Aglycon specificity profiling of alpha-glucosidases using synthetic probes

We designed and synthesized hydrogen bond based probes 1-8 with the exception of known glycosidase inhibition mechanisms, and aglycon specificity of 11 different sources of alpha-glucosidases were investigated using their probes. Probe 4 (2,6-anhydro-1-deoxy-1-[(1-oxopentyl-5-hydroxy)amino]-D-glycero-D-ido-heptitol) showed a potent inhibition of S. cerevisiae alpha-glucosidase among all alpha-glucosidases. Probe 4 was found to be a competitive inhibitor for S. cerevisiae alpha-glucosidase with Ki 0.13 mM.     

Synthetic studies on oligosaccharides composed of 5-thioglucopyranose units

Glycosylation reactions of 5-thioglucopyranosyl trichloroacetimidates bearing ethereal protective groups at the 2-O-position 14-15, and 37 proceed smoothly to give alpha-glycosides stereoselectively by using a catalytic amount of silyl triflate. This methodology allowed us to achieve syntheses of sulfur-substituted isomaltotetraoside 2 and maltotetraoside 3. These studies also revealed that benzoyl-protected 5-thioglucopyranosyl trichloroacetimidate 12 underwent beta-selective glycosylation with C6-OH glucopyranosyl acceptors upon activation by BF3OEt2. This was applied for preparation of sulfur-substituted gentiobiosides 1 and 46.     

Inactivation of endotoxin by human plasma gelsolin

Septic shock from bacterial endotoxin, triggered by the release of lipopolysaccharide (LPS) molecules from the outer wall of Gram-negative bacteria, is a major cause of human death for which there is no effective treatment once the complex inflammatory pathways stimulated by these small amphipathic molecules are activated. Here we report that plasma gelsolin, a highly conserved human protein, binds LPS from various bacteria with high affinity. Solid-phase binding assays, fluorescence measurements, and functional assays of actin depolymerizing effects show that gelsolin binds more tightly to LPS than it does to its other known lipid ligands, phosphatidylinositol 4,5-bisphosphate and lysophosphatidic acid. Gelsolin also competes with LPS-binding protein (LBP), a high-affinity carrier for LPS. One result of gelsolin-LPS binding is inhibition of the actin binding activity of gelsolin as well as the actin depolymerizing activity of blood serum. Simultaneously, effects of LPS on cellular functions, including cytoskeletal actin remodeling, and collagen-induced platelet activation by pathways independent of toll-like receptors (TLRs) are neutralized by gelsolin and by a peptide based on gelsolin residues 160-169 (GSN160-169) which comprise part of gelsolin's phosphoinositide binding site. Additionally, TLR-dependent NF-kappaB translocation in astrocytes appears to be blocked by gelsolin. These results show a strong effect of LPS on plasma gelsolin function and suggest that some effects of endotoxin in vivo may be mediated or inhibited by plasma gelsolin.     

Quinapril inhibits progression of heart failure and fibrosis in rats with dilated cardiomyopathy after myocarditis

The cardioprotective properties of quinapril, an angiotensin-converting enzyme inhibitor, were studied in a rat model of dilated cardiomyopathy. Twenty-eight days after immunization of pig cardiac myosin, four groups rats were given 0.2 mg/kg (Q0.2, n = 11), 2 mg/kg (Q2, n = 11) or 20 mg/kg (Q20, n = 11) of quinapril or vehicle (V, n = 15) orally once a day. After 1 month, left ventricular end-diastolic pressure (LVEDP), ±dP/dt, area of myocardial fibrosis, and myocardial mRNA expression of transforming growth factor (TGF)-1, collagen-III and fibronectin were measured. Four of 15 (27%) rats in V and two of 11 (18%) in Q0.2 died. None of the animals in Q2 or Q20 died. The LVEDP was higher and ±dP/dt was lower in V (14.1 ± 2.0 mmHg and +2409 ± 150/–2318 ± 235 mmHg/sec) than in age-matched normal rats (5.0 ± 0.6 mmHg and +6173 ± 191/–7120 ± 74 mmHg/sec; all p < 0.01). After quinapril treatment, LVEDP was decreased and ±dP/dt was increased in a dose-dependent manner (10.8 ± 1.8 mmHg and +3211 ± 307/–2928 ± 390 mmHg/sec in Q0.2, 9.4 ± 1.5 mmHg and +2871 ± 270/–2966 ± 366 mmHg/sec in Q2, and 6.6 ± 1.5 mmHg, and +3569 ± 169/–3960 ± 203 mmHg/sec in Q20). Increased expression levels of TGF-1, collagen-III and fibronectin mRNA in V were reduced in Q20. Quinapril improved survival rate and cardiac function in rats with dilated cardiomyopathy after myocarditis. Furthermore, myocardial fibrosis was regressed and myocardial structure returned to nearly normal in animals treated with quinapril.     

