The intersectional hybrid between Weigela hortensis and W. maximowiczii (Caprifoliaceae)

Morphologically intermediate plants between Weigela hortensis (Siebold & Zucc.) K.Koch and W. maximowiczii (S.Moore) Rehder have been found in Miyagi and Yamagata Pref., northern Japan. Quantitative character analyses of flowers, pollen stainability and molecular analyses indicated that the intermediate plants were hybrids of those two species. This is the first record of an intersectional hybrid with W. maximowiczii (sect. Weigelastrum ) as one of the parent species. The morphological differences among hybrid individuals imply the possibility of backcrosses or formation of second or later generations of hybrids, although those may be quite rare because of a low frequency of viable pollen grains. Causes of hybridization between two distantly-related species in Weigela are discussed. © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 138 , 369–380.     

Yeast Glucan in the Cyst Wall of Pneumocystis carinii

Ultrastructurally, the cyst wall of Pneumocystis carinii consists of an electron-dense outer layer, an electron-lucent middle layer, and an innermost plasmalemma. This is similar in appearance to the cell wall of some yeasts, e.g. Saccharomyces cerevisiae , which consists of an outer dense layer of mannan, a middle lucent layer of β−1,3-glucan (yeast glucan) and an innermost plasmalemma. The cyst wall P. carinii , as well as the cell wall of S. cerevisiae , can be labeled by a variety of methods which stain polysaccharides, such as Gomori's methenamine silver (GMS) and by Aniline blue, a dye which selectively stains β-1,3-glucan. The treatment of P. carinii cysts with Zymolyase, which the key enzyme is β,3-gIucan laminari-pentaohydrolase, results in lysis of the outer 2 layers of the cyst wall and the loss of positive staining by both GMS and Aniline blue. The lysis of elements of the cyst wall of P. carinii is achieved under the same conditions and concentration at which Zymolyase lyses the outer 2 layers of the cell wall of viable cells of S. cerevisiae . These observations indicate that a major component of the cyst wall of P. carinii is β-1,3-glucan.     

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.     

Possible Involvement of ERK 1/2 in UVA‐Induced Melanogenesis in Cultured Normal Human Epidermal Melanocytes

UV‐induced melanogenesis is a well known physiological response of human skin exposed to solar radiation; however, the signaling molecules involved in the stimulation of melanogenesis in melanocytes following UV exposure remain unclear. In this study we induced melanogenesis in vitro in normal human epidermal melanocytes using a single irradiation with UVA at 1 kJ/m² and examined the potential involvement of mitogen‐activated protein kinases (MAPK) as UVA‐responsive signaling molecules in those cells. UVA irradiation did not affect the proliferation of melanocytes, but it did increase tyrosinase mRNA expression, which reached a maximum level 4 hr after UVA irradiation. The amount of tyrosinase protein, as quantitated by immunoblotting, was also increased at 24 hr following UVA irradiation. Among the MAPK examined, extracellular signal‐related kinase (ERK) 1/2 was phosphorylated within 15 min of UVA irradiation, but no such phosphorylation was observed for c‐Jun N‐terminal kinases (JNK) or p38. Accordingly, the activity of ERK1/2 was also increased shortly after UVA irradiation. These responses of ERK1/2 to UVA irradiation were markedly inhibited when cells were pre‐treated with N‐acetyl‐l ‐cysteine, an antioxidant, or with suramin, a tyrosine kinase receptor inhibitor. The formation of (6‐4)photoproducts or cyclobutane pyrimidine dimers was not detected in cellular DNA after UVA irradiation. These findings suggest that a single UVA irradiation‐induced melanogenesis is associated with the activation of ERK1/2 by upstream signals that originate from reactive oxygen species or from activated tyrosine kinase receptors, but not from damaged DNA.     

Change in CO2 balance under a series of forestry activities in a cool-temperate mixed forest with dense undergrowth

To evaluate the effects on CO₂ exchange of clearcutting a mixed forest and replacing it with a plantation, 4.5 years of continuous eddy covariance measurements of CO₂ fluxes and soil respiration measurements were conducted in a conifer-broadleaf mixed forest in Hokkaido, Japan. The mixed forest was a weak carbon sink (net ecosystem exchange, −44 g C m⁻² yr⁻¹), and it became a large carbon source (569 g C m⁻² yr⁻¹) after clearcutting. However, the large emission in the harvest year rapidly decreased in the following 2 years (495 and 153 g C m⁻² yr⁻¹, respectively) as the gross primary production (GPP) increased, while the total ecosystem respiration (RE) remained relatively stable. The rapid increase in GPP was attributed to an increase in biomass and photosynthetic activity of Sasa dwarf bamboo, an understory species. Soil respiration increased in the 3 years following clearcutting, in the first year mainly owing to the change in the gap ratio of the forest, and in the following years because of increased root respiration by the bamboo. The ratio of soil respiration to RE increased from 44% in the forest to nearly 100% after clearcutting, and aboveground parts of the vegetation contributed little to the RE although the respiration chamber measurements showed heterogeneous soil condition after clearcutting.     

