Prooxidant Action of Maltol: Role of Transition Metals in the Generation of Reactive Oxygen Species and Enhanced Formation of 8-hydroxy-2′-deoxyguanosine Formation in DNA

Maltol (3-hydroxy-2-methyl-4-pyrone) produced reactive oxygen species as a complex with transition metals. Maltol/iron complex inactivated aconitase the most sensitive enzyme to oxidative stress. The inactivation of aconitase was iron-dependent, and prevented by TEMPOL, a scavenger of reactive oxygen species, suggesting that the maltol/iron-mediated generation of superoxide anion is responsible for the inactivation of aconitase. Addition of maltol effectively enhanced the ascorbate/copper-mediated formation of 8-hydroxy-2′-deoxyguanosine in DNA. Oxidation of ascorbic acid by CuSO₄ was effectively stimulated by addition of maltol, and the enhanced oxidation rate was markedly inhibited by the addition of catalase and superoxide dismutase. These results suggest that maltol can stimulate the copper reduction coupled with the oxidation of ascorbate, resulting in the production of superoxide radical which in turn converts to hydrogen peroxide and hydroxyl radical. Cytotoxic effect of maltol can be explained by its prooxidant properties: maltol/transition metal complex generates reactive oxygen species causing the inactivation of aconitase and the production of hydroxyl radical causing the formation of DNA base adduct.     

Extracellular Production of l-Lysine α-Oxidase in Wheat Bran Culture of a Strain of Trichoderma viride

A mold strain Y244-2 capable of producing l-lysine α-oxidase, a new enzyme catalyzing the α-oxidative deamination of l-lysine, was identified as Trichoderma viride. Among strains belonging to the genus Trichoderma tested, only Trichoderma viride Y244-2 produced the enzyme in wheat bran culture. The maximum enzyme production by the mold grown on wheat bran was observed after 10 and 14 days incubation with and without NaN0₃, respectively. Addition of NaN0₃, NH₄N0₃, adenine, purine nucleosides, l-histidine, glycine or l-glutamine to wheat bran stimulated the production of the enzyme. In the liquid culture, the enzyme was produced extracellulary under the aerobic conditions, although the production was much lower than that in the wheat bran culture.     

A novel and simple method for quantification of small,dense LDL

A preponderance of small, dense (sd) LDL is strongly associated with the development of coronary heart disease, but the method for the measurement of sd LDL is too laborious for clinical use. We report a simple method for the quantification of sd LDL that is applicable to an autoanalyzer. This method consists of two steps: first, to precipitate the lipoprotein of density (d) <1.044 g/ml using heparin-magnesium; and second, to measure LDL-cholesterol in the supernatant by the homogeneous method or apolipoprotein B (apoB) by an immunoturbidometric assay. The cholesterol and apoB values obtained by the precipitation method (45 +/- 26 and 33 +/- 20 mg/dl, respectively) were similar to those obtained in the lipoprotein (d = 1.044-1.063) separated by ultracentrifugation (42 +/- 22 and 31 +/- 17 mg/dl, respectively), and there was an excellent correlation between the two methods for sd LDL-cholesterol (y = 1.05X + 1, r = 0.88, n = 69) and apoB (y = 1.07X, r = 0.90). Sd LDL values had a significant inverse correlation with LDL size. A high correlation was found between sd LDL-cholesterol and apoB values (r = 0.94). Sd LDL value was related to triglyceride, apoB, and LDL-cholesterol, but not to the buoyant LDL level. These results suggest that this precipitation method is a simple and rapid method for the measurement of sd LDL concentration.     

The usefulness of CHAPS as a non-cytotoxic stabilizing agent in purification of growth factors

Among several detergents, a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), was found to be least cytotoxic for cultured mammalian cells. CHAPS improved the activity recovery and elution profile of crude and purified fibroblast growth factors (FGFs) during chromatographies. Diluted preparations of FGFs were stabilized by CHAPS against the loss during storage. Amino acid sequence analysis was not disturbed by CHAPS. CHAPS was removable by reversed-phase high-performance liquid chromatography. These results indicate that CHAPS is useful as a non-cytotoxic stabilizing agent in purification of various kinds of bioactive polypeptides.Abbreviations -MEM Alpha Modification of Eagle's Minimal essential medium - CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate - CHAPSO 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate - CS Calf Serum - EGF Epidermal Growth Factor - FGF Fibroblast Growth Factor - HPLC High-Performance Liquid Chromatography - NGF Nerve Growth Factor - NOG 1-O-n-octyl--D-glucopyranoside - NP-40 Nonidet P-40 - PBS Phosphate-Buffered Saline - SB 12 3-(dodecylmethylammonio)-1-propane sulfonate - SDS Sodium Dodecyl Sulfate - TGF- and Transforming Growth Factor type and      

Cytologic diagnosis of laryngeal and hypopharyngeal squamous cell carcinoma in sputum

A prospective study of the value of sputum cytology in the diagnosis of squamous cell carcinoma of the larynx and hypopharynx is reported. Sputum cytology established the diagnosis in 63.5% of the patients with laryngeal lesions and in 77.4% of the patients with hypopharyngeal lesions. In laryngeal cancer, a positive diagnosis by sputum cytology was related to clinical T factors (according to the TNM classification): while only 29.4% of T1 lesions were positively detected by sputum cytology, 63.3% of T2 lesions, 69.7% of T3 lesions and 79.2% of T4 lesions were so detected. In hypopharyngeal cancer, there was no discernible relationship between sputum cytodiagnosis and clinical T factors. Generally, there was only a small number of cancer cells present in the sputum in these cases. Some of the squamous cancer cells were not very conspicuous and would require careful screening of the sputum specimens to be detected.     

