DORMANCY OF GLADIOLUS CORMS VI. EFFECTS OF TEMPERATURE TREATMENT ON BREAKING DORMANCY OF GLADIOLUS CORMS STORED IN A STORAGE ROOM AND OF THOSE GROWN UNDER DIFFERENT DAY-LENGTH

A high temperature treatment at 35? for 5 days followed by alow temperature treatment at 0-5? for 25 days was given at fifteen-dayintervals to five groups each of two varieties (White Gold andCardinal Prince) stored in a storage room from September 27to November 26. The temperature treatment was apparently effectivefor sprouting of corms of both varieties at earlier period,but rather inhibitory at later period of dormancy. The plants of two varieties (Early Red and Spot Light) grownunder short day-length (9 hr) showed slightly earlier floweringand corm-forming than those grown under natural long day-length.These plants were harvested from July 1 to Septmber 1 at twentydayintervals, and sprouting of their corms with and without temperaturetreatment similar to the previous method was investigated. SpotLight corms did not sprout irrespective of temperature treatment.Early Red corms always sprouted earlier in the temperature-treatedlots, whereas they sprouted a little later in the non-treatedlots. The temperature treated corms grown under the short day-lengthshowed delaying and low percentage of sprouting. On the contrary,in the non-treated lots, sprouting of corms grown under thelong day-length was slower than those grown under the shortday-length. ¹Contribution No. 24 from Laboratory of Horticulture (Olericultureand Floriculture), Kyoto University. (Received May 23, 1960; )     

Studies on germination of chrysanthemum pollen II. Occurrence of a germinationpromoting substance

The in vitro germination of chrysanthemum pollen is promotedby adding floral organs, excepting anther, to a basal mediumconsisting of sucrose and boric acid. Some other plant organs,such as young fruit of tomato and onion bulb, are also effective.In Chrysanthemum morifolium, the percentage of pollen germinationin the presence of such plant tissues is two to three timesas high as in the control (10 %). In Ch. leucanthemum, it ishigher than 50 %, in contrast to the control in which no germinationis noted. This promotion of germination may be due to a substance extractablefrom the tissues with water, ether or methanol. The promoting substance is not identical with several knowngrowth regulators or with the Ca ion. (Received November 21, 1967; )     

Functional Properties of Cloned Melanogenic Proteins

Several genes critical to the regulation of melanin production in mammals have recently been cloned and characterized. They map to the albino, brown, and slaty loci in mice, and encode proteins with similar structures and features, but with distinct catalytic capacities. The albino locus encodes tyrosinase, an enzyme with three distinct catalytic activities—tyrosine hydroxylase, 3,4-dihydroxyphenylalanine (DOPA) oxidase and DHI (5,6-dihydroxyindole) oxidase. The brown locus encodes TRP-l (tyrosinase-related protein-I), which has the same, but greatly reduced, catalytic potential. The slaty locus encodes TRP-2, another tyrosinase related-protein, which has DOPAchrome tautomerase activity. In this study we have examined the enzymatic interactions of these proteins, and their regulation by a novel melanogenic inhibitor. We observed that tyrosinase activity is more stable in the presence of TRP-l and/or TRP-2, but that the catalytic function of TRP-2 is not affected by the presence of TRP-1 or tyrosinase. Other factors also may influence melanogenesis and a unique melanogenic inhibitor suppresses tyrosinase and DOPAchrome tautomerase activities, but does not affect the spontaneous rate of DOPAchrome decarboxylation to DHI. The results demonstrate the catalytic functions of these proteins and how they stably interact within a melanogenic complex in the melanosome to regulate the quantity and quality of melanin synthesized by the melanocyte.     

Changes in Endogenous Growth Regulators in the Gladiolus Corm during Dormancy

Changes in endogenous growth regulators in gladiolus corms during dormancy were studied using paper and column chromatography followed by a bioassay with the test for straight growth of Avena coleoptile. Corms were grown in the field or in a glass room of a phytotron at 20°C in the light. Another lot was grown in a dark room at 20°C in the dark. Half of the daughter corms in each lot were cold-treated for about one month and the other half were stored at room temperature after harvest. The earliest sprouting was seen in dark grown corms with cold treatment, and the latest sprouting in light grown corms without cold. This pattern was similar in each cultivar over a period of three years. Corms from both lots contained considerable amounts of inhibiting substance just after harvest. However, dark grown corms treated with cold showed a rapid decrease in inhibitor activity and an increase in promoter activity. On the other hand, in light grown corms without cold treatment there was inhibitor activity found consistently even after two months. —There appear to be two inhibiting zones in the chromato-grams. One of these contains two inhibitory substances, one of which was assumed to be abscisic acid.     

