Masayuki Arakawa

Drug-induced readthrough of premature stop codons leads to the stabilization of laminin α2 chain mRNA in CMD myotubes

The most common form of congenital muscular dystrophy is caused by a deficiency in the α2 chain of laminin‐211, a protein of the extracellular matrix. A wide variety of mutations, including 20 to 30% of nonsense mutations, have been identified in the corresponding gene, LAMA2. A promising approach for the treatment of genetic disorders due to premature termination codons (PTCs) is the use of drugs to force stop codon readthrough.

Preparation and stability of liposome-type artificial red blood cells stabilized with carboxymethylchitin

Liposome-type artificial red blood cells stabilized with carboxymethylchitin (mean diameter 310 nm) were prepared by a two-step emulsification technique. Sheep haemolysate was dispersed as fine droplets in a lecithin solution in dichloromethane to yield a W/O-type emulsion. The W/O emulsion thus obtained was then dispersed in an aqueous carboxymethylchitin solution to give a W/O/W-type complex emulsion. Removal of the organic solvent by evaporation from the complex emulsion left an aqueous suspension of the artificial red blood cells. The haemoglobin-trapping efficiency of the cells was found strongly dependent on the pH of the carboxymethylchitin solution used. The artificial red blood cells underwent disintegration by the action of surfactants. When a comparison was made among those surfactants which have the same alkyl chain length, the degree of cell disintegration was in the increasing order, anionic greater than cationic greater than nonionic. Globulin and fibrinogen produced no disintegration of the cells while albumin disrupted the cells to a slight extent.

A New Terrein Glucoside, a Novel Inhibitor of Angiogenin Secretion in Tumor Angiogenesis

Angiogenesis is a critical step for the tumor therapy. Many angiogenic factors are involved in the tumor angiogenesis. In the course of our screening for inhibitors of angiogenin secretion, one of angiogenic factors, we have isolated a new terrein glucoside (1) and terrein (2) from the fermentation broth of fungal strain Aspergillus sp. PF1381. The structure and absolute stereochemistry of 1 was determined to be (4S,5R)-5-[(α-D-glucopyranosyl)oxy]-4-hydroxy-3-(E-1-propenyl)-2-cyclopenten-1-one on the basis of spectral and enzymatic analyses. Compounds 1 and 2 equally inhibited angiogenin secretion from androgen-dependent prostate cancer cells, LNCaP-CR, with IC50 values of 13 μM. However, both compounds did not affect VEGF secretion, another angiogenic factor. Furthermore, both compounds inhibited tube formation of human umbilical vein endothelial cells (HUVEC). These results suggested that 1 and 2 act as angiogenesis inhibitors through the inhibition of angiogenin secretion.

Cloning of cDNA Encoding a Regeneration-Associated Muscle Protease Whose Expression Is Attenuated in Cell Lines Derived from Duchenne Muscular Dystrophy Patients

In the dystrophin-mutant mdx mouse, an animal model for Duchenne muscular dystrophy (DMD), damaged skeletal muscles are efficiently regenerated and thus the animals thrive. The phenotypic differences between DMD patients and the mdx mice suggest the existence of factors that modulate the muscle wasting in the mdx mice. To identify these factors, we searched for mRNAs affected by the mdx mutation by using cDNA microarrays with newly established skeletal muscle cell lines from mdx and normal mice. We found that in the mdx muscle cell line, 12 genes, including L-arginine:glycine amidinotransferase and thymosin beta4, are up-regulated, whereas 7 genes, including selenoprotein P and a novel regeneration-associated muscle protease (RAMP), are down-regulated. Northern blot analysis and in situ hybridization revealed that RAMP mRNA is predominantly expressed in normal skeletal muscle and brain, and its production is enhanced in the regenerating area of injured skeletal muscle in mice. RAMP expression was much lower in individual muscle cell lines derived from biopsies of six DMD patients compared to a normal muscle cell line. These results suggest that RAMP may play a role in the regeneration of skeletal muscle and that its down-regulation could be involved in the progression of DMD in humans.

A new method of preparing monocored water-loaded micro capsules using interfacial polymer deposition process

A new method was proposed to prepare monocored water-loaded microcapsules with diameters of 50 microns or larger by making use of the process of interfacial polymer deposition. A solution of ethylcellulose or polystyrene in dichloromethane was added dropwise to an O/W emulsion in which n-hexane was dispersed as fine droplets in aqueous gelatin solution. Successive evaporation of dichloromethane at 40 degrees C and n-hexane at 80 degrees C gave monocored water-loaded ethylcellulose or polystyrene microcapsules. Monocored water-loaded ethylcellulose/polystyrene composite microcapsules were similarly prepared using mixed solutions of the two polymers in dichloromethane instead of ethylcellulose or polystyrene solution. When those mixed solutions which exhibit phase separation were used, the composite microcapsules obtained had a patchwork-like structure in which polystyrene-rich islands are dispersed in the ethylcellulose-rich sea.

