Fukiko Kojima

Purification by affinity chromatography using amastatin and properties of aminopeptidase A from pig kidney.

1. Amastatin, a specific inhibitor of aminopeptidase A (L-alpha-aspartyl(L-alpha-glutamyl)-peptide hydrolase, EC 3.4.11.7), was linked to an agarose matrix and by this affinity chromatography aminopeptidase A of pig kidneys was purified as a single protein shown by acrylamide gel electrophoresis. 2. Aminopeptidase A which was purified 710-fold, hydrolyzed only acidic amino acid beta-naphthylamide. The optimum pH and the optimum temperature was 7.5 and 45-50 degrees C, respectively. 3. The molecular weight was approx. 300 000 as determined by Sephadex G-200 gel filtration. 4. The activity of aminopeptidase A was not affected by sulfhydryl agents, S-S dissociating agents and serine proteinase inhibitor, but was inhibited strongly by metal chelating agents, and enhanced by alkaline earth metals. 5. Amastatin inhibited aminopeptidase A in a competitive manner with L-glutamic acid beta-naphthylamide, and the Ki value was calculated to be 2.5 x 10(-7) M. The inhibitory effect of amastatin on aminopeptidase A was not reversed by addition of Ca2+.

Bequinostatins C and D, new inhibitors of glutathione S-transferase, produced by Streptomyces sp. MI384-DF12.

Benastatins have been isolated as part of a program designed to find microorganism-produced inhibitors of glutathione S-transferase from Streptomyces sp. MI384-DF12. They were purified by chromatography of reversed-phase silica gel, silica gel and Capcell Pak C18 (HPLC) followed by solvent extraction and then isolated as yellow powders. Benastatins A and B have the molecular formulae, C30H28O7 and C30H30O7, respectively. They were competitive with 3,4-dichloronitrobenzene as the substrate, and the inhibition constants (Ki) of benastatins A and B were 5.0 x 10(-6) and 3.7 x 10(-6), respectively.

A facile synthesis of D-glucose-type gem-diamine 1-N-iminosugars: a new family of glucosidase inhibitors.

gem-Diamine 1-N iminosugars of D-glucose-type, a new type of glycosidase inhibitors, have been synthesized from siastatin B, isolated from Streptomyces culture. 2-Trifluoroacetamido-1-N-iminosugar, (2S,3R,4R,5R)-2-trifluoroacetamido-5-hydroxymethylpiperidine -3,4-diol was proved to be a potent inhibitor for alpha-D- and beta-D-glucosidases (IC50 1.9x10(-7) and 4.2x10(-7) M, respectively). 2-Acetamido-1-N-iminosugar, (2S,3R,4R,5R)-2-acetamido-5-hydroxymethylpiperidine-3,4-diol also affected these enzymes (IC50 2.9x10(-6) and 5.4x10(-6) M, respectively).

Pyrostatins A and B, new inhibitors of N-acetyl-beta-D-glucosaminidase, produced by Streptomyces sp. SA-3501.

Pyrostatins A and B, new inhibitors of N-acetyl-beta-D-glucosaminidase (GlcNAc-ase), have been purified from the culture broth of Streptomyces sp. SA-3501 isolated from a marine environment. They were purified by chromatography on Dowex 50W, silica gel and Capcell Pak C18 (HPLC) followed treatment with active carbon and then isolated as white powders. The structures of pyrostatins A and B were determined by NMR studies to be 4-hydroxy-2-imino-1-methylpyrrolidine-5-carboxylic acid and 2-imino-1-methylpyrrolidine-5-carboxylic acid, respectively. They were competitive with the substrate, and the inhibition constants (Ki) of pyrostatins A and B were 1.7 x 10(-6) M and 2.0 x 10(-6) M respectively.

Decalpenic acid, a novel small molecule from Penicillium verruculosum CR37010, induces early osteoblastic markers in pluripotent mesenchymal cells

Osteoblasts are the cells responsible for bone formation during embryonic development and adult life. Small compounds that could induce osteoblast differentiation might be promising sources of therapies for bone diseases such as osteoporosis. During screening for inducers of osteoblast differentiation of mouse pluripotent mesenchymal C3H10T1/2 cells, we isolated a small compound from the fermentation broth of Penicillium verruculosum CR37010. This compound, named decalpenic acid, bears a decalin moiety with a tetraenoic acid side chain. Treatment of C3H10T1/2 cells with decalpenic acid alone induced the expression of early osteoblast markers, such as alkaline phosphatase activity and osteopontin mRNA, but did not induce the late osteoblast marker osteocalcin mRNA or adipocyte markers under our experimental conditions.

