Melanin Granules Prevent the Cytotoxic Effects of l‐DOPA on Retinal Pigment Epithelial Cells in Vitro by Regulation of NO and Superoxide Radicals

Inasmuch as the nitrogen cycle elicits the direct reduction of N₂ to NH₃ through enzymatic reactions, and inasmuch as l ‐DOPA ( l ‐dihydroxyphentlalamine), a catecholamine, can be a source of nitric oxide (NO), it is possible that melanin granules in the eye affect the generation of NO, which causes damage to the retinal pigment epithelial (RPE) cells during the oxidation of l ‐DOPA. In order to confirm this possibility, we analyzed the correlations of NO generation, cell growth, and superoxide dismutase (SOD) activities in two types (melanotic and amelanotic) of bovine RPE cells following exposure to l ‐DOPA. NO generation from l ‐DOPA was determined using an NO detector that is reliant on redox currents. The concentration of NO was measured in terms of diffusion currents run between a working electrode and a counter electrode, both being set in culture medium placed in a Petri dish. For the assays, l ‐DOPA was added to the medium at various concentrations (5, 29.9, 79.4, 152.7 or 249 μM), and 6 min after addition, an NO‐trapping agent 2,4‐carboxyphenyl‐4,4,5,5‐tetramethylimidazole‐1‐oxyl 3‐oxide (carboxy‐PTIO) was also added. The melanotic and amelanotic types of RPE cells were cultured separately in medium with l ‐DOPA under an atmosphere containing 20, 10 or 5% oxygen. Cell numbers were counted using a Coulter counter, and SOD activities were determined following incubation for 24, 48 or 72 hr using a modification of the luminol assay. The results obtained indicated that: (a) NO was produced from l ‐DOPA in a concentration‐dependent manner and was trapped quantitatively by carboxy‐PTIO; (b) the generation of NO was inhibited more markedly in the melanotic cell line than in the amelanotic one, suggesting an increased tolerance to l ‐DOPA‐derived cytotoxicity in the former; and (c) the SOD activities were more affected by oxygen concentration in the melanotic cells than in the amelanotic ones. From these results, it is concluded that melanin granules in RPE cells have a role in preventing the cytotoxicity derived from l ‐DOPA and in regulating the generation of NO and superoxide radicals.     

Ultracytochemical localization of H＋—adenosine triphosphatase activity in autophagic vacuoles induced by vinblastine in rat liver

H^-adenosine triphosphatase (H^ -ATPase) activity was demonstrated cytochemically in autophagic vacuoles(AVs) of rat hepatocytes using a modification of the method for the demonstration of neutral p-nitrophenyl phosphatase(p-NPPase) activity[1].When an inhibitor of H^ -ATPase,N-ethylmaleimide (NEM) or 4,4‘-diisothiocyanostilbene-2,2‘disulfonic acid,disodium salt (DIDS) was included in the incubation medium the enyzme activity was abolished indicating that p-NPPase demonstrated in this study represents H^ -ATPase.Autophagy was induced by a single intraperitoneal injection of vinblastine sulfate(VBL).The number of AVs increased remarkably in hepatocytes from 40 min after VBL treatment.H^ -ATPase activity was observed mainly on the membranes of lysosomes and AVs.However,early forms of AVs containing only incompletely digested material showed no H^ -ATPase activity.Most AVs revealing a positive reaction seemed to be in advanced stages of development.Acid phosphatase acticity was demonstrable in mature but not in early forms of AVs.The present investigation showed that membranes of advanced stage AVs possess an H^ -ATPase which may be derived from lysosomal membranes.     

L-Tyrosine carboxy-lyase of barley roots

_L-Tyrosine carboxy-lyase (E.C. 4. 1. 1. 25) was isolated fromroots of germinating barley (Hordeum vulgare). The enzyme requirespyridoxal phosphate for maximum activity. The optimum pH foractivity is about 7.0. The enzyme is inhibited by p-chloromercuribenzoateand hydroxylamine at 10^–3 M. Enzyme activity is foundin extracts from young roots, especially from those in earlystages of development, but not in extracts from shoots of thesame plant. Localization and changes in the amounts of _L-tyrosinecarboxy-lyase and aromatic amines in developing barley seedlingswere measured. Participation of carboxy-lyase in the formationof aromatic amines in barley roots is suggested. (Received July 17, 1970; )     

Prothoracicotropic Hormone Bioassay: Pupal-Adult Bombyx Assay

Blockage of adult development by brain removal and its resumption by application of the prothoracicotropic hormone (PTTH) were studied using pupae of a racial hybrid J-122 × C-115 of Bombyx mori . A log-linear dose-response relationship was obrained after injection of a PTTH solution. The Bombyx -unit of PTTH has been defined from this dose-response curve.     

