Plant regeneration from protoplasts of Gentiana by embedding protoplasts in gellan gum

A protoplast-to-plant system was developed in Gentiana using a gellan gum-embedding culture. Viable protoplasts could be routinely isolated from in vitro-grown plantlets, and they were embedded in 0.2% gellan gum-solidified B5 medium containing 2 mg l^-1 NAA, 0.1 mg l^-1 TDZ, 0.1 M sucrose and 0.4 M mannitol. Weekly addition of fresh liquid medium was essential for preventing cell browning. Colony growth was promoted by lowering mannitol concentration of the culture media after one month, and visible colonies were produced after 2 months of culture. Shoot regeneration from protoplast-derived calluses was stimulated by 1 to 10 mg l^-1 TDZ in combination with 0.1 mg l^-1 NAA. Protoplast-derived plants were recovered following rooting of the shoots in plant growth regulator-free medium and they were successfully transferred to soil.Abbreviations BA benzylaminopurine - FDA fluorescein diacetate - FW fresh weight - MES 2-N-morpholinoethane sulfonic acid - NAA -naphthaleneacetic acid - TDZ N-1,2,3-thiadiazol-5-yl-N-phenylurea (also called thidiazuron)     

Immunocytochemical localization of thrombomodulin in the aqueous humor passage of the rat eye

 This report describes the distribution and localization of thrombomodulin (TM) in the rat eye by light and electron microscopic immunocytochemistry. In addition to the endothelium of the entire vasculature, TM was found on the non-vascular structures lining the cavities of the posterior and anterior chambers and the limbus. TM was localized on the basal, lateral, and apical plasma membranes of the inner and outer ciliary epithelium, and the posterior iris epithelium in which there was no polarized expression of TM. In the anterior chamber, TM was localized on the luminal surface of the corneal endothelium, but was negative on the anterior border layer of the iris, which is composed of a discontinuous layer of fibroblasts and collagen fibers. Thus, TM was present at sites of cell-to-cell contact. TM was also present on the endothelia of the trabecular meshwork and the Schlemm’s canal in the limbus. TM was localized not only on the luminal plasma membrane, but also on the cytoplasmic giant vacuoles in the endothelial cells of the Schlemm’s canal. These findings extend the importance of anticoagulant mechanisms to the systems of secretion, circulation, and drainage of the aqueous humor. Accepted: 18 March 1997     

Importance of Fe2+-ADP and the relative unimportance of OH in the mechanism of mitomycin C-induced lipid peroxidation

The mechanism of mitomycin C-induced lipid peroxidation has been studied at pH 7.5, using systems containing phospholipid membranes (liposomes) and an Fe3+-ADP complex with purified NADPH-cytochrome P-450 reductase. Both O2- and H2O2 are generated during the aerobic enzyme-catalyzed reaction in the presence of mitomycin C. Hydroxyl radical is formed in the reaction by the reduction of H2O2. This is catalyzed by the Fe2+-ADP complex in a phosphate buffer or to a lesser extent when in a Tris-HCl buffer. The reduction of Fe3+-ADP to Fe2+-ADP is mainly achieved by O2-. The resulting Fe2+-ADP in the presence of O2 forms a perferryl ion complex which is a powerful stimulator of lipid peroxidation. However, the formation of such an iron-oxygen complex is strongly inhibited by phosphate ions, which do not interfere with the generation of OH radicals. These findings suggest that, since lipid peroxidation occurs in a Tris-HCl buffer (but not in a phosphate buffer), the OH radical is unlikely to be involved in the observed lipid peroxidation process.     

Predominance of beta-structure in solubilized zona pellucida from porcine ova

The isolated zona pellucida from porcine ova was effectively solubilized in water at 60 degrees C within one hour. The circular dichroic spectra of zona in water with and without dithiothreitol showed the beta-form. Although sodium dodecyl sulfate partially induced helical structure, the beta-form was considerably retained. These results indicate that the zona glycoproteins have a structure-forming potential for the beta-structure and the hydrogen bonds of the beta-structure stabilize the supramolecular complex of the zona pellucida. The beta-form was also detected in zona solubilized by tryptic digestion. Porcine acrosin, however, did not solubilize the zona.     

