Galantamine modulates nicotinic receptor and blocks Abeta-enhanced glutamate toxicity

Galantamine is a plant alkaloid that is used in the treatment of Alzheimer's disease. We have studied the effects of galantamine on beta-amyloid-enhanced glutamate toxicity using primary rat cultured cortical neurons. Nicotine and galantamine alone, and in combination, protected neurons against this neurotoxicity. The protection was not blocked by alpha4beta2 nicotinic acetylcholine receptor (nAChR) antagonists, but was partially blocked by alpha7 nAChR antagonists. Galantamine induced phosphorylation of Akt, an effector of phosphatidylinositol 3-kinase (PI3K), while PI3K inhibitors blocked the protective effect and Akt phosphorylation. The antibody FK1, which selectively blocks the allosterically potentiating ligand site on nAChR, significantly reduced the galantamine-induced protection and Akt phosphorylation. Furthermore, suppression of alpha7 nAChR using an RNA interference technique reduced Akt phosphorylation induced by galantamine. Our data suggest that neuroprotection by galantamine is mediated, at least in part, by alpha7 nAChR-PI3K cascade.     

Potent hypocholesterolemic activity of the yeast Kluyveromyces marxianus YIT 8292 in rats fed a high cholesterol diet

The hypocholesterolemic activities of 81 yeast strains were examined in rats fed a high cholesterol diet (HCD). Male Wistar rats were fed an HCD or an HCD supplemented with 10% yeast for 7 d. It was found that the hypocholesterolemic activities of the yeasts varied remarkably between strains. Kluyveromyces marxianus YIT 8292 exhibited the most potent hypocholesterolemic activity among the yeasts that were tested. K. marxianus YIT 8292 significantly decreased not only plasma total cholesterol but also liver total cholesterol when administered as a dietary admixture at a concentration of 3%. In contrast, brewer's yeast and baker's yeast, which have been predominantly used for food, did not exhibit hypocholesterolemic activity even when administered at a concentration of 10%. These results suggest that K. marxianus YIT 8292 may be utilized as a novel food material with the ability to contribute to the prevention of hypercholesterolemia.     

Efficient and chemoselective N-acylation of 10-amino-7-ethyl camptothecin with poly(ethylene glycol)

A new poly(ethylene glycol) (PEG) conjugate of 10-amino-7-ethyl camptothecin, a potent antitumor analogue of camptothecin, has been synthesized and preliminary in vivo tests have been performed. Successful chemoselective N-acylation of 10-amino-7-ethyl camptothecin was accomplished using phenyl dichlorophosphate, a coupling reagent used in esterification of alcohols, while other coupling methods failed, due to the low nucleophilicity of the amino group in position 10. The conjugate was tested against P388 murine leukemia cell lines and resulted equipotent to CPT-11, a camptothecin analogue already in clinical use.     

Tetrahydrobiopterin is synthesized from 6-pyruvoyl-tetrahydropterin by the human aldo-keto reductase AKR1 family members

Tetrahydrobiopterin (BH(4)) is a cofactor for aromatic amino acid hydroxylases and nitric oxide synthase. The biosynthesis includes two reduction steps catalyzed by sepiapterin reductase. An intermediate, 6-pyruvoyltetrahydropterin (PPH(4)) is reduced to 1(')-oxo-2(')-hydroxypropyl-tetrahydropterin (1(')-OXPH(4)) or 1(')-hydroxy-2(')-oxopropyl-tetrahydropterin (2(')-OXPH(4)), which is further converted to BH(4). However, patients with sepiapterin reductase deficiency show normal urinary excretion of pterins without hyperphenylalaninemia, suggesting that other enzymes catalyze the two reduction steps. In this study, the reductase activities for the tetrahydropterin intermediates were examined using several human recombinant enzymes belonging to the aldo-keto reductase (AKR) family and short-chain dehydrogenase/reductase (SDR) family. In the reduction of PPH(4) by AKR family enzymes, 2(')-OXPH(4) was formed by 3 alpha-hydroxysteroid dehydrogenase type 2, whereas 1(')-OXPH(4) was produced by aldose reductase, aldehyde reductase, and 20 alpha-hydroxysteroid dehydrogenase, and both 1(')-OXPH(4) and 2(')-OXPH(4) were detected as the major and minor products by 3 alpha-hydroxysteroid dehydrogenases (types 1 and 3). The activities of aldose reductase and 3 alpha-hydroxysteroid dehydrogenase type 2 (106 and 35 nmol/mg/min, respectively) were higher than those of the other enzymes (0.2-4.0 nmol/mg/min). Among the SDR family enzymes, monomeric carbonyl reductase exhibited low 1(')-OXPH(4)-forming activity of 5.0 nmol/mg/min, but L-xylulose reductase and peroxisomal tetrameric carbonyl reductase did not form any reduced product from PPH(4). Aldose reductase reduced 2(')-OXPH(4) to BH(4), but the other enzymes were inactive towards both 2(')-OXPH(4) and 1(')-OXPH(4). These results indicate that the tetrahydropterin intermediates are natural substrates of the human AKR family enzymes and suggest a novel alternative pathway from PPH(4) to BH(4), in which 3 alpha-hydroxysteroid dehydrogenase type 2 and aldose reductase work in concert.     