Existence of Sixteen 2′→5′ Dinucleotides in Nuclease P1 (Penicillium Nuclease) Digest of Technical Grade Yeast RNA

Sixteen 2′→5′ dinucleotides; (2′–5′)pA-A, pA-G, pA-C, pA-U, pG-A, pG-G, pG-C, pG-U, pC-A, pC-G, pC-C, pC-U, pU-A, pU-G, pU-C, and pU-U were detected in nuclease P₁ digest of a technical grade yeast RNA by means of gel filtration on Sephadex G-10, DEAE-Sephadex A-25 column chromatography in the presence of 7 m urea, paper electrophoresis and paper chromatography. Content of each dinucleotide was about 0.1 to 0.6% of the digest. As the sixteen 2′→5′ dinucleotides were found in all of the digests of technical grade RNA preparations tested, each polynucleotide chain in the preparations may be concluded to contain several per cent of the 2′–5′ minor phosphodiester linkages in addition to the 3′–5′ major phosphodiester linkages.     

The influence of experimentally produced oligohydramnios on lung growth and pulmonary surfactant content in fetal rabbits.

To study the effect of oligohydramnios on lung growth and biochemical lung development in fetal rabbits, amniotic fluid was drained through a tube inserted into the maternal peritoneal cavity on the 23 day of gestation. Littermate fetuses without an amniotic shunt were used as controls. The fetuses were delivered abdominally on the 28 day of gestation. In a total of 8 pregnant does, 17 fetuses underwent amniotic shunting and 22 fetuses were used as controls. The amniotic shunt produced a significant reduction in the amniotic fluid volume. There were no differences in the wet weights of the fetal body, liver or brain between the two groups. However, the amniotic shunt significantly decreased the wet weight of the fetal lung, fetal lung wet weight/body weight ratio, and protein concentration per lung as compared to the control fetuses. In the fetal liver and brain tissues, no changes were found in the concentrations of total phospholipids, phosphatidylcholine (PC) or disaturated phosphatidylcholine (DSPC, the main component of lung surfactant) per g of wet tissue and per mg of protein. However, the lungs of the fetuses with amniotic shunts contained significantly more PC and DSPC, and the L/S ratio was higher than in the control fetuses. These results suggest that the oligohydramnios produced by an amniotic shunt causes pulmonary hypoplasia, but raises the pulmonary surfactant content of fetal rabbit lung.     

Facile synthesis of 2′-substituted lactoses

Isopropylidenation of lactose with 2,2-dimethoxypropane in the presence ofp-toluenesulfonic acid gave two products, which were identified by¹H- and¹³C-NMR as 2,35,63,4-tri-O-isopropylidenelactose dimethyl acetal (1) and its 6-O-(2-methoxy)-isopropyl derivative (2). These products were used for the synthesis of 2-O-methyllactose (7), 2,6-di-O-methyllactose (9) and 2-O-benzyllactose (13).     

Regulation of chicken erythrocyte AMP deaminase by phytic acid.

AMP deaminase [EC 3.5.6.4] purified from chicken erythrocytes was inhibited by phytic acid (inositol hexaphosphate), which is the principal organic phosphate in chicken red cells. Kinetic analysis has indicated that this inhibition is of an allosteric type. The estimated Ki value was within the normal range of phytic acid concentration, suggesting that this compound acts as a physiological effector. Divalent cations such as Ca2+ and Mg2+ were shown to affect AMP deaminase by potentiating inhibition by lower concentrations of phytic acid, and by relieving the inhibition at higher concentrations of phytic acid. These results suggests that Ca2+ and Mg2+ can modify the inhibition of AMP deaminase by phytic acid in chicken red cells.     

Regulation of ammonia homeostasis by the ammonium transporter AmtA in Dictyostelium discoideum

Ammonia has been shown to function as a morphogen at multiple steps during the development of the cellular slime mold Dictyostelium discoideum; however, it is largely unknown how intracellular ammonia levels are controlled. In the Dictyostelium genome, there are five genes that encode putative ammonium transporters: amtA, amtB, amtC, rhgA, and rhgB. Here, we show that AmtA regulates ammonia homeostasis during growth and development. We found that cells lacking amtA had increased levels of ammonia/ammonium, whereas their extracellular ammonia/ammonium levels were highly decreased. These results suggest that AmtA mediates the excretion of ammonium. In support of a role for AmtA in ammonia homeostasis, AmtA mRNA is expressed throughout the life cycle, and its expression level increases during development. Importantly, AmtA-mediated ammonia homeostasis is critical for many developmental processes. amtA(-) cells are more sensitive to NH(4)Cl than wild-type cells in inhibition of chemotaxis toward cyclic AMP and of formation of multicellular aggregates. Furthermore, even in the absence of exogenously added ammonia, we found that amtA(-) cells produced many small fruiting bodies and that the viability and germination of amtA(-) spores were dramatically compromised. Taken together, our data clearly demonstrate that AmtA regulates ammonia homeostasis and plays important roles in multiple developmental processes in Dictyostelium.