Characterization of Acid-Cyclodextrin Glycosyltransferase of an Alkalophilic Bacillus sp.

Three kinds of lectins (LOL-I, II and III) were isolated from seeds of Lathyrus odoratus (sweet pea) in a homogenous form. The three fractions agglutinated the erythrocytes of laying hens, and the agglutination was strongly inhibited by α-methyl d-mannoside and d-mannose. However, they did not agglutinate those of the males and nonlyaing hens, differing from concanavalin A which showed a similar binding specificity for monosaccharide to LOL and agglutinated all types of erythrocytes derived from chicken in this study. LOL–I and II had a molecular weight of 52,000 and both consisted of two large (20,000 daltons) and two small subunits (6000 daltons). LOL–III had a molecular weight of 55,000, and its subunit structure was different from those of LOL–I and II. The amino acid compositions of the three fractions were very similar. They contained large amounts of aspartic acid, threonine, serine and valine, but no cysteine or methionine. Circular dichroism measurements indicated that β-structure was a major secondary structure of these lectins. The addition of α-methyl d-mannoside or d-mannose had significant effects on the CD spectra in the near-ultraviolet region, but no detectable change was observed in the 200~250 nm region. LOL–I had two binding sites for d-mannose, and the association constant was about 1000 liters per mol.     

Purification and Properties of Neutral-cyclodextrin Glycosyl-transferase of an Alkalophilic Bacillus sp.

Neutral-cyclodextrin glycosyltransferase (EC 3.2.1.19) of alkalophilic Bacillus sp. (ATCC 21783) was purified by starch adsorption, DEAE-cellulose chromatography and Sephadex G–150 gel filtration chromatography followed by preparative polyacrylamide gel electrophoresis. Molecular weight of the purified enzyme was 85,000-88,000 by SDS-disc gel electrophoresis. The enzyme was most active at pH 7 and 50°C, and stable up to 60°C at pH 7 and in the range of pH 6~8 at 60°C by 30 min incubation. The apparent V_max and Km values for α- and β-cyclodextrin at a constant concentration of sucrose were 417, 70 µmoles glucose/min · mg protein and 10, 0.83 nm, respectively. About 85~90% of amylose, 75~80% of potato starch, 65~70% of amylopectin, 55~60% of glycogen, 45~50% of amylopectin β-limit dextrin, 20~25% of maltotriose and 10~15% of maltose were converted to cyclodextrins with 0.5~1% (w/v) of each substrate.

Schardinger β-dextrin was preferentially produced from starch, and α- or γ-dextrin was gradually formed after prolonged incubation. After 20 min incubation, about 0.4, 14 and 2.5% of α-, β- and γ-dextrin were formed from starch, respectively.     

Estimation of hydroxyl radical generation by salicylate hydroxylation method in multiple organs of mice exposed to whole-body X-ray irradiation

Appropriate experimental conditions for the estimation of hydroxyl radical generation by salicylate hydroxylation were determined for multiple organs of X-irradiated mice in vivo. The in vitro experiments showed that there were significant correlations between the salicylic acid (SA) concentration, the amount of 2,3-dihydroxy benzoic acid (2,3-DHBA) and the X-ray exposure dose, and we obtained two linear-regression equations to calculate the amounts of hydroxyl radicals generated by the X-irradiation. The optimum dosage of SA and the appropriate sampling time for in vivo experiments was determined, and significant increases in the ratio of 2,3-DHBA to SA were detected in several organs of mice after X-irradiation. The hydroxyl radical equivalents of the 2,3-DHBA increases were also calculated. Our results clearly demonstrated the usefulness of the salicylate hydroxylation method in estimating hydroxyl radical generation in multiple organs in vivo.     

