RBEs of Thermal Neutron Capture Therapy and 10B(n,α)7 Li Reaction on Melanoma-Bearing Hamsters

Using Greene's melanoma transplanted into Syrian (golden) hamsters, we determined the relative biological effectiveness (RBE) of thermal neutron capture therapy (TNCT) using ¹⁰B-paraboronophenylalanine (¹⁰B-BPA) in comparison with a 9-MeV electron beam. We also obtained the RBE of the ¹⁰B(n,α)⁷Li reaction by calculation based on summed dose data from TNCT. Throughout this study, the Kyoto University Research Reactor was used as the source for thermal neutrons; the reactor was specially altered to attain a low contamination level both for gamma-rays and fast neutrons. ¹⁰B-BPA was administered 8 hours before thermal neutron irradiation to the hamsters with melanoma. The tumor was then irradiated at 5 MW for 90 minutes. The absorbed dose from this TNCT was calculated by the method of Fairchild and Goodman (Phys. Med. Biol. 1966; 2:15–30). The RBEs of the TNCT and the ¹⁰B(n,α)⁷Li reaction obtained by the tumor growth delay time (TGDT) method were 2.22 and 2.51, respectively, at 10.5 days of TGDT. These RBE values varied with TGDT and the absorbed dose. The RBE value of TNCT had a peak at 7.0 days of TGDT; that of the ¹⁰B(n,α)⁷Li reaction was higher at a low absorbed dose level and lower at a high absorbed dose level.     

Enhanced Melanogenesis Induced by Tyrosinase Gene-Transfer Increases Boron-Uptake and Killing Effect of Boron Neutron Capture Therapy for Amelanotic Melanoma

Specific and powerful cancer killing effect for melanoma by boron neutron capture therapy (BNCT) using DOPA analogue, ¹⁰B-p-boronophenylalanine (¹⁰B-BPA), has been established, but amelanotic melanoma is insufficiently responsive to ¹⁰B-BPA BNCT in comparison with actively melanin-producing melanoma. Although the accumulation mechanism of ¹⁰B-BPA within melanoma was not established, we have recently obtained findings suggesting that melanin monomers, key intermediates for melanin polymer formation, play a critical role in ¹⁰B-BPA accumulation. In addition, there are some kinds of human amelanotic melanomas, such as MEL2A, in which expression of tyrosinase is repressed or lacking though tyrosinase-related protein (TRP)-l and TRP-2 are well expressed. Thus, by using a similarly tyrosinase-lacking mouse amelanotic melanoma cell line, A1059, we constructed TA1059 cells by transfecting human tyrosinase-cDNA into these cells. TA1059 cells acquired higher DOPA-oxidase and DOPAchrome tautomerase activity as well as eumelanin content at even higher levels than those of B16F10 cells. TA1059 cells showed about 2.5 times higher ^p-boronophenylalanine (BPA) uptake than A1059 cells in culture. In animal experiments, by using these cell lines, tumor growth of TA1059 was significantly suppressed by ¹⁰B-BPA BNCT as compared with A1059. These findings indicate that the induction of active melanin biosynthesis by melanogenic gene-transfer effectively improves the treatment of amelanotic melanoma by BNCT.     

Description and ecological traits of a new species of Pitydiplosis (Diptera: Cecidomyiidae) that induces leaf galls on Pueraria (Fabaceae) in East Asia,with a possible diversification scenario of intraspecific groups

A gall midge that induces thick lenticular galls on leaflets of Pueraria species (Fabaceae) in Japan, mainland China, Taiwan and South Korea is described as Pitydiplosis puerariae sp. nov. (Diptera: Cecidomyiidae). Tanaostigmodes puerariae (Hymenoptera: Tanaostigmatidae), described earlier from mainland China as an inducer of the lenticular gall, is regarded to be an inquiline. Pitydiplosis puerariae is distinguishable from the only known congener, the Nearctic Pitydiplosis packardi, by the male genitalia with entire aedeagus and with hypoproct that is as long as cerci and bilobed with a U‐shaped emargination. DNA sequencing data indicate the existence of three genetically different intraspecific groups: (i) “YNT‐montana group” induces galls on Pueraria montana on the Yaeyama Islands, Japan and in northern Taiwan; (ii) “CT‐montana group” on P. montana in central Taiwan; (iii) and “JCK‐lobata group” on Pueraria lobata in mainland China, South Korea and Japan north of Okinoerabu Island. A possible diversification scenario of the three groups is hypothesized based on DNA sequencing data and geohistorical information. A distribution gap of the gall midge on five islands between Tokunoshima and Ishigaki Islands, Japan was confirmed by intensive field surveys. Ecological traits and adult behavior of Pity. puerariae are also described. Its possibility as a potential biological control agent against P. lobata seems counter‐indicated.     

