Preparation of biologically active Ascaris suum mitochondrial tRNAMet with a TV-replacement loop by ligation of chemically synthesized RNA fragments.

Ascaris suum mitochondrial tRNA Met lacking the entire T stem was prepared by enzymatic ligation of two chemically synthesized RNA fragments. The synthetic tRNA could be charged with methionine by A.suum mitochondrial extract, although the charging activity was considerably low compared with that of the native tRNA, probably due to lack of modification. Enzymatic probing of the synthetic tRNA showed a very similar digestion pattern to that of the native tRNA Met, which has already been concluded to take an L-shape-like structure [Watanabe et al. (1994) J. Biol. Chem., 269, 22902-22906]. These results suggest that the synthetic tRNA possesses almost the same conformation as the native one, irrespective of the presence or absence of modified residues. The method of preparing the bizarre tRNA used here will provide a useful tool for elucidating the tertiary structure of such tRNAs, because they can be obtained without too much difficulty in the amounts necessary for physicochemical studies such as NMR spectroscopy.     

Natural disturbance and tree species coexistence in an old-growth beech - dwarf bamboo forest,southwestern Japan

Abstract. The structure and composition of a cool-temperate old-growth beech (Fagus crenata) - dwarf bamboo (Sasa spp.) forest, partially affected by landslide disturbance, in the Daisen Forest Reserve of southwestern Japan, were investigated in relation to forest floor and canopy conditions. All stems ≥ 4 cm DBH were mapped on a 4-ha plot and analyses were made of population structure, spatial distribution and spatial association of major tree species. The dominant species, F. crenata, which had the maximum DBH among the species present, had the highest stem density. However, for other species, larger-sized species had lower stem density with few smaller stems or saplings, while smaller-sized species had higher stem density with many smaller stems or saplings. Canopy trees of F. crenata were distributed randomly in the plot, while its stems in the other layers and all other species were distributed patchily. Small patches represent gap-phase regeneration. Larger patches correlate with landslide disturbance, difference in soil age, or the presence/absence of Sasa. Cluster analysis for spatial associations among species and stems in the different layers revealed that the forest community consists of several groups. One main group was formed on sites not covered with Sasa. This group contained a successional subgroup (from Betula grossa to Acer mono and/or F. crenata) initiated by landslide disturbance and a subgroup of tree species that avoid Sasa. Another group was formed on sites with mature soils covered largely with Sasa. This contained associations of canopy trees of F. crenata and smaller-sized tree species such as Acanthopanax sciadophylloides and Acer japonicum. It is found that the community of this old-growth beech forest is largely organized by natural disturbance and heterogeneous conditions of the forest floor (difference in soil age and presence/absence of Sasa). The existence of these different factors and the different responses of species to them largely contribute to the maintenance of tree species diversity in this forest.; Keywords: Cluster analysis; Fagus crenata; Forest dynamics; Gap; Landslide; Spatial pattern.     

Isolation and identification of (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid from anAmanita of the sectionRoanokenses (Amanitaceae)

A chlorine-containing non-protein amino acid which was recently discovered from the fruit bodies ofAmanita gymnopus (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid, was isolated and crystallized for the first time from the fruit bodies of an unknown member ofAmanita belonging to the sectionRoanokenses, subsectionSolitariae. The results of elementary analyses, determination of optical rotations,¹H- and¹³C-NMR-spectra, and some chemical reactions supported an earlier proposed structure.Part 24 in the series Biochemical studies of nitrogen compounds in fungi. for Part 23, see Hatanaka, S. I. et al. 1994. this journal35: 391–394.     

Gap regeneration in primary evergreen broad-leaved forests with or without a major canopy tree,Distylium racemosum,southwestern Japan: A comparative analysis

Gap regeneration was studied in a typical primary evergreen broad-leaved forest withoutDistylium racemosum, at the Kasugayama Forest Reserve, southwestern Japