce lipophilicity to levels for which calculated logD values were lower than that of compound 1b. While evaluating the pyrazole derivatives, introduction of a methyl substituent at the 3-position of the pyrazole ring led to increased GPR52 agonistic activity. Moreover, additional methyl substituent at the 5-position of the pyrazole and further introduction of hydroxy group to lower logD led to significant improvement of solubility while maintaining the activity. As a result, we identified 3-methyl-5-hydroxymethyl-1H-pyrazole derivative 17 (GPR52 EC₅₀?=?21?nM, E_max?=?103%, logD?=?2.21, Solubility at pH 6.8?=?21?μg/mL) with potent GPR52 agonistic activity and good solubility compared to compound 1b. Furthermore, this compound 17 dose-dependently suppressed methamphetamine-induced hyperlocomotion in mice.     

Conformational changes of recombinant monoclonal antibodies by limited proteolytic digestion,stable isotope labeling,and liquid chromatography–mass spectrometry

Limited proteolytic digestion is a method with a long history that has been used to study protein domain structures and conformational changes. A method of combining limited proteolytic digestion, stable isotope labeling, and mass spectrometry was established in the current study to investigate protein conformational changes. Recombinant monoclonal antibodies with or without the conserved oligosaccharides, and with or without oxidation of the conserved methionine residues, were used to test the newly proposed method. All of the samples were digested in ammonium bicarbonate buffer prepared in normal water. The oxidized deglycosylated sample was also digested in ammonium bicarbonate buffer prepared in ¹⁸O-labeled water. The sample from the digestion in ¹⁸O–water was spiked into each sample digested in normal water. Each mixed sample was subsequently analyzed by liquid chromatography–mass spectrometry (LC–MS). The molecular weight differences between the peptides digested in normal water versus ¹⁸O–water were used to differentiate peaks from the samples. The relative peak intensities of peptides with or without the C-terminal incorporation of ¹⁸O atoms were used to determine susceptibility of different samples to trypsin and chymotrypsin. The results demonstrated that the method was capable of detecting local conformational changes of the recombinant monoclonal antibodies caused by deglycosylation and oxidation.     

Anti-proliferative activity of oversulfated fucoidan from commercially cultured Cladosiphon okamuranus TOKIDA in U937 cells

Fucoidan from Cladosiphon okamuranus and its sulfate derivatives were prepared. Sulfate contents of native and oversulfated fucoidan were estimated to be 13.5% and 32.8%, respectively. The results of (1)H NMR suggest that 2,4-di-O-sulfo-, 2-mono-O-sulfo- and 4-mono-O-sulfo-l-fucopyranose were involved in oversulfated fucoidan and 4-mono-O-sulfo-l-fucopyranose was involved in native fucoidan. The oversulfated fucoidan reduced the proliferation of U937 cells in a dose-dependent manner, but the activity of native fucoidan was weak. The sulfate content and substituting position of sulfate group might be important factors of anti-proliferative activity in U937 cells. To examine whether the anti-proliferative activity of oversulfated fucoidan was caused by induction of apoptosis, apoptosis assay, caspase-3 activity assay and Western blotting analysis were performed. These results indicated that the oversulfated fucoidan induced apoptosis via caspase-3 and -7 activation-dependent pathway.     

Biochemical Characterization of the Nicotinic Cholinergic Receptors in Human Brain: Binding of (—)-[3H]Nicotine

(-)-[3H]Nicotine was found to bind specifically to membranes of human brains obtained at autopsy. The binding was stereospecific, (-)-nicotine being 40 times more potent than (+)-nicotine in displacing labeled (-)-nicotine. Saturation binding studies revealed the presence of two binding sites with dissociation constant (KD) values of 8.1 and 86 nM, and maximum binding capacity (Bmax) values of 36 and 90 fmol/mg protein, respectively. In competition studies, nicotinic agonists were 1,000 times more potent than ganglionic, neuromuscular, and muscarinic blocking drugs in displacing labeled (-)-nicotine. IC50 values for cholinergic drugs of (-)-[3H]nicotine binding were as follows: (-)-nicotine, 0.51 nM; acetylcholine, 12.6 nM; (+)-nicotine, 19.9 nM; cytisine, 27.3 nM; and carbachol, 527 nM. IC50 values of alpha-bungarotoxin, hexamethonium, d-tubocurarine, and atropine were larger than 50 microM. (-)-[3H]Nicotine binding was highest in the nucleus basalis of Meynert and thalamus and lowest in the cerebral cortex and caudate in the brain regions tested. These results suggest that nicotinic cholinergic receptors are present in human brain and that there are regional differences in the density of these receptors.     

Biochemical studies on monoamine contents in spinal cord of patients with multiple system atrophy

Monoamine contents were measured in the cervical spinal cord of patients with multiple system atrophy (MSA) by high-performance liquid chromatography with electrochemical detection. The concentrations of noradrenaline (NA) and its metabolite 4-methyl-4-hydroxyphenylglycol (MHPG) were highest in ventral horn compared with other regions of the spinal cord in controls. Both NA and MHPG contents were reduced in all regions in 4 MSA patients. But in one case (case 5), which did not show an autonomic dysfunction, NA as well as MHPG level was similar to controls. Similarly, the concentrations of 5-hydroxytryptamine (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were highest in ventral horn and reduced in all regions in 4 MSA patients who showed mild motor weakness. In one case (case 5), which revealed clinical motor weakness associated with fasciculation and areflexia and pathological degeneration of ventral horn, 5-HT content showed higher values than controls whereas the 5-HIAA level was lower than controls. These results probably indicate that the cell loss of supraspinal monoaminergic nuclei may be one of the causes responsible for neurological dysfunction such as autonomic failures and motor weakness in MSA.     

Changes in β-Adrenergic Receptor Subtypes in Alzheimer-Type Dementia

Using ligand binding techniques, we studied beta-adrenergic receptor subtypes in brains obtained at autopsy from seven histologically normal controls and seven histopathologically verified cases with Alzheimer-type dementia (ATD). Inhibition of [3H]dihydroalprenolol [( 3H]DHA) binding by the selective beta 1 antagonist, metoprolol, results in nonlinear Hofstee plots, suggesting the presence of the two receptor subtypes in the human brain. The calculated ratios of beta 1/beta 2-adrenergic receptors in control brains are as follows: frontal cortex, 49:51; temporal cortex, 31:69; hippocampus, 66:34; thalamus, 23:77; putamen, 70:30; caudate, 48:52; nucleus basalis of Meynert (NbM), 43:57; cerebellar hemisphere, 25:75. Compared with the controls, total concentrations of beta-adrenergic receptors were significantly reduced only in the thalamus of the ATD brains. beta 1-Adrenergic receptor concentrations were significantly reduced in the hippocampus and increased in the NbM and cerebellar hemisphere, whereas beta 2-adrenergic receptor concentrations were significantly reduced in the thalamus, NbM, and cerebellar hemisphere and increased in the hippocampus and putamen of the ATD brains. These results suggest that beta 1- and beta 2-adrenergic receptors are present in the human brain and that there are significant changes in both receptor subtypes in selected brain regions in patients with ATD.