Function of the loop residue Thr792 in human DNA topoisomerase II alpha

We studied the mutation effect of one of the putative loop residues Thr792 in human DNA topoisomerase II alpha (TOP2 alpha). Thr792 mutants were expressed from high or low copy plasmids in a temperature sensitive yeast strain deficient in TOP2 (top2-1). When expressed from a high copy plasmid, mutants with small side chains complemented the yeast defect; however, from a low copy plasmid, only wild-type, Ser, and Cys substitution mutants complemented the yeast defect. Interestingly, at the permissive temperature other mutants (e.g., Val, Gly, and Glu substitutions) showed the dominant negative effect to the top2-1 allele, which was not observed by the control alpha 4-helix mutants. T792E mutant was 10-fold less active than wild-type and the T792P had no decatenation activity in vitro. These results suggest that Thr792 in human TOP2 alpha is involved in enzyme catalysis.     

Adrenomedullin provokes endothelial Akt activation and promotes vascular regeneration both in vitro and in vivo

We previously reported that adrenomedullin (AM), a vasodilating hormone secreted from blood vessels, promotes proliferation and migration of human umbilical vein endothelial cells (HUVECs). In this study, we examined the ability of AM to promote vascular regeneration. AM increased the phosphorylation of Akt in HUVECs and the effect was inhibited by the AM antagonists and the inhibitors for protein kinase A (PKA) or phosphatidylinositol 3-kinase (PI3K). AM promoted re-endothelialization in vitro of wounded monolayer of HUVECs and neo-vascularization in vivo in murine gel plugs. These effects were also inhibited by the AM antagonists and the inhibitors for PKA or PI3K. The findings suggest that AM plays significant roles in vascular regeneration, associated with PKA- and PI3K-dependent activation of Akt in endothelial cells, and possesses therapeutic potential for vascular injury and tissue ischemia.     

Induction of PYPAF1 during in vitro maturation of mouse mast cells

Coculture of mouse bone marrow-derived immature mast cells (BMMC) with Swiss 3T3 fibroblasts in the presence of stem cell factor (SCF) promotes morphological and functional maturation toward a connective tissue mast cell (CTMC)-like phenotype, which is accompanied by increased expression of several unique genes. Here we report the molecular identification of one of them, mast cell maturation-associated inducible gene (MMIG)-1. The MMIG-1 cDNA encodes a 117-kDa cytosolic protein that comprises an N-terminal PYRIN domain, a central nucleotide-binding domain, and nine C-terminal leucine-rich repeats. MMIG-1 shows >85% sequence similarity to human cryopyrin/PYPAF1, a causal gene for familial cold urticaria and Muckle-Wells syndrome. MMIG-1 was distributed in the cytosol of CTMC-like differentiated BMMC. MMIG-1 underwent alternative splicing in the leucine-rich repeats and each variant was induced differently in BMMC during coculture. Moreover, its expression was increased in the ears of mice with experimental atopic dermatitis. Thus, MMIG-1, a likely mouse PYPAF1 ortholog, may play a role in mast cell-directed inflammatory diseases.     

Origin of mannose-binding lectin-associated serine protease (MASP)-1 and MASP-3 involved in the lectin complement pathway traced back to the invertebrate,amphioxus

Mannose-binding lectin-associated serine proteases (MASPs) are involved in complement activation through the lectin pathway. To elucidate the phylogenetic origin of MASP and a primordial complement system, we cloned two MASP cDNAs from amphioxus (Branchiostoma belcheri) of the cephalochordates, considered to be the closest relative of vertebrates. The two sequences, orthologues of mammalian MASP-1 and MASP-3, were produced by alternative processing of RNA from a single gene consisting of a common H chain-encoding region and two L chain-encoding regions, a structure which is similar to that of the human MASP1/3 gene. We also isolated two MASP genes from the ascidian Halocynthia roretzi (urochordates) and found that each of them consists simply of an H chain-encoding region and a single L chain-encoding region. The difference in structure between the ascidian MASP genes and the amphioxus/mammalian MASP genes suggests that a prototype gene was converted to the MASP1/3-type gene possessing two L chain-encoding regions at an early stage of evolution before the divergence of amphioxus. This conclusion is supported by the presence of MASP-1 and MASP-3 homologues in almost all vertebrates, as demonstrated by the cloning of novel cDNA sequences representing lamprey (cyclostomes) MASP-1 and Xenopus MASP-3. The ancient origin of MASP-1 and MASP-3 suggests that they have crucial functions common to all species which emerged after cephalochordates.