Estimation of hydroxyl radical generation by salicylate hydroxylation method in kidney of mice exposed to ferric nitrilotriacetate and potassium bromate

Hydroxyl radical (·OH) generation in the kidney of mice treated with ferric nitrilotriacetate (Fe-NTA) or potassium bromate (KBrO₃) in vivo was estimated by the salicylate hydroxylation method, using the optimal experimental conditions we recently reported. Induction of DNA lesions and lipid peroxidation in the kidney by these nephrotoxic compounds was also examined. The salicylate hydroxylation method revealed significant increases in the ·OH generation after injection of Fe-NTA or KBrO₃ in the kidneys. A significant increase in 8-hydroxy-2′-deoxyguanosine in nuclei of the kidney was detected only in the KBrO₃ treated mice, while the comet assay showed that the Fe-NTA and KBrO₃ treatments both resulted in significant increases in DNA breakage in the kidney. With respect to lipid peroxidation, the Fe-NTA treatment enhanced lipid peroxidation and ESR signals of the alkylperoxy radical adduct. These DNA breaks and lipid peroxidation mediated by ·OH were diminished by pre-treatment with salicylate in vivo. These results clearly demonstrated the usefulness of the salicylate hydroxylation method as well as the comet assay in estimating the involvement of ·OH generation in cellular injury induced by chemicals in vivo.     

Haploinsufficient and predominant expression of multiple endocrine neoplasia type 1 (MEN1)-related genes, MLL, p27 and p18 in endocrine organs

Multiple endocrine neoplasia type 1 (MEN1) is a rare autosomal dominantly inherited syndrome characterized by parathyroid, gastro-entero-pancreatic and anterior pituitary tumors. Although the tissue selectivity of tumors in specific endocrine organs is the very essence of MEN1, the mechanisms underlying the tissue-selectivity of tumors remain unknown. The product of the Men1 gene, menin, and mixed lineage leukemia (MLL) have been found to cooperatively regulate p27^Kip1/CDKN1B (p27) and p18^Ink4C/CDKN2C (p18) genes. However, there are no reports on the tissue distribution of these MEN1-related genes. We investigated the expression of these genes in the endocrine and non-endocrine organs of wild-type, Men1 knockout and MLL knockout mice. Men1 mRNA was expressed at a similar level in endocrine and non-endocrine organs. However, MLL, p27 and p18 mRNAs were predominantly expressed in the endocrine organs. Notably, p27 and MLL mRNAs were expressed in the pituitary gland at levels approximately 12- and 17-fold higher than those in the liver. The heterozygotes of Men1 knockout mice the levels of MLL, p27 and p18 mRNAs did not differ from those in the wild-type mice. In contrast, heterozygotes of MLL knockout mice showed significant reductions in p27 mRNA as well as protein levels in the pituitary and p27 and p18 in the pancreatic islets, but not in the liver. This study demonstrated for the first time the predominant expression MEN1-related genes, particularly MLL and p27, in the endocrine organs, and a tissue-specific haploinsuffiency of MLL, but not menin, may lead to a decrease in levels of p27 and p18 mRNAs in endocrine organs. These findings may provide basic information for understanding the mechanisms of tissue selectivity of the tumorigenesis in patients with MEN1.     

Genetic ablation and chemical inhibition of IP3R1 reduce mutant huntingtin aggregation

Huntington's disease (HD) is a dominantly inherited neurodegenerative disease caused by an expansion of the polyglutamine (polyQ) stretch in huntingtin (htt). Previously, it has been shown that inhibition of the inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) activity reduced aggregation of pathogenic polyQ proteins. Experimentally, this effect was achieved by modification of the intracellular IP3 levels or by application of IP3R1 inhibitors, such as 2-aminoethyl diphenylborinate (2-APB). Unfortunately, there are certain concerns about the 2-APB specificity and cytotoxicity. Moreover, a direct link between IP3R1 and polyQ aggregation has not been shown yet. In this study we show, that down-regulation of the IP3R1 levels by shRNA reduced the aggregation of mutant htt. We tested 2-APB analogs in an attempt to identify less toxic and more IP3R1-specific compounds and found that the effect of these analogs on the reduction of the mutant htt aggregation did weakly correlate with their inhibitory action toward the IP3-induced Ca(2+) release (IICR). Their effect on aggregation was not correlated with the store-operated Ca(2+) entry (SOCE), which is another target of the 2-APB related compounds. Our findings suggest that besides functional contribution of the IP3R inhibition on the mutant htt aggregation there are additional mechanisms for the anti-aggregation effect of the 2-APB related compounds.     

