Comparison of ecophysiological responses to heavy snow in two varieties ofAucuba japonica with different areas of distribution

Aucuba japonica varieties are common evergreen understory shrubs in Japan.Aucuba japonica var.borealis is distributed on the Sea of Japan side of Honshu and Hokkaido where heavy snow cover lasts for more than 3 months in winter.Aucuba japonica var.japonica is distributed in areas with shallow or no snow on the Pacific Ocean side of Honshu and Shikoku. The ecophysiological characteristics of var.borealis were compared with those of var.japonica to examine the effects of heavy and long-term snow cover on the life cycle of var.borealis. Shoots of both varieties were shaded in crushed ice for 110 days, but their photosynthetic activities, chlorophyll contents and the chlorophylla/b ratio was not affected. The leaves of var.borealis were no less frost tolerant than those of var.japonica. In spite of the difference in environmental factors, both varieties had similar characteristics in seasonal changes of photosynthesis, respiration and chlorophylla/b ratio. These results suggest that var.japonica could survive in areas with heavy snow where it does not normally occur. Leaf net production (LNP) was estimated based on the microclimatic data and seasonal photosynthetic and respiration rates. The difference in the annual LNP between the two varieties was equivalent to the difference in the LNP during the snow season. One of the major effects of snow cover is to interrupt and reduce the production period of var.borealis.     

Involvement of Cell Wall-Bound Diferulic Acid in Light-Induced Decrease in Growth Rate and Cell Wall Extensibility of Oryza Coleoptiles

Irradiation of white fluorescent light (5 W m^–2) inhibitedthe growth of Oryza coleoptiles. Light irradiation increasedstress-relaxation parameters of coleoptile cell walls, minimumstressrelaxationtime and relaxation rate, and decreased cellwall extensibility (strain/load). Under light conditions, thecontents of ferulic and diferulic acids ester-linked to thehemicellulosic arabinose residue in cell walls increased andcorrelated with the modification of the cell wall mechanicalproperties. These results suggest that light irradiation enhancesthe formation of diferulic acid bridges in hemicelluloses, makingcell walls mechanically rigid and thus inhibits cell elongationin rice coleoptiles. Also, irrespective of coleoptile age orthe presence of light, the ratio of diferulic acid to ferulicacid was almost constant, suggesting that the rate limitingstep in the formation of diferulic acid bridges in Oryza cellwalls is in the step of feruloylation. (Received September 24, 1991; Accepted December 3, 1991)     

Search for substrate-based inhibitors fitting the S2' space of malarial aspartic protease plasmepsin II.

Plasmepsin (Plm) has been identified as an important target for the development of new antimalarial drugs, since its inhibition leads to the starvation of Plasmodium falciparum. A series of substrate-based dipeptide-type Plm II inhibitors containing the hydroxymethylcarbonyl isostere as a transition-state mimic were synthesized. The general design principle was provision of a conformationally restrained hydroxyl group (corresponding to the set residue at the P2' position in native substrates) and a bulky unit to fit the S2' pocket.     

Concanavalin A Inhibits Auxin-Induced Elongation and Breakdown of (1 -> 3), (1 -> 4)-{beta}-D-Glucans in Segments of Rice Coleoptiles

Concanavalin A (Con A) suppresses auxin-induced elongation ofsurface-abraded segments from both dicotyledonous and poaceousplants. In coleoptile segments of rice (Oryza sativa L.), theauxin-induced decrease in the minimum stress-relaxation timeand increase in the mechanical extensibility of the cell wallswere also inhibited by Con A, indicating that the lectin suppresseselongation by inhibiting the cell wall loosening. Auxin causeda decrease in the level of (1 3), (1 4)-ß-D-glucansin the cell walls of rice coleoptile segments, and this decreasewas also inhibited by the lectin. Con A suppressed the autolytichydrolysis of the glucans, as well as their breakdown in vitroby a protein fraction that had been extracted from the cellwalls of rice coleoptiles with 1 M NaCl. Furthermore, most ofthe glucan-hydrolyzing activity of the wall proteins bound toa Con A-Sepharose column, suggesting that glycoprotein enzymesare involved in the hydrolysis. Although Con A also affectedthe hydrolysis of other wall polysaccharides, the present data,when considered in combination with the inhibitory effects ofglucan-specific or glucanasespecific antibodies, support theview that the breakdown of (1 3),(1 4)-ß-D-glucansis associated with the cell wall loosening that is responsiblefor auxin-induced elongation in Poaceae. (Received August 17, 1994; Accepted February 15, 1995)     

