The Gene Encoding Flavanone 3-Hydroxylase is Expressed Normally in the Pale Yellow Flowers of the Japanese Morning Glory Carrying the speckled Mutation Which Produce Neither Flavonol nor Anthocyanin but Accumulate Chalcone, Aurone and Flavanone

The Japanese morning glory carrying the recessive mutable speckledallele with the dominant speckled-activator bears colorlessflowers with fine and round colored spots distributed over thecorolla whereas the plant without the speckled-activator producespale yellow flowers. Previous chemical analysis has indicatedthat a mutation in the gene for flavanone 3-hydroxylase (F3H)is a likely candidate for the speckled allele. However, theF3HmRNA without sequence alteration accumulates normally inthe pale yellow flowers, indicating that the speckled alleleis neither the F3H gene nor a regulatory gene acting on theF3H gene expression. (Received April 4, 1997; Accepted June 2, 1997)     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A new haptenic compound, a muramyl dipeptide (MDP) derivative (designated as L4-MDP-ONB) cross-reactive with Bacillus Calmette Guerin (BCG) was synthesized. The cross-reactivity of L4-MDP hapten to BCG was demonstrated from the following evidence; (a) lymph node cells from BCG-primed C3H/HeN mice exhibited appreciable L4-MDP-specific proliferative responses to the in vitro stimulation of L4-MDP-modified syngeneic cells (L4-MDP-self); (b) inoculation of L4-MDP-self into footpads of BCG-primed C3H/HeN mice elicited ample delayed type-hypersensitivity (DTH) responses in vivo as measured by footpad swelling; and (c) BCG-primed mice contained L4-MDP-reactive helper T cell activity which functions to augment the generation of effector T cell responses to cell surface antigens. This crossreactivity between L4-MDP hapten and BCG as measured by the helper T cell activity was applied to enhanced induction of tumor-specific immunity. When BCG-primed C3H/HeN mice were immunized with L4-MDP-modified syngeneic X5563 tumor cells, these mice could generate augmented tumor-specific in vivo protective (tumor neutralizing) immunity as well as in vitro cytotoxic T cell responses. These results indicate the effectiveness of L4-MDP hapten in augmenting tumor-specific immunity. The present approach is discussed in the context of potential advantages of this new hapten for its future application to clinical tumor systems.     

Monoclonal antibodies specific to a Ca2(+)-bound form of lipocortin I distinguish its Ca2(+)-dependent phospholipid-binding ability from its ability to inhibit phospholipase A2.

Lipocortin I, a Ca2(+)-and phospholipid-binding protein without EF-hand structures, has many biological effects in vitro. Its actual role in vivo, however is unknown. We obtained and characterized five monoclonal antibodies to lipocortin I. Two of these monoclonal antibodies (L2 and L4-MAbs) reacted with the Ca(+)-bound form of lipocortin I, but not with the Ca2(+)-free form, both in vivo and in vitro. Lipocortin I required greater than or equal to 10 microM-Ca2+ to bind the two antibodies, and this Ca2+ requirement was not affected by phosphatidylserine. L2-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I and inhibited its binding to Escherichia coli membranes and to phosphatidylserine in vitro. L4-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I, but did not affect its binding to E. coli membranes or to phosphatidylserine. These findings indicated that the inhibition of phospholipase A2 by lipocortin I was not simply due to removal or capping of the substrates in E. coli membranes. Furthermore, an immunofluorescence study using L2-MAb showed the actual existence of Ca2(+)-bound form of lipocortin I in vivo.     

Comparative structures of the apopolysialoglycoproteins from unfertilized and fertilized eggs of salmonid fishes

The complete amino acid sequence of the major polysialoglycoproteins (PSGPs) from two genera of salmonid fish eggs, Salvelinus and Oncorhynchus, has been determined. The occurrence of tandem repeats of a genus-specific dodeca- and tridecapeptide was found for the apoPSGP of Salvelinus leucomaenis pluvius (Slp) and Oncorhynchus masou ishikawai (Omi), respectively, their amino acid sequences being highly homologous with that of rainbow trout [Salmo gairdneri (Sg)] apoPSGP (*denotes the glycosylation site; mean value of N = approximately 25): H-PSGP(Slp): (Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-)N H-PSGP(Omi): (Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Ser-)N H-PSGP(Sg): (Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Gly-)N Within 5-7 min following fertilization H-PSGP is converted to the low-molecular-mass PSGP (L-PSGP) by a specific protease (PSGPase). We have purified L-PSGP from the fertilized eggs of S. leucomaenis pluvius and Oncorhynchus keta (chum salmon) and compared it with rainbow trout egg L-PSGP(Sg) by analysis of their amino acid sequence: L-PSGP(Slp): Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Asp L-PSGP(Ok): Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Ser L-PSGP(Sg): Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Gly The data support the conclusion that H-PSGP is degraded in vivo 5-7 min after fertilization to L-PSGP by proteolytic cleavage at the position two residues C-terminally to the Pro residue, i.e., -Pro-Ser-Xaa-Asp-(Xaa = either Gly, Ser, or Asp) by the action of PSGPase.     