Hyper-mobility of water around actin filaments revealed using pulse-field gradient spin-echo H NMR and fluorescence spectroscopy

This paper reports that water molecules around F-actin, a polymerized form of actin, are more mobile than those around G-actin or in bulk water. A measurement using pulse-field gradient spin-echo ¹H NMR showed that the self-diffusion coefficient of water in aqueous F-actin solution increased with actin concentration by ∼5%, whereas that in G-actin solution was close to that of pure water. This indicates that an F-actin/water interaction is responsible for the high self-diffusion of water. The local viscosity around actin was also investigated by fluorescence measurements of Cy3, a fluorescent dye, conjugated to Cys 374 of actin. The steady-state fluorescence anisotropy of Cy3 attached to F-actin was 0.270, which was lower than that for G-actin, 0.334. Taking into account the fluorescence lifetimes of the Cy3 bound to actin, their rotational correlation times were estimated to be 3.8 and 9.1 ns for F- and G-actin, respectively. This indicates that Cy3 bound to F-actin rotates more freely than that bound to G-actin, and therefore the local water viscosity is lower around F-actin than around G-actin.     

Haploinsufficient and predominant expression of multiple endocrine neoplasia type 1 (MEN1)-related genes, MLL, p27Kip1 and p18Ink4C in endocrine organs

Multiple endocrine neoplasia type 1 (MEN1) is a rare autosomal dominantly inherited syndrome characterized by parathyroid, gastro-entero-pancreatic and anterior pituitary tumors. Although the tissue selectivity of tumors in specific endocrine organs is the very essence of MEN1, the mechanisms underlying the tissue-selectivity of tumors remain unknown. The product of the Men1 gene, menin, and mixed lineage leukemia (MLL) have been found to cooperatively regulate p27(Kip1)/CDKN1B (p27) and p18(Ink4C)/CDKN2C (p18) genes. However, there are no reports on the tissue distribution of these MEN1-related genes. We investigated the expression of these genes in the endocrine and non-endocrine organs of wild-type, Men1 knockout and MLL knockout mice. Men1 mRNA was expressed at a similar level in endocrine and non-endocrine organs. However, MLL, p27 and p18 mRNAs were predominantly expressed in the endocrine organs. Notably, p27 and MLL mRNAs were expressed in the pituitary gland at levels approximately 12- and 17-fold higher than those in the liver. The heterozygotes of Men1 knockout mice the levels of MLL, p27 and p18 mRNAs did not differ from those in the wild-type mice. In contrast, heterozygotes of MLL knockout mice showed significant reductions in p27 mRNA as well as protein levels in the pituitary and p27 and p18 in the pancreatic islets, but not in the liver. This study demonstrated for the first time the predominant expression MEN1-related genes, particularly MLL and p27, in the endocrine organs, and a tissue-specific haploinsuffiency of MLL, but not menin, may lead to a decrease in levels of p27 and p18 mRNAs in endocrine organs. These findings may provide basic information for understanding the mechanisms of tissue selectivity of the tumorigenesis in patients with MEN1.     

2-Methoxycinnamaldehyde inhibits tumor angiogenesis by suppressing Tie2 activation

Blood vessels are mainly composed of intraluminal endothelial cells (ECs) and mural cells adhering to the ECs on their basal side. Immature blood vessels lacking mural cells are leaky; thus, the process of mural cell adhesion to ECs is indispensable for stability of the vessels during physiological angiogenesis. However, in the tumor microenvironment, although some blood vessels are well-matured, the majority is immature. Because mural cell adhesion to ECs also has a marked anti-apoptotic effect, angiogenesis inhibitors that destroy immature blood vessels may not affect mature vessels showing more resistance to apoptosis. Activation of Tie2 receptor tyrosine kinase expressed in ECs mediates pro-angiogenic effects via the induction of EC migration but also facilitates vessel maturation via the promotion of cell adhesion between mural cells and ECs. Therefore, inhibition of Tie2 has the advantage of completely inhibiting angiogenesis. Here, we isolated a novel small molecule Tie2 kinase inhibitor, identified as 2-methoxycinnamaldehyde (2-MCA). We found that 2-MCA inhibits both sprouting angiogenesis and maturation of blood vessels, resulting in inhibition of tumor growth. Our results suggest a potent clinical benefit of disrupting these two using Tie2 inhibitors.