Reorientation of Cortical Microtubules in the Sub-Apical Region during Tuberization in Single-Node Stem Segments of Potato in Culture

The initial events in tuberization were examined in single-nodestem segments of potato, in which the tuberization was easilyregulated in culture. The addition of 8% sucrose to the culturemedium caused the cessation of elongation of lateral shootsand the swelling of the sub-apical region of each shoot. Swellingwas first induced by lateral cell expansion, which was followedby periclinal cell division. The divided cells then expandedlaterally. The alteration in the direction of growth was accompaniedby the reorientation of arrays of cortical microtubules (MTs),which was monitored by immunofluorescence microscopy. Cellsin the sub-apical region of elongating shoots had prominenttransverse arrays of MTs. The MTs in swelling cells were orientedlongitudinally with respect to the axis of the shoot. Finally,the arrays of MTs became completely disorganized. By contrast,the elongation of lateral shoots continued in GA₃-treated segmentsand the cells in the sub-apical region of such shoots retainedconspicuous transverse arrays of MTs during culture, even inthe presence of a high concentration (8%) of sucrose. (Received July 2, 1994; Accepted May 19, 1995)     

Characterization of a Dopamine-Releasing Action of 6R-l-erythro-Tetrahydrobiopterin: Comparison with a 6S-Form

Abstract: 6R-l -erythro-Tetrahydrobiopterin (6R-BH₄) is a cofactor for aromatic l -amino acid hydroxylases and nitric oxide synthase. Recently, we have reported that independently of its cofactor activities, 6R-BH₄ acts from the outside of neurons in the brain to enhance the release of monoamine neurotransmitters such as dopamine. To characterize the pharmacological properties of the action, we examined the effects of 6S-BH₄, a diastereoisomer of 6R-BH₄, on dopamine release in the rat striatum by using brain microdialysis and compared its effects with those of 6R-BH₄. Perfusion of 6S-BH₄ or 6R-BH₄ through the dialysis probe increased extracellular dopamine levels (an index of in vivo dopamine release) concentration dependently; the maximal increase by 6S-BH₄, was one-sixth of that by 6R-BH₄. 6S-BH₄ increased extracellular DOPA levels in the presence of NSD 1015, an inhibitor of aromatic l -amino acid decarboxylase (an index of in vivo tyrosine hydroxylase activity), to an extent similar to the increase induced by 6R-BH₄. The increase in the DOPA levels induced by either of the pteridines was abolished after pretreatment of rats with α-methyl-p-tyrosine (an inhibitor of tyrosine hydroxylase). Under the same conditions, the 6S-BH₄-induced dopamine release was abolished, but most of the 6R-BH₄-induced increase persisted. Coadministration of 6S-BH₄ with 6R-BH₄ inhibited the increase in dopamine release induced by 6R-BH₄ alone. These results show that 6R-BH₄ stimulates dopamine release by acting at the specific recognition site on the neuronal membrane, and that 6S-BH₄ acts as an antagonist of 6R-BH₄ at this site, although it has cofactor activities.     

Elimination of heparan sulfate by heparitinases induces abnormal mesodermal and neural formation in Xenopus embryos

The expression of heparan sulfate glycosaminoglycan (HS-GAG) was examined in Xenopus embryos during the developmental stages. Chemical analysis showed the existence of HS-GAG in the ³⁵S-labeled embryos. By western blot analysis using a specific anti-HS monoclonal antibody, HS-GAG related epitope was found after the neurulation on two protein bands, whose molecular weights were approximately 90 kDa and 100 kDa, respectively. Immunohistochemistry revealed that HS-GAG occurred exclusively in the animal hemisphere in early gastrulae, and then appeared predominantly on the sheath of the neural tube, the notochord and epithelium. To address whether HS-GAG chains contribute to Xenopus embryonic development, we eliminated the embryonic HS-GAG by injecting purified Flavobacterium heparitinases (HSase) into their blastocoels. Most of the injected embryos were aberrant in mesodermal and neural formation, and became acephalic. Histological examination showed that these embryos were completely devoid of the central nervous system and the mesodermal tissues. Neither heat-inactivated heparitinase nor chondroitinase showed such abnormality. The HS-GAG-eliminated embryos showed decreased expression of both muscular and neural-specific markers. These results suggest that HS-GAG plays an indispensable role in establishing the fundamental body plan during early Xenopus development.     