Glycosidase inhibitors of gem-diamine 1-N-iminosugars: Structures in media of enzyme assays

The relationships between structures and inhibitory activities of glycosidase inhibitors of gem-diamine 1-N-iminosugars in media of enzyme assays have been investigated. It has been proved that gem-diamine 1-N-iminosugar smoothly undergoes a structural change to a hydrated ketone or its derivative via a hemiaminal in the media (pH 5.0-6.3), and that the products generated in the media as well as the parent gem-diamine 1-N-iminosugars potently inhibit glycosidases.

gem-Diamine 1-N-iminosugars of l-fucose-type, the extremely potent l-fucosidase inhibitors

An efficient route from D-ribono-gamma-lactone to gem-diamine 1-N-iminosugars of L-fucose-type, a new family of glycosidase inhibitor, has been developed in a formation of a gem-diamine 1-N-iminopyranose ring by the Mitsunobu reaction of an aminal as a key step. The analogues were proved to be the extremely potent inhibitors against alpha-L-fucosidase (IC50 approximately 3 ng mL(-1), Ki approximately 5 x 10(-9) M). The present study has shown that a cyclic methanediamine generated in media affects glycosidases as a real active-form of the gem-diamine 1-N-iminosugars of L-fucose-type.

Two different modes of enzymatic changes in serum with progression of Duchenne muscular dystrophy

Nineteen serum enzymes from patients with Duchenne muscular dystrophy and asthma, and normal subjects were studied. These enzymes include aminopeptidases, cathepsin C, angiotensin-converting enzyme, serine proteinase, sulphatase, phosphatase, esterases and ribonuclease. The enzymatic changes in dystrophic patients were related to two parameters: severity of the disease as judged from symptomatology, and duration of the disease. Most of the enzyme levels tested were increased in milder cases, but they tended to decrease with severity of the disease. On the other hand, there was a group of enzymes showing just opposite tendencies: serine proteinase, cathepsin C and ribonuclease. Even when viewed from the relationship to duration of the disease, the above mentioned grouping of enzymes was generally valid. Most of the enzyme levels, including those routinely applied as clinical parameters, tended to decrease, logarithmically, with an increase in duration of the disease. On the contrary, some others, including serine proteinase, cathepsin C and ribonuclease, tended to increase toward their control levels. Such tendencies were not found in the patients with asthma. The discrepancy between the above two groups of enzymes may have some implications for the process of protein degradation in dystrophic patients.

Synthesis and inhibitory activity of 8-substituted 2-deoxy-β-KDO against CMP-KDO synthetase

3-Deoxy-D-manno-octulosonate cytidyltransferase (CMP-KDO synthetase) is involved in the biosynthesis of lipopolysaccharide (LPS) which is an essential component of the outer membrane of gram-negative bacteria. New CMP-KDO synthetase inhibitors, 8-substituted derivatives of 2-deoxy-β-KDO (2) have been prepared. Compounds 8, 11, 15 and 16 in which the 8-hydroxyl group of 2 is replaced by guanidine, di(carbamoylethyl)amino, p-methoxy- or p-nitro-benzyloxycarbonylamino, respectively affect moderately the CMP-KDO synthetase activity.

Decalpenic acid induces early osteoblastic markers in pluripotent mesenchymal cells via activation of retinoic acid receptor γ.

Decalpenic acid is a natural small molecule previously isolated from the fermentation broth of fungi that induces early osteoblastic markers in pluripotent mesenchymal cells. Treatment of mouse pluripotent mesenchymal C3H10T1/2 cells with decalpenic acid gave rise to a morphological change similar to that induced by the treatment with retinoic acid, i.e. the cells adopted a more elongated spindle shape. Using a retinoic acid response element reporter and receptor activity assays, we show that decalpenic acid is a new retinoid with selectivity towards retinoic acid receptors γ and α. The induction of early osteoblastic markers by decalpenic acid was significantly inhibited by treatment with the retinoid antagonist, LE540, or with small interfering RNA-mediated knockdown of retinoic acid receptor γ. These results demonstrated that decalpenic acid induces early osteoblastic markers in pluripotent mesenchymal cells through activation of retinoic acid receptor γ.