Structure of forest canopies as related to their primary productivity

Some structural features of forest canopy were analysed in relationto their role in photosynthetic production by forest communitieswhich are thought to produce more organic matter than herbaceouscommunities under the same environment. 1) Leaf area density was found to be much smaller in forestthan in herbaceous canopies. 2) Light extinction in forest canopy followed the BEER-LAMBERT'Slaw as was found for herbaceous canopy, though the coefficientof light extinction (K) was relatively small in the former. 3) A geometrical model was proposed to account for the smallvalue of K and the resultant large leaf area index, based onthe characteristically clustered distribution of tree leavesin forest canopy. 4) Stratification in forest community was interpreted as theuneven vertical distribution of leaf area density. 5) Deterioration of leaf functions, such as photosynthesis andrespiration, toward the bottom of forest canopy was noticed. 6) An attempt to estimate total canopy photosynthesis is presentedtaking this into consideration. 7) A new method for estimating total canopy respiration is proposedand discussed. ¹Contributions from JIBP-PT No. 36. The essential parts of thispaper were presented by KIRA to the IBP symposium The BiologicalBasis of Productivity held at Varna, Bulgaria, on April 4, 1968. (Received August 15, 1968; )     

ON THE NATURE OF MOSS OXALIC ACID OXIDASE

Moss oxalic acid oxidase freed from catalase by boiling is stronglyinhibited by the "metal-complexing" compounds such as thiocyanate,azide, diethyldithiocarbamate, and hydrosulfite. Inactivatedby dialysis against thiocyanate or azide, the enzyme can bereactivated to a considerable extent by the addition of ferricsalt, cytochrome-c or hemoglobin, not by other metal ions, suchas Cu²⁺, Zn²⁺ , Mn²⁺, and Fe²⁺. Nitrate, chlorate, monoiodoacetate,and iodide also act as strong inhibitors towards moss oxalicacid oxidase. Some enzyme fractions which were obtained by the sodium sulfateprecipitation method were stimulated by Fe³⁺, but not by cytochrome-cor by other metallic ions. This stimulation was inhibited bythiocyanate, azide and monoiodoacetate. ¹ Present address: Biological Institute, University of Toyama,Toyama     

THE DISTRIBUTION OF FORMYLTETRAHYDROFOLATE SYNTHETASE IN PLANTS, AND THE PURIFICATION AND PROPERTIES OF THE ENZYME FROM PEA SEEDLINGS

1. Formyltetrahydrofolate synthetase (E. C. 6. 3. 4. 3) was foundto be widely distributed in higher plants and the high enzymeactivity was observed in green leaves of Brassica and Alliumspecies, spinach, and in pea seedlings. In pea seedlings, theenzyme activity changed during the course of germination, andmost of the enzyme activity was located in a soluble fractionof the cytoplasm.
2. The enzyme was labile and lost the activityrapidly, even whenstored at 5 in the presence of 0.1 M mercaptoethanol.It was,however, found that ammonium sulfate was very effectivein stabilizingthe enzyme activity.
3. The enzyme has been purifiedapproximately 500-fold from extractsof pea seedlings by treatmentswith ammonium sulfate, protaminesulfate, hydroxylapatite, calciumphosphate gel, and DEAE-cellulosecolumn chromatography.
4. Thepurified enzyme was specific for formate, ATP and FAH₄,andthe Michaelis constants for these reactants were 2.1 10^–2M, 5.1 10^–4 M, and 5.6 10^–3 M, respectively.
5. The optimum pH was found to be 8.0, and the optimal temperaturewas observed at 37. Both NH₄^$ and a divalent cation (Mg^SS orMn^SS) were required for the optimal activity.

¹ Studies on the Enzymatic Synthesis and Metabolism of FolateCoenzymes in Plants. II. (For the previous paper see reference(8)) A part of this paper was presented at the Meeting of theKansai Division of the Agricultural Chemical Society of Japan,Kyoto, January 29, 1966.     

Change in the Triglyceride Level in Sea Urchin Eggs and Embryos During Early Development

Triglycerides in the embryos of the sea urchin, Anthocidaris crassispina , analyzed by gas-liquid chromatography, distributed in a range of carbon numbers between 42 and 58 in the sum of three fatty acid residues. During the development until gastrulation, the levels of triglycerides with 48, 56 and 58 carbon numbers decreased at constant rates and the levels of the others decreased at specific stages different with one another, respectively. Thereafter, the amounts of all triglycerides decreased simultaneously. The amount of oxygen consumed in the embryos is enough for the oxidation of mobilized triglycerides during post-hatching period but is not during pre-hatching period. The levels of neutral glycerides increased gradually during pre-hatching period and thereafter decreased. The fatty acid level also increased during pre-hatching and post-hatching period. These suggest that the cleavage of triglycerides and the oxidation of their cleavage-products occur during whole span of early development. During pre-hatching period, the break down of triglycerides is probably higher in its rate than the rate of their oxidation, resulting in the increase in the levels of neutral glycerides, as well as fatty acids.