Differential Localization of the γ3 and γ12 Subunits of G Proteins in the Mammalian Brain

Abstract: The localization of two forms of the γ subunit of G proteins, γ₃ and γ₁₂, was examined in the mammalian brain. Concentrations of these two γ subunits increased markedly, as did those of glial fibrillary acidic protein, during postnatal development in the rat cerebral cortex. In aged human brains, by contrast, the concentration of γ₃ tended to decrease with age, whereas that of γ₁₂ in the temporal cortex increased slightly. An immunohistochemical study of human brains revealed that γ₃ was abundant in the neuropil, whereas γ₁₂ was localized in glial cells. In the hippocampal formation of aged human brains, levels of γ₁₂-positive cells, as well as levels of glial fibrillary acidic protein- and vimentin-positive astrocytes, increased, in particular in the CA1 subfield and the prosubiculum, in which there was a decrease in the number of pyramidal cells. The appearance of γ₁₂-positive cells associated with the loss of pyramidal cells was also observed in the hippocampus of rats that had been treated with kainic acid. These results indicate that γ₁₂ is strongly expressed in reactive astrocytes. In a study of cultured neural cells, we found that γ₁₂ was predominant in glioma cells, such as C6 and GA-1 cells, in contrast with the specific localization of γ₃ in PC12 pheochromocytoma cells, which are neuron-like cells. Taken together, the results indicate that γ₃ and γ₁₂ are selectively expressed in neuronal and glial cells, respectively, and that concentrations of γ₃ and γ₁₂ in the brain are related to the numbers and/or extent of maturation of these cells.     

Auxin-Induced Changes in the Molecular Weight Distribution of Cell Wall Xyloglucans in Avena Coleoptiles

The effect of auxin on the molecular weight (Mw) distributionof cell wall xyloglucans was investigated by gel permeationchromatography using coleoptile segments of Avena sativa L.cv. Victory, and the following results were obtained.

1. The water-insoluble hemicellulose (HC-A) mainly consisted ofxyloglucans. Iodine staining method revealed that relativelylarge amounts of xyloglucans were present in the water-solublehemicellulose (HC-B) and water-soluble polysaccharide (WS) fractions.
2. IAA did not cause remarkable changes in xyloglucan contentsin the hemicellulose, but significantly increased the xyloglucancontent in the WS fraction.
3. IAA substantially decreased theweight-average Mw of HC-A. Thiseffect became apparent within30 min of the incubation period,and was not affected by the0.15 M mannitol or 2% sucrose applied.Hydrogen ions also causeda decrease in the weight-average Mwof HC-A; its effect beingreversible.
4. Neither IAA nor hydrogen ions caused any remarkablechangesin the weightaverage Mw of water-soluble xyloglucansin theHC-B.

These results suggest that cell wall xyloglucans have an importantrole in auxininduced cell wall loosening in oat coleoptile cells. (Received May 10, 1984; Accepted August 20, 1984)     

Different metastatic modes of malignant melanoma implanted in the ear of young and old mice

Summary An attempt has been made to determine (a) whether aging plays an important role in resistance against metastasis and (b) whether dithiothreitol, an effective in vitro mitogenic potentiator of splenic cells of young and old mice, can modulate the occurrence of pulmonary metastasis. B16-F10 melanoma cells were injected into the outer ear of young and old female C57BL/6 mice; and the growth of the primary tumor, the palpable size of the cervical lymph node, and the number of lung metastases were then determined at various intervals. The ear was amputated when the primary tumor reached 4 mm in mean diameter. The following results were obtained. (a) The growth rate of the primary tumor in young mice is comparable to that in old mice. (b) Enlargement of the cervical lymph node occurs earlier in old than in young mice. (c) Old mice are more vulnerable to pulmonary metastases, but small metastasized pulmonary colonies are more prominent in old than in young mice. (d) Dithiothreitol (100 g) injected every 2 days after the inoculation of tumor cells is effective in reducing the incidence of pulmonary metastases in old mice.