FLAVOPROTEINS IN THE LEAVES OF HIGHER PLANTS CATALYZING THE OXIDATION OF L-GLUTAMATE

1. Two forms of enzyme capable of catalyzing the oxidation of L-glutamate(and L-aspartate) were isolated from the leaves of spinach andseparated from each other by column-chromatographic purificationon calcium phosphate and anion exchangers. They were distinguishedas GD₁ (L-glutamate dehydrogenase 1) and GD₂ (L-glutamate dehydrogenase2). The purification procedures and some fundamental propertiesof the partially purified enzymes were investigated.
2. It wasdiscovered that the enzymes did not require any cofactor,ie., neither dialysis nor precipitation with ammonium sulfatecaused a fall in enzyme activities and the addition of DPN andTPN to the reaction mixture did not accelerate the reactionrate
3. From the results of spectroscopic investigation GD₁ andGD₂were shown to be flavoproteins, although their prostheticgrouphas not yet been identified The activity of GD₁ was enhancedby the addition of FAD or FMN, while GD₂ was not acceleratedby these factors.
4. The characteristics of the two enzymes includingsubstrate specificity,MICHAELIS constant, optimum pH of thereaction and specificityfor electron acceptors were compared.
5. From the stoichiometric study of the oxidation of L-glutamatewith these enzymes, it was confirmed that the reaction is representedby the following equation: L-glutamate+oxidized dye+h₂o
6. Among various inhibitors tested,molecular oxygen which couldfunction as electron acceptor ofL-glutamate oxidation in thepresence of GD₁ was found to causea strong inhibition uponthe same reaction with TTC as el acceptor.The inhibition wasconfirmed to be due to hydrogen peroxideproduced as a resultof the aerobic oxidation of L-glutamate.

(Received July 25, 1962; )     

Reductive carboxylation and amination of keto acids by spinach chloroplasts

1. In spinach chloroplasts, the occurrence of malic enzyme,isocitrate dehydrogenase, glutamate dehydrogenase and alaninedehydrogenase was confirmed. 2. In the presence of ammonia, pyruvate and -ketoglutarate andpyruvate were photoreductively aminated to glutamate and alanine,respectively. 3. In the absence of ammonia, pyruvate and -ketoglutarate werephotoreductively carboxylated to malate and isocitrate, respectively. 4. Photoreductive carboxylation of pyruvate and -ketoglutaratewas suppressed by molecular oxygen. Inhibition was partly dueto oxidation of photoreduced NADP or NAD. (Received August 4, 1969; )     

Studies on flower color in Chrysanthemum morifolium RAMAT. I. Anthocyanins

Isolation and characterization of anthocyanins from the flowersof two cultivars of Chrysanthemum morifolium RAMAT. are reported.The main pigment is a new glucoside of cyanidin. ¹Cultivated at the Kyoto University Agricultural ExperimentalFarm (Received October 25, 1969; )     

Effect of midazolam and butorphanol premedication on inhalant isoflurane anesthesia in mice

Isoflurane is a representative inhalant anesthesia used in laboratory animals. However, isoflurane mediates respiratory depression and adverse clinical reactions during induction. In the present study, we established a novel balanced anesthesia method in mice that combined isoflurane anesthesia with midazolam and butorphanol (MB). Thirty-four male C57BL/6J mice received either isoflurane alone or isoflurane with an intra-peritoneal MB premedication (3 mg/kg midazolam and 4 mg/kg butorphanol). The minimum alveolar concentration (MAC) in each group was evaluated. Induction time and adverse clinical reactions were recorded in each group. Core body temperature, heart rate, respiratory rate, and oxygen saturation (SPO₂) were assessed before and for 1 h after induction. Premedication with MB achieved a significant reduction in MAC compared with isoflurane monoanesthesia (isoflurane, 1.38 ± 0.15%; isoflurane with MB, 0.78 ± 0.10%; P<0.05). Induction time was significantly shortened with MB premedication, and adverse reactions such as excitement or incontinence were observed less frequently. Furthermore, isoflurane anesthesia with MB premedication caused increase of respiratory rates compared to isoflurane monoanesthesia. No significant decrease of SPO₂ was observed in MBI anesthesia, while a decrease in SPO₂ was apparent with isoflurane monoanesthesia (baseline, 98.3% ± 1.1; 10 min after induction, 91.8 ± 6.4%; P<0.05). In conclusion, premedication with MB was effective for the mitigation of respiratory depression induced by isoflurane in mice, with rapid induction and fewer adverse clinical reactions.     

Tissue Distribution of an Immunogenic Melanoma Associated Antigen,D-1

A human melanoma-associated antigen immunogenic in patients was recently identified by screening an expression cDNA library constructed from cultured human melanoma cell line with sera from patients with melanoma. The nucleic acid sequence of the cloned D-1 cDNA has no significant homology with previously reported mammalian genes. The cDNA D-1 encodes a peptide of about 37 kDa, which showed fivefold higher reactivity with sera from patients with melanoma than with sera from normal donors. In order to detect D-1 gene expression in vivo, in-situ hybridization and immunostaining with cRNA probe and murine anti-D-1 sera were carried out on surgically removed tissues. Digoxigenin-labeled cRNA D-1 was exclusively hybridized with mRNA in the cytoplasm of melanoma cells but not with keratinocytes and fibroblasts adjacent to melanoma nests. Polyclonal anti-D-1 antibodies were obtained by immunization of Balb/c mice with recombinant D-1 peptide and clearly reacted with melanoma cells but not with keratinocytes and fibroblasts, similar to the results of in-situ hybridization. The above information will help to assess the suitability of recombinant D-1 peptide to implement active specific immunotherapy in patients with melanoma.     

The role of a glyoxylate-glycolate system in chloroplasts of higher plants

Photoreduction of glyoxylate and oxidation of glycolate wereinvestigated using unwashed chloroplasts from spinach leaves.A glyoxylateglycolate system operated in light and under aerobicconditions. Accompanying the photoreduction of glyoxylate andthe oxidation of glycolate, was the disappearance of inorganicphosphate. Photoreduction of oxalate was also observed in illuminatedchloroplasts. The reaction rate was, however, much lower thanthat for the photoreduction of glyoxylate. (Received August 4, 1969; )