Negamycin restores dystrophin expression in skeletal and cardiac muscles of mdx mice

The ability of aminoglycoside antibiotics to promote read-through of nonsense mutations has attracted interest in these drugs as potential therapeutic agents in genetic diseases. However, the toxicity of aminoglycoside antibiotics may result in severe side effects during long-term treatment. In this paper, we report that negamycin, a dipeptide antibiotic, also restores dystrophin expression in skeletal and cardiac muscles of the mdx mouse, an animal model of Duchenne muscular dystrophy (DMD) with a nonsense mutation in the dystrophin gene, and in cultured mdx myotubes. Dystrophin expression was confirmed by immunohistochemistry and immunoblotting. We also compared the toxicity of negamycin and gentamicin, and found negamycin to be less toxic. Furthermore, we demonstrate that negamycin binds to a partial sequence of the eukaryotic rRNA-decoding A-site. We conclude that negamycin is a promising new therapeutic candidate for DMD and other genetic diseases caused by nonsense mutations.     

Deciphering the molecular basis of the broad substrate specificity of alpha-glucosidase from Bacillus sp. SAM1606

The alpha-glucosidase of Bacillus sp. strain SAM1606 is a member of glycosyl hydrolase family 13, and shows an extraordinarily broad substrate specificity and is one of very few alpha-glucosidases that can efficiently hydrolyze the alpha-1,1-glucosidic linkage of alpha,alpha'-trehalose (trehalose). Phylogenetic analysis of family-13 enzymes suggests that SAM1606 alpha-glucosidase may be evolutionally derived from an alpha-1,6-specific ancestor, oligo-1,6-glucosidase (O16G). Indeed, replacement of Pro(273*) and Thr(342*) of B. cereus O16G by glycine and asparagine (the corresponding residues in the SAM1606 enzyme), respectively, was found to cause 192-fold enhancement of the relative catalytic efficiency for trehalose, suggesting that O16G may easily "evolved" into an enzyme with an extended substrate specificity by substitution of a limited number of amino acids, including that at position 273* (an asterisk indicates the amino-acid numbering of the SAM1606 sequence). To probe the role of the amino acid at position 273* of alpha-glucosidase in determination of the substrate specificity, the amino acid at position 273 of SAM1606 alpha-glucosidase was replaced by all other naturally occurring amino acids, and the resultant mutants were kinetically characterized. The results showed that substitution of bulky residues (e.g., isoleucine and methionine) for glycine at this position resulted in large increases in the K(m) values for trehalose and maltose, whereas the affinity to isomaltose was only minimally affected by such an amino-acid substitution at this position. Three-dimensional structural models of the enzyme-substrate complexes of the wild-type and mutant SAM1606 alpha-glucosidases were built to explore the mechanism responsible for these observations. It is proposed that substitution by glycine at position 273* could eliminate steric hindrance around subsite +1 that originally occurred in parental O16G and is, at least in part, responsible for the acquired broad substrate specificity of SAM1606 alpha-glucosidase.     

Ventricular-specific expression of angiotensin II type 2 receptors causes dilated cardiomyopathy and heart failure in transgenic mice

The angiotensin II type 2 (AT2) receptor is upregulated in the left ventricle in heart failure, but its pathophysiological roles in vivo are not understood. In the present study, AT2 receptors were expressed in transgenic (TG) mice using the ventricular-specific myosin light-chain (MLC-2v) promoter. In TG compared with nontransgenic (NTG) mice, in vivo left ventricular (LV) systolic pressure and peak +dP/dt were depressed while LV diastolic pressure was elevated (P < 0.05). Echocardiography showed severely depressed LV fractional shortening, increased systolic and diastolic dimensions, and wall thinning (P < 0.05). Confocal and electron microscopy studies revealed an increase in the size of myocytes and interstitial spaces as well as an increase in interstitial collagen, disruption of the Z-band, and changes in cytochrome c localization. The changes were most prominent in the highest-expressing TG line, which implies a dose-response relationship. AT2 overexpression was also directly associated with the increase of phosphorylated protein levels of PKC-alpha, PKC-beta, and p70S6 kinase. These data demonstrate that ventricular myocyte-specific expression of AT2 receptors promotes the development of dilated cardiomyopathy and heart failure in vivo.     

Betacellulin improves glucose metabolism by promoting conversion of intraislet precursor cells to beta-cells in streptozotocin-treated mice

Betacellulin (BTC) induces differentiation of pancreatic beta-cells and promotes regeneration of beta-cells in experimental diabetes. The present study was conducted to determine if BTC improved glucose metabolism in severe diabetes induced by a high dose of streptozotocin (STZ) in mice. Male ICR mice were injected with 200 microg/g ip STZ, and various doses of BTC were administered daily for 14 days. The plasma glucose concentration increased to a level of >500 mg/dl in STZ-injected mice. BTC (0.2 microg/g) significantly reduced the plasma glucose concentration, but a higher concentration was ineffective. The effect of BTC was marked by day 4 but became smaller on day 6 or later. The plasma insulin concentration and the insulin content were significantly higher in mice treated with 0.1 and 0.2 microg/g BTC. BTC treatment significantly increased the number of beta-cells in each islet as well as the number of insulin-positive islets. Within islets, the numbers of 5-bromo-2-deoxyuridine/somatostatin-positive cells and pancreatic duodenal homeobox-1/somatostatin-positive cells were significantly increased by BTC. These results indicate that BTC improved hyperglycemia induced by a high dose of STZ by promoting neoformation of beta-cells, mainly from somatostatin-positive islet cells.     

