Protein S-guanylation by the biological signal 8-nitroguanosine 3',5'-cyclic monophosphate

The signaling pathway of nitric oxide (NO) depends mainly on guanosine 3',5'-cyclic monophosphate (cGMP). Here we report the formation and chemical biology of a nitrated derivative of cGMP, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), in NO-mediated signal transduction. Immunocytochemistry demonstrated marked 8-nitro-cGMP production in various cultured cells in an NO-dependent manner. This finding was confirmed by HPLC plus electrochemical detection and tandem mass spectrometry. 8-Nitro-cGMP activated cGMP-dependent protein kinase and showed unique redox-active properties independent of cGMP activity. Formation of protein Cys-cGMP adducts by 8-nitro-cGMP was identified as a new post-translational modification, which we call protein S-guanylation. 8-Nitro-cGMP seems to regulate the redox-sensor signaling protein Keap1, via S-guanylation of the highly nucleophilic cysteine sulfhydryls of Keap1. This study reveals 8-nitro-cGMP to be a second messenger of NO and sheds light on new areas of the physiology and chemical biology of signal transduction by NO.     

Morphological behavior of acidic and neutral liposomes induced by basic amphiphilic alpha-helical peptides with systematically varied hydrophobic-hydrophilic balance

Lipid-peptide interaction has been investigated using cationic amphiphilic alpha-helical peptides and systematically varying their hydrophobic-hydrophilic balance (HHB). The influence of the peptides on neutral and acidic liposomes was examined by 1) Trp fluorescence quenched by brominated phospholipid, 2) membrane-clearing ability, 3) size determination of liposomes by dynamic light scattering, 4) morphological observation by electron microscopy, and 5) ability to form planar lipid bilayers from channels. The peptides examined consist of hydrophobic Leu and hydrophilic Lys residues with ratios 13:5, 11:7, 9:9, 7:11, and 5:13 (abbreviated as Hels 13-5, 11-7, 9-9, 7-11, and 5-13, respectively; Kiyota, T., S. Lee, and G. Sugihara. 1996. Biochemistry. 35:13196-13204). The most hydrophobic peptide (Hel 13-5) induced a twisted ribbon-like fibril structure for egg PC liposomes. In a 3/1 (egg PC/egg PG) lipid mixture, Hel 13-5 addition caused fusion of the liposomes. Hel 13-5 formed ion channels in neutral lipid bilayer (egg PE/egg PC = 7/3) at low peptide concentrations, but not in an acidic bilayer (egg PE/brain PS = 7/3). The peptides with hydrophobicity less than Hel 13-5 (Hels 11-7 and Hel 9-9) were able to partially immerse their hydrophobic part of the amphiphilic helix in lipid bilayers and fragment liposome to small bicelles or micelles, and then the bicelles aggregated to form a larger assembly. Peptides Hel 11-7 and Hel 9-9 each formed strong ion channels. Peptides (Hel 7-11 and Hel 5-13) with a more hydrophilic HHB interacted with an acidic lipid bilayer by charge interaction, in which the former immerses the hydrophobic part in lipid bilayer, and the latter did not immerse, and formed large assemblies by aggregation of original liposomes. The present study clearly showed that hydrophobic-hydrophilic balance of a peptide is a crucial factor in understanding lipid-peptide interactions.     

Suppressive effect of a hot water extract of adzuki beans (Vigna angularis) on hyperglycemia after sucrose loading in mice and diabetic rats

A hot water extract obtained by boiling adzuki beans (Vigna angularis) to produce bean paste for Japanese cake showed inhibitory activity against alpha-glucosidase, alpha-amylase, maltase, sucrase, and isomaltase after HP-20 column chromatography. The IC(50) values for each hydrolylase were 0.78 mg/ml (alpha-amylase), 2.45 mg/ml (maltase), 5.37 mg/ml (sucrase), and 1.75 mg/ml (isomaltase). The active fraction showed potential hypoglycemic activity in both normal mice and streptozotocin (STZ)-induced diabetic rats after an oral administration of sucrose, but did not show any effect on the blood glucose concentration after glucose administration, suggesting that the active fraction suppressed the postprandial blood glucose level by inhibiting alpha-glucosidase and alpha-amylase, irrespective of the endogenous blood insulin level.     

The prostacyclin analogue beraprost sodium prevents development of arterial stiffness in elderly patients with cerebral infarction

Prostacyclin (PGI(2)) inhibits platelet aggregation, smooth muscle cell proliferation, and vasoconstriction. Arterial stiffness assessed by pulse wave velocity (PWV) predicts mortality in various cardiovascular diseases. To study the preventive effects of a prostacyclin analogue, beraprost sodium, on arterial PWV values in elderly patients with cerebral infarction. Forty-four patients with a history of cerebral infarction received beraprost sodium (120 microg/day p.o.) or no beraprost sodium (control) for 3 months. Arterial PWV and ankle brachial indices (ABI) were determined prior to starting the medication and after 3 months of medication. Initially, there were no differences in age, blood pressure, and body mass index. Further, PWV or ABI did not differ between the beraprost sodium group (n = 22) and the control group (n = 22). After 3 months, PWV in beraprost sodium group was significantly reduced (-123 +/- 282) when compared with the control group (147 +/- 274)(P = 0.006). ABI was not significantly different when comparing the two groups at 3 months. Long-term administration of beraprost sodium prevents the decline in arterial biomechanics in elderly patients with cerebral infarction.     