Amycolamicin: A Novel Broad‐Spectrum Antibiotic Inhibiting Bacterial Topoisomerase

The abuse of antibacterial drugs imposes a selection pressure on bacteria that has driven the evolution of multidrug resistance in many pathogens. Our efforts to discover novel classes of antibiotics to combat these pathogens resulted in the discovery of amycolamicin (AMM). The absolute structure of AMM was determined by NMR spectroscopy, X-ray analysis, chemical degradation, and modification of its functional groups. AMM consists of trans-decalin, tetramic acid, two unusual sugars (amycolose and amykitanose), and dichloropyrrole carboxylic acid. The pyranose ring named as amykitanose undergoes anomerization in methanol. AMM is a potent and broad-spectrum antibiotic against Gram-positive pathogenic bacteria by inhibiting DNA gyrase and bacterial topoisomerase IV. The target of AMM has been proved to be the DNA gyrase B subunit and its binding mode to DNA gyrase is different from those of novobiocin and coumermycin, the known DNA gyrase inhibitors.

Disintegration by surfactants of egg yolk phosphatidylcholine vesicles stabilized with carboxymethylchitin

Abstract Disintegration by surfactants of egg yolk phosphatidylcholine vesicles stabilized with carboxymethylchitin was investigated by measuring the amount released of a marker dye from the vesicles. In solutions of pH around 7, anionic and nonionic surfactants caused vesicle disintegration at very low concentrations, while cationic surfactants produced a breakdown of the vesicles at rather high concentrations. Increase in the alkyl chain-length of surfactant molecules brought about decrease in the surfactant concentration at which vesicle disintegration starts. As the length of the polyoxyethylene chain in nonionic surfactant molecules increased, the tendency of vesicle disintegration to occur decreased. Both anionic and cationic surfactants gave clear solutions above their critical micelle concentrations when they acted on the phospholipid vesicles, whereas nonionic surfactants left ghost cell-like debris consisting of carboxymethylchitin molecules in their micellar solutions. The effect of pH on vesicle disintegration was notable for ionic surfactants but not for nonionic surfactants. Thus, anionic surfactants increased the degree of disintegration as pH increased, while cationic surfactants produced an identical vesicle disintegration curve below pH 8 above which the curve started to shift toward the lower concentration region of the agents. These findings were explained in terms of surfactant penetration into phospholipid bilayers and solubilization of phospholipid molecules by surfactant micelles.

Permeability of poly(1-4-piperazinediylterephthaloyl) microcapsules towards sodium chloride

Abstract Water-loaded poly(1,4-piperazinediylterephthaloyl) (PPiP) microcapsules were prepared by an interfacial polycondensation technique using piperazine and terephthaloyl dichloride as water-soluble and water-insoluble monomers, respectively. The organic solvents used were mixtures of cyclohexane and chloroform and n-hexane and chloroform in various volume ratios. The microcapsules obtained were washed successively with n-hexane, acetone, and water and fractionated into several fractions of different sizes. A suspension of each fraction and a solution of sodium chloride were mixed and the change with time in electroconductivity of the mixture was measured to estimate the rate of salt entry into the microcapsules. The permeability coefficient for the salt was calculated from the rate of salt entry. It was found that the permeability increased as the capsule size increased and was higher for the capsules prepared with n-hexane—chloroform mixtures than for those prepared with cyclohexane—chloroform mixtures. These findings were explained in terms of the density and thickness of the capsule membrane.

Human first-trimester chorionic villi have a myogenic potential

First-trimester chorionic-villi-derived cells (FTCVs) are the earliest fetal material that can be obtained for prenatal diagnosis of fetal disorders such as Duchenne muscular dystrophy (DMD). DMD is a devastating X-linked disorder characterized by the absence of dystrophin at the sarcolemma of muscle fibers. Currently, a limited number of treatment options are available for DMD, although cell therapy is a promising treatment strategy for muscle degeneration in DMD patients. A novel candidate source of cells for this approach is FTCVs taken between the 9th and 11th weeks of gestation. FTCVs might have a higher undifferentiated potential than any other tissue-derived cells because they are the earliest fetal material. We examined the expression of mesenchymal stem cell and pluripotent stem cell markers in FTCVs, in addition to their myogenic potential. FTCVs expressed mesenchymal stem cell markers and Nanog and Sox2 transcription factors as pluripotent stem cell markers. These cells efficiently differentiated into myotubes after myogenic induction, at which point Nanog and Sox2 were down-regulated, whereas MyoD, myogenin, desmin and dystrophin were up-regulated. To our knowledge, this is the first demonstration that FTCVs can be efficiently directed to differentiate in vitro into skeletal muscle cells that express dystrophin as the last stage marker of myogenic differentiation. The myogenic potential of FTCVs reveals their promise for use in cell therapy for DMD, for which no effective treatment presently exists.

Disintegration of poly(Nα, Nϵ-terephthaloyl-l-lysine) microcapsules by alkylpyridinium chlorides

Abstract Poly(Nα, Nϵ-terephthaloyl- l -lysine) (PPL) microcapsules were found to undergo disintegration in aqueous medium by the action of alkylpyridinium chlorides when the surfactant cations existed in such amounts that they could solubilize PPL molecules constituting the microcapsules, which were dependent on the pH of the medium and alkyl chain length of the surfactant cation. The solubilized PPL molecule behaved as a polyelectrolyte. Low concentrations of the surfactant cations caused aggregation of the microcapsules. Crosslinking with glutaraldehyde of the terminal amino groups prevented the capsules from disintegrating.