The excess light energy that is neither utilized in photosynthesis nor dissipated by photoprotective mechanisms determines the rate of photoinactivation in photosystem II

Photoinactivation of PSII is thought to be caused by the excessive light energy that is neither used for photosynthetic electron transport nor dissipated as heat. However, the relationship between the photoinactivation rate and excess energy has not been quantitatively evaluated. Chenopodium album L. plants grown under high-light and high-nitrogen (HL-HN) conditions show higher tolerance to photoinactivation and have higher photosynthetic capacity than the high-light and low-nitrogen (HL-LN)- and low-light and high-nitrogen (LL-HN)-grown plants. The rate of photoinactivation in the LL-HN plants was faster than that in the HL-LN, which was similar to that in the HL-HN plants, while the LL-HN and HL-LN plants had similar photosynthetic capacities [Kato et al. (2002b) Funct. Plant Biol. 29: 787]. We quantified partitioning of light energy between the electron transport and heat dissipation at the light intensities ranging from 300 to 1,800 micromol m(-2) s(-1). The maximum electron transport rate was highest in the HL-HN plants, heat dissipation was greatest in the HL-LN plants, and the excess energy, which was neither consumed for electron transport nor dissipated as heat, was greatest in the LL-HN plants. The first-order rate constant of the PSII photoinactivation was proportional to the magnitude of excess energy, with a single proportional constant for all the plants, irrespective of their growth conditions. Thus the excess energy primarily determines the rate of PSII photoinactivation. A large photosynthetic capacity in the HL-HN plants and a large heat dissipation capacity in the HL-LN plants both contribute to the protection of PSII against photoinactivation.     

Non-random features of loop-size chromatin fragmentation

Upon isolation of DNA from normal eukaryotic cells by standard methods involving extensive proteolytic treatment, a rather homogeneous population of loop-size, double-stranded DNA fragments is regularly obtained. These DNA molecules can be efficiently end-labeled by the DNA polymerase I Klenow fragment, as well as by a 3'- to -5'-exonuclease-free Klenow enzyme, but not by terminal transferase (TdT) unless the ends have been filled up by Klenow, suggesting that dominantly 5' protruding termini are generated upon fragmentation. The filled-up termini were used for cloning the distal parts of the approximately 50 kb fragments. BLAST analysis of the sequence of several clones allowed us to determine the sequence of the non-cloned side of the breakpoints. Comparison of 25, 600 bp-long breakpoint sequences demonstrated prevalence of repetitive elements. Consensus motives characteristic of the breakpoint sequences have been identified. Several sequences exhibit peculiar computed conformational characteristics, with sharp transition or center of symmetry located exactly at the breakpoint. Our data collectively suggest that chromatin fragmentation involves nucleolytic cleavages at fragile/hypersensitive sites delimiting loop-size fragments in a non-random manner. Interestingly, the sequence characteristics of the breakpoints are reminiscent of certain breakpoint cluster regions frequently subject to gene rearrangements.     

SNP detection in transforming growth factor-beta1 gene using bacterial magnetic particles

A single nucleotide polymorphism (SNP) within the transforming growth factor-beta1 (TGF-beta1) gene was detected by hybridization-based method using bacterial magnetic particles (BMPs). TGF-beta1 is commonly associated with a single base change resulting in a Leu(10)-->Pro (T(869)-->C) polymorphism and is a genetic marker for susceptibility to osteoporosis. Short (9 bases) and specific probes were designed to detect SNP in TGF-beta1. Detection probes were immobilized on BMPs using cross-linking reagents. TGF-beta1 PCR products (139 bp) were labeled with the fluorescent dye coumarin and hybridized with detection probes on BMPs. Complementary hybridized targets gave over four times higher fluorescent intensities, compared with one base mismatched hybridizations. The SNP genotype was successfully discriminated using this technique.     

Gibberellin induces alpha-amylase gene in seed coat of Ipomoea nil immature seeds

Two full-length cDNAs encoding gibberellin 3-oxidases, InGA3ox1 and InGA3ox2, were cloned from developing seeds of morning glory (Ipomoea nil (Pharbitis nil) Choisy cv. Violet) with degenerate-PCR and RACEs. The RNA-blot analysis for these clones revealed that the InGA3ox2 gene was organ-specifically expressed in the developing seeds at 6-18 days after anthesis. In situ hybridization showed the signals of InGA3ox2 mRNA in the seed coat, suggesting that active gibberellins (GAs) were synthesized in the tissue, although no active GA was detected there by immunohistochemistry. In situ hybridization analysis for InAmy1 (former PnAmy1) mRNA showed that InAmy1 was also synthesized in the seed coat. Both InGA3ox2 and InAmy1 genes were expressed spatially overlapped without a clear time lag, suggesting that both active GAs and InAmy1 were synthesized almost simultaneously in seed coat and secreted to the integument. These observations support the idea that GAs play an important role in seed development by inducing alpha-amylase.     

A new prenylated flavonoid from propolis collected in Okinawa, Japan

The new prenylflavonoid, isonymphaeol-B (1), together with three known compounds, nymphaeol-A (2), nymphaeol-B (3), and nymphaeol-C (4), were isolated from propolis collected in Okinawa, the southern-most prefecture of Japan. The structure of each compound was determined by spectral methods, including mass spectrometry and 2D NMR. Each compound had 1,1-diphenyl-2-picryl-hydrazyl radical-scavenging activity.