Identification of beta-tubulin isoform V as an autoantigen in allergic rhinitis by a proteomic approach

Autoantibodies to IgE and beta2-adrenergic receptor have been reported in patients with allergic rhinitis. To investigate whether autoimmunity in allergic rhinitis is directed to such limited molecules or directed to a wide range of self proteins, we here attempted to survey autoantigens/autoantibodies comprehensively, using proteomics. Specifically, we separated proteins extracted from peripheral blood mononuclear cells by 2-dimensional electrophoresis and then detected autoantigens by subsequent western blotting with sera from patients with allergic rhinitis. As a result, we detected multiple autoantigens, some of which were further identified by mass fingerprinting. Next, we confirmed antigenicity of one of the identified autoantigens, beta-tubulin isoform V (beta-tubV), using a recombinant protein and then measured prevalence of the anti-beta-tubV autoantibodies. As a result, 52% of the tested patients with allergic rhinitis were found to possess anti-beta-tubV autoantibodies. Our study indicates that autoimmunity is a common phenomena and beta-tubV is one of the major autoantigens in allergic rhinitis.     

The significant effect of the carbohydrate structures on the DNA photocleavage of the quinoxaline-carbohydrate hybrids

The novel DNA interactive quinoxaline-carbohydrate hybrids possessing disaccharides as the carbohydrate moieties were designed and synthesized, and their DNA photocleaving abilities were evaluated in order to examine the effect of the disaccharide structures. The configurations of the glycosidic bonds in the disaccharide strongly affected the DNA photocleaving ability, and two beta-glycosidic bond linkages were very effective for the DNA photocleavage. Furthermore, the quinoxaline-disaccharide hybrids exhibited selective cytotoxicity against cancer cells with photoirradiation.     

Direct observation of poly(3-hydroxybutyrate) depolymerase adsorbed on polyester thin film by atomic force microscopy

Poly[(R)-3-hydroxybutyrate] (PHB) depolymerases adsorbed on poly(L-lactide) (PLLA) thin film were directly observed by atomic force microscopy (AFM). A PLLA thin film of 100 nm thickness was prepared on a silicon wafer by spin-cast method. The PLLA thin film was treated at 220 degrees C and quenched to room temperature, resulting in the formation of a completely amorphous film with a smooth surface. Then, the PHB depolymerases from Pseudomonas stutzeri YM1006 and Ralstonia pickettii T1 were dispersed on the amorphous PLLA thin film. Direct AFM observation has revealed that the PHB depolymerases bind in an elliptic shape on the surface of the PLLA thin film and that a small ridge is created around each enzyme molecule. After removal of the enzymes with 40% ethanol aqueous solution, small hollows were found on the PLLA thin film. These results suggest that a PHB depolymerase interacts with polyester molecules during their adsorption to make a hollow on the substrate surface.     

C17,20-lyase inhibitors I. Structure-based de novo design and SAR study of C17,20-lyase inhibitors

Novel nonsteroidal C(17,20)-lyase inhibitors were synthesized using de novo design based on its substrate, 17 alpha-hydroxypregnenolone, and several compounds exhibited potent C(17,20)-lyase inhibition. However, in vivo activities were found to be short-lasting, and in order to improve the duration of action, a series of benzothiophene derivatives were evaluated. As a result, compounds 9h, (S)-9i, and 9k with nanomolar enzyme inhibition (IC(50)=4-9 nM) and 9e (IC(50)=27 nM) were identified to have powerful in vivo efficacy with extended duration of action. The key structural determinants for the in vivo efficacy were demonstrated to be the 5-fluoro group on the benzothiophene ring and the 4-imidazolyl moiety. Superimposition of 9k and 17 alpha-hydroxypregnenolone demonstrated their structural similarity and enabled rationalization of the pharmacological results. In addition, selected compounds were also identified to be potent inhibitors of human enzyme with IC(50) values of 20-30 nM.     

Synthesis and evaluation of 6-methylene-bridged uracil derivatives. Part 2: optimization of inhibitors of human thymidine phosphorylase and their selectivity with uridine phosphorylase

A series of novel 6-methylene-bridged uracil derivatives have been optimized for clinical use as the inhibitors of human thymidine phosphorylase (TP). We describe their synthesis and evaluation. Introduction of a guanidino or an amidino group enhanced the in vitro inhibitory activity of TP comparing with formerly reported inhibitor 1. Their selectivity for TP based on uridine phosphorylase inhibitory activity was also evaluated. Compound 2 (TPI) has been selected for clinical evaluation based on its strong TP inhibition and excellent modulation of 2'-deoxy-5-(trifluoromethyl)uridine (F(3)dThd) pharmacokinetics. As a result, TAS-102 (a combination of F(3)dThd and TPI) is currently in phase 1 clinical studies.