Isolation and characterization of microsatellite loci from Quercus mongolica var. crispula

Microsatellites were isolated and characterized for the Japanese oak species, Quercus mongolica var. crispula, distributed in temperate deciduous forests of Japan. Eleven of the 48 primer pairs designed successfully amplified unambiguous and polymorphic single loci among 67 Q. mongolica var. crispula individuals within a plot in southwestern Japan. The observed and expected heterozygosities of the 11 microsatellite markers ranged from 0.522 to 0.896 and from 0.536 to 0.882, respectively. These polymorphic microsatellite markers are useful for estimating pollen‐mediated gene flow in Q. mongolica var. crispula.     

Effect of Adenine on Paramecium multimicronucleatum*

SYNOPSIS. The addition of adenine to the culture medium of Paramecium multimicronucleatum caused an inhibition of conjugation and fission and also an increase in the number of food vacuoles. The inhibition of conjugation was observed at the stage of adhesion of cell membranes. With adenine, the duration of fission was prolonged and L-shaped cells were formed. Adenine apparently repressed the excretion of food vacuoles and thus caused an accumulation of food vacuoles within the cells. All these effects were observed at similar concentrations of adenine higher than 2 mM. The addition of uridine to the adenine-containing medium reversed the inhibition of fission; conjugation and vacuole accumulation remained unaffected.     

CYTOKINE- AND NEUROPEPTIDE-MEDIATED DIFFERENTIATION IN RETINAL PIGMENT EPITHELIAL CELLS IN VITRO

To determine the mechanism of growth and differentiation of retinal pigment epithelial (RPE) cells it is important to understand the pathogenesis of several retinal diseases. Recently it has been reported that several cytokines and neuropeptides regulate the growth of RPE cells. In this study, the role of cytokines and neuropeptides in melanin synthesis, which is one indication of the RPE cell differentiation, was examined using chick RPE cells in vitro IL-1β, TNF-α, substance P, β-endorphin and methionine-enkephalin stimulated the melanin synthesis of RPE cells in a dose-dependent manner. The most effective concentrations of these agents on RPE cell melanin synthesis were not the same as that for RPE cell proliferation. These results indicate that cytokines and neuropeptides play an important role not only for the growth but also for the differentiation of RPE cells.     

Newer Melanogenesis Control and Melanoma Eradication

Further advances leading to more sophisticated and effective suppression of melanogenesis and melanoma growth based on clarification and utilization of common vital factors involved in both processes are reviewed. Induction of depigmentation has been achieved by both glycosylation and its processing inhibitors, which have been found to be critical for the maturation and transport of tyrosinase from ribosomes through GERL-coated vesicles into premelanosomes. Kojic acid, a copper chelating melanogenic inhibitor, can induce inhibition of isolated tyrosinase activity as well as melanization in living pigment cells in in vitro and in vivo systems. This depigmenting effect was found to be due to a concurrent decrease in both eu- and pheomelanin formation. Malignant melanoma principally has accentuated melanosome genesis, which has been utilized to accumulate selectively ¹⁰B into melanoma cells using ¹⁰B-dopa analogue. Subsequent thermal neutron irradiation induces ¹⁰B(n, α)⁷Li reaction which releases high LET particles within a range of 10–14 μm thus erradicating selectively melanoma at the cellular level. This new therapy has been applied to a human melanoma lesion for the first time, and a successful therapeutic effect on melanoma has been obtained.     

Isolation and Characterization of High Molecular Weight Melanogenic Inhibitors Naturally Occurring in Melanoma Cells

Intrinsic melanogenic inhibitors with high molecular weights have been isolated from Greene's amelanotic hamster melanoma by DEAE ion-exchange and gel filtration chromatographies. The native molecular weights of two partially purified inhibitors have been determined to be 15 kDa (β-type) and 67 kDa (γ-type), respectively, using HPLC gel filtration. Both types of inhibitors, despite their inability to directly inhibit isolated tyrosinase, have been shown to markedly inhibit melanin formation in cultured B16 cells. In contrast to the β-type inhibitor, the γ-type inhibitor can induce depigmentation in B16 cells without abolishing their internal tyrosinase activity. Further, it has been determined that both inhibitors contain various amounts of unsaturated fatty acids, C15:1, C18:1, C18:2, C18:3, C20:3, and C20:4, which exhibit depigmentary activities on cultured B16 cells. C15:1 is low in the β-type, but high in the γ-type whereas C18:3 is high in the β-type but low in the γ-type. These results suggest that the differential action of these inhibitors is most likely due to the composition of the unsaturated fatty acids.