Effective Elimination of Fibroblasts in Cultures of Melanocytes by Lowering Calcium Concentration in TPA Depleted Medium Following Geneticin Treatment

Melanocytes grow well in culture media supplemented with phorbol ester. However, phorbol ester is metabolically stable and has prolonged effects on multiple cellular responses. Different groups have reported that they could cultivate melanocytes without phorbol ester. However, in medium without phorbol esters contaminating fibroblasts are often difficult to eliminate. We have developed an improved method for eliminating fibroblasts using geneticin in serum free medium with bFGF, endothelin-1 and no phorbol ester. Treatment with a low concentration of geneticin effectively eliminated contaminating fibroblasts and was not harmful to melanocytes if geneticin was added initially in a high calcium media (2 mM) followed by a change to a low (0.09 mM) calcium media immediately after removal of the geneticin. With this technique we could easily establish pure melanocyte culture. This culture method will provide a useful tool for studies on melanocyte cellular response without the influence of phorbol ester.     

Origin of the disjunct distribution of flower colour polymorphism within Limonium wrightii (Plumbaginaceae) in the Ryukyu Archipelago

The sea lavender, Limonium wrightii , has six morphs of flower colour variation. The geographical distribution of flower colour morphs is disjunct; the distribution of the pink flower morph is divided into two subregions, and that of the yellow flower morph intervenes between them. The present study aimed to examine the origin of this apparent distribution pattern of flower colour in L. wrightii . Two main hypotheses (i.e. past dispersal events and phenotypic changes by natural selection and/or stochastic processes) have been proposed to account for the origin of leapfrog distribution patterns. To determine which hypothesis was applicable, we conducted a molecular phylogenetic analysis using sequence variation in chloroplast DNA (three regions of intergenic spacers, trnG - trnfM , trnV - trnM , and psbA-trnH ). We sequenced 58 accessions of L. wrightii frin 28 islands in the Ryukyu Archipelago and the Izu-Ogasawara Islands, located south of the Japanese mainland, and 12 accessions of four congeneric species. Within L. wrightii , we obtained four lineages of ten haplotypes. These lineages and haplotypes did not correlate with the different flower colours. These results indicate that the formation processes of populations are complex. The haplotypes of the pink flower morph did not show a sister relationship between the two disjunct subregions, indicating that the disjunct populations of the pink flower morphs are unlikely to share the pink flower colour as a result of common ancestry. We conclude that the observed leapfrog distribution pattern is caused by natural selection and/or stochastic processes.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 709–717.     

Adult eclosion rhythm of the Indian meal moth Plodia interpunctella: response to various thermocycles with different means and amplitudes

In addition to photoperiod, thermoperiod (or thermocycle) might be an important Zeitgeber for entraining the circadian oscillator controlling adult eclosion rhythm in the Indian meal moth Plodia interpunctella Hübner (Lepidoptera: Pyralidae). This is confirmed by exposing larvae receiving diapause‐preventing treatments to various thermocycles with different means and amplitudes of temperature. The thermocycles investigated in the present study are TC 8 : 16 h, TC 12 : 12 h, TC 16 : 8 h and TC 20 : 4 h, where T and C represent thermophase (30 °C) and cryophase (20 °C), respectively. For all thermocycles, the peak of adult eclosion rhythm occurs at around the mid‐thermophase. This indicates that the larvae use both ‘temperature‐rise’ and ‘temperature‐fall’ signals to adjust the eclosion phase in each thermocycle. The absence (DD) or presence (LL) of light affects this time‐keeping system slightly under the given thermocycle. The rhythmic adult eclosion noted after exposure of larvae to 30 °C DD for 14 days is recorded in the thermocycles (TC 12 : 12 h, DD; mean temperature = 25 °C) with different amplitudes of 27.5/22.5 °C, 26.5/23.5 °C and 25.5/24.5 °C. The peak in adult eclosion advances in time as the amplitude of the temperature cycle decreases. In the temperature cycle of 25.5/24.5 °C, a peak occurs at the end of the cryophase, 2 h before the temperature‐rise. The adult eclosion rhythm is also observed under various thermocycles (TC 12 : 12 h, DD) consisting of different temperature levels (30 to 20 °C) with different amplitudes. It is found that the temporal position of the peak advances significantly when the amplitude of the thermocycle becomes lower.