Effects of Nutrient Limitation and {gamma}-Irradiation on Tracheary Element Differentiation and Cell Division in Single Mesophyll Cells of Zinnia elegans

The effects of nutrient limitation and -irradiation on trachearyelement differentiation and cell division were investigatedusing single cells isolated from the mesophyll of Zinnia elegans.When the phosphate concentration of the medium was reduced to10 µM (1/50 of Fukuda and Komamine's medium, 1980a), thefrequency of cell division during 4 days of culture decreased,while the frequency of tracheary element differentiation wasunaffected. -Irradiation with a dose of 92 Gy at 36 h of culturepreferentially and thoroughly suppressed cell division withoutreducing the number of tracheary elements formed. The appearanceof secondary cell wall thickenings was delayed by irradiation,but synchrony was maintained. Thus the Zinnia system previouslyreported [Fukuda and Komamine (1980a) Plant Physiol. 65: 57]was improved to give a more useful system for the study of cytodifferentiation,in which tracheary element formation occurred from single cellswithout cell division. ¹Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai, Miyagi 980, Japan. (Received November 28, 1985; Accepted February 22, 1986)     

Induction of ovarian maturation and development of eggs,larvae and juveniles of the tonguefish,Cynoglossus abbreviates,reared in the laboratory

Cynoglossus abbreviatus spawns from mid-March to mid-April in the Sea of Shimabara in Kyushu. During the spawning season ovarian maturation was successfully induced by injection of the pituitary homogenate ofHypophthalmichthys molitrix. The dose of the aceton-dried pituitary homogenate was 6.5 mg/kg body weight ofC. abbreviatus. It took about 2 days for ovulation after injection at a water temperature of 14 to 16°C. Artificial fertilizations were accomplished on March 29, 1974 and again on April 7, 1984, using the females matured by hormone injection in the latter case only. The larvae were reared on the rotifers,Artemia nauplii,Tigriopus japonicus and copepods collected from the sea over a period of 113 days in 1974 and 58 days in 1984. The eggs were pelagic, spherical, 1.19–1.23 mm in diameter and had 30–50 oilglobules of 0.068–0.095 mm in diameter, and the perivitelline space was narrow. The incubation period was 90–98 hours at a water temperature of 14 to 16°C. The newly hatched larvae were 3.18–3.45 mm TL and had 61–64 myomeres. The larvae had many melanophores and xanthophores on the body, forming three bands on the caudal region, but were lacking chromatophores on the finfolds. The yolk was completely absorbed when the larvae attained a size of 4.7–5.6 mm TL 8 days after hatching. A single elongated dosai fin ray developed on the head in the 8-day old larvae. The ray was reduced in size as long as the other rays 1 or 2 days after metamorphosis. The rudiment of pectoral fins were found on the both sides of the body in the 2-day old larvae, but two of them disappeared after metamorphosis. A pelvic fin first appeared as a ventral bud just anterior to the gut in the larva of 8.39 mm TL. The full count of 4 rays was observed on the larva of 10.83 mm TL. Metamorphosis began 22 days after hatching when the larvae were 11.20 mm TL. The right eye began to shift the left side of the head at night and reached to the final place after 8.5 hours. It took about 36 hours to complete the metamorphosis, including the eye movement and fusion of the hole in the rostral beak. At the last stage of metamorphosis, the dosal, caudal, anal and ventral fins became confluent. The larvae reached the juvenile stage at a size of 13.5–14.0 mm TL, approximately 28 days after hatchling. The growth of larvae reared in 1974 is expressed by the following equations: Y₁ = 3.448 · 1.0507^x (8≦X≦28) Y₂ = 6.3322 · 1.0275^x (28≦X≦75) where Y is the total length (mm) and X is the number of days after hatching. Growth rate changed after metamorphosis.     

A Comparison of Tomato Fruit Chloroplast and Chromoplast DNAs as Analyzed with Restriction Endonucleases

Chromoplast DNA was isolated from ripe-red tomato fruits, andits structure compared with that of chloroplast DNAs from maturegreen fruits and leaves. There was a good correspondence betweenthe structures analyzed by BamHI or EcoRI digestion and by hybridizationwith a probe for the gene of the ribulose 1,5-bisphosphate carboxylase/oxygenaselarge subunit. ¹ Present address: Koryo International College, Nisshin, Aichi470-01, Japan. (Received November 5, 1984; Accepted February 6, 1985)