Monodehydroascorbate Reductase in Spinach Chloroplasts and Its Participation in Regeneration of Ascorbate for Scavenging Hydrogen Peroxide

The primary reaction product of chloroplast ascorbate peroxidaseactivity was shown to be monodehydroascorbate radical (MDA).MDA reductase (EC 1.6.5.4 [EC] ) was localized in spinach chloroplaststroma. The MDA reductase activity of spinach chloroplasts,using NAD(P)H as electron donor, could account for the regenerationof ascorbate from MDA produced by ascorbate peroxidase activity.In the absence of MDA reductase, MDA disproportionated to ascorbate(AsA) and dehydroascorbate (DHA). The DHA was reduced to AsAby DHA reductase (EC 1.8.5.1 [EC] ) in chloroplasts. Both NADH andNADPH served as the electron donor of partially purified MDAreductase from spinach leaves. (Received September 24, 1983; Accepted January 23, 1984)     

Mechanism of thyroxine-mediated oxidation of reduced nicotinamide adenine dinucleotide in peroxidase-H2O2 system.

The oxidation of reduced nicotinamide adenine dinucleotide (NADH) by the horseradish peroxidase (HRP)-H2O2 system is greatly increased by the addition of thyroxine or related compounds. On the basis of a study of the rate of NADH oxidation in the presence of various concentrations of thyroxine, it is clear that thyroxine acts as a catalyst for NADH oxidation. Spectral changes of a HRP-H2O2 complex (compound I) indicate that thyroxine acts as an electron donor to both compounds I and II. The rate of electron donation from thyroxine is much faster than that from NADH. The HRP-H2O2 system requires 0.83 mol of O2 for the oxidation of 1 mol of NADH. Ferricytochrome c is reduced to ferrocytochrome c by the system, and causes an inhibition of O2 consumption which can be abolished by superoxide dismutase. JUDGING FROM THE INHIBITION OF O2 uptake by ferricytochrome c, about 54% of the total flux of electrons from NADH to oxygen appears to proceed by way of O2-. These results suggest that the initial step of thyroxine-mediated NADH oxidation by HRP and H2O2 is the formation of oxidized thyroxine, a phenoxy radical, which attacks NADH to produce NAD.     

Mammalian collagenase increases in early alcoholic liver disease and decreases with cirrhosis

To determine if alterations in collagen degradation may contribute to the pathogenesis of fibrosis and cirrhosis, we studied the hepatic collagenase activity of 20 baboons given alcohol containing diets or control diets under carefully controlled experimental conditions. We also studied 28 patients whose livers were biopsied for diagnostic purposes. Hepatic collagenase activity was significantly increased in baboons with fatty liver compared to levels in normal, control fed animals [(1.98 +/- 0.19 vs 1.20 +/- 0.08 units (microgram collagen digested/hour/mg liver protein) +/- S.E.M., p less than 0.001)]. The increase in hepatic collagenase activity persisted at the stage of fibrosis when compared to the activity in control baboons (2.16 +/- 0.07 vs 1.20 +/- 0.08 units +/- S.E.M., p less than 0.001). A single cirrhotic baboon available for study had an hepatic collagenase activity of 1.58 units. Patients with hepatic fibrosis had significantly higher hepatic collagenase activity than those with fatty livers [(9.12 +/- 0.94 (n =14) vs 4.52 +/- 0.54 (n = 7) units +/- S.E.M., p less than 0.001)]. However, in the group with cirrhosis, hepatic collagenase was lower [(3.92 +/- 0.61 (n = 7) units +/- S.E.M., p less than 0.001)] than in the group with fibrosis. Our findings suggest that the development of cirrhosis is coincident with, or favored by a failure of hepatic collagen degradative enzymes to keep pace with hepatic collagen synthesis.