Super agonist actions of clothianidin and related compounds on the SAD beta 2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes

To compare the actions of clothianidin, a neonicotinoid acting on insect nicotinic acetylcholine receptors, and related compounds with that of imidacloprid, the compounds were tested on the Drosophila SAD-chicken beta2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. The maximum response of the SAD beta 2 nicotinic receptor to clothianidin was larger than that observed for acetylcholine. Ring breakage of the imidazolidine ring of imidacloprid resulting in the generation of a guanidine group was critical for this super agonist action.     

(6R)-Tetrahydrobiopterin Increases the Activity of Tryptophan Hydroxylase in Rat Raphe Slices

The effects of (6R)- and (6S)-tetrahydrobiopterin (BPH4), tetrahydroneopterin, and 6-methyltetrahydropterin on the activity of tryptophan hydroxylase were investigated in rat raphe slices. The activity of tryptophan hydroxylase was estimated by measurement of 5-hydroxytryptophan (5-HTP) formation under inhibition of aromatic L-amino acid decarboxylase with use of HPLC-fluorometric detection. (6R)-BPH4 (the naturally occurring form) at 42 microM, tetrahydroneopterin at 50 microM, and 6-methyltetrahydropterin at 100 microM increased tryptophan hydroxylase activity to 350, 145, and 146% of control values, respectively. (6S)-BPH4, however, had no significant effects on tryptophan hydroxylase activity. These results suggest that tryptophan hydroxylase is subsaturating in vivo for the naturally occurring cofactor, (6R)-BPH4, and that the concentration of (6R)-BPH4 may play an important role for the regulation of tryptophan hydroxylase activity in vivo.     

Cloning of cDNA for human T-cell replacing factor (interleukin-5) and comparison with the murine homologue.

We have cloned cDNA for T-cell replacing factor (interleukin-5), which replaces T-cell helper function for normal B cells which secrete immunoglobulin, from human T cell leukemia line, ATL-2, using mouse interleukin-5 cDNA as probe. Total nucleotide sequence of the cDNA (816 base pairs) was determined and compared with that of mouse interleukin-5 cDNA. The cloned cDNA encoded the interleukin-5 precursor of 134 amino acids containing an N-terminal signal sequence. Although the human interleukin-5 precursor is one amino acid longer than the murine homologue, the sizes of the mature proteins appear similar. The nucleotide and amino acid sequence homologies of the coding regions of human and murine interleukin-5 are 77% and 70%, respectively. Human interleukin-5 synthesized by the direction of the cloned cDNA induced immunoglobulin synthesis in human B cells stimulated by Staphylococcus aureus mitogen.     

Isolation of U1-snRNP(s) from mouse teratocarcinoma cells using immunochemical and biochemical procedures: high proportion of U1a-snRNP to U1b-snRNP

An attempt was made to immunochemically and biochemically purify and characterize the U1-snRNP(s) of mouse embryonal carcinoma cells. The results obtained by RNA analysis of U1-snRNP(s) purified immunochemically from embryoid bodies, F9 cells and PYS-2 cells indicated that the U1-snRNP(s) in these cells consisted of U1a-snRNP and U1b-snRNP. The proportion of U1a-snRNP to U1b-snRNP was also found to be high in the embryoid bodies and F9 cells. The U1a-snRNP predominance in U1-snRNP population was also detected in PYS-2 cells. The immunochemically purified U1-snRNP population from liver nuclei of 129 syngeneic male mouse (129/sv), a host mouse for transplantable tetratocarcinoma OTT6050, and ICR male mouse, contained approximately equal levels of the two U1-snRNP species (U1a- and U1b-snRNP). Partially purified U1-snRNP from embryoid bodies was also obtained by elution from a DEAE-Sepharose column at around 0.18 M NH4Cl or by fractionation by 5-20% linear sucrose gradient centrifugation. The electrophoretic RNA profiles of the partially purified U1-snRNP of embryoid bodies were similar to those obtained immunochemically.     

Reconstitution and poly(ADP-ribosyl)ation of proteolytically fragmented poly(ADP-ribose) synthetase

Calf thymus poly(ADP-ribose) synthetase (Mr = 120,000) is cleaved with papain into two fragments of M(r) = 74,000 and 46,000 and also split with chymotrypsin into two fragments of M(r) = 66,000 and 54,000. Each fragment purified to homogeneity is enzymatically inactive, but combined incubation of the 74,000 and 46,000 fragments in the presence of DNA restored 20% of the enzyme activity. In contrast, combined incubation of the 66,000 and 54,000 fragments does not restore any enzyme activity. In the former incubation, autopoly(ADP-ribosyl)ation reaction occurs exclusively on the 74,000 fragment. When each fragment is incubated with [adenine-U-14C]NAD in the presence of DNA and a catalytic amount of the native enzyme, poly(ADP-ribosyl)action occurs in the overlapped portion (22,000) of the 66,000 fragment and the 74,000 fragment. Nevertheless, the purified 22,000 fragment is a poor acceptor for poly(ADP-ribosyl)ation. The degree of poly(ADP-ribosyl)ation of the proteolytic fragments is significantly reduced by increasing NaCl concentration, probably due to the lack of the interaction between the enzyme fragments and DNA. These results, taken together, indicate that DNA is indispensable for the reconstitution of the catalytic activity as well as the poly(ADP-ribosyl)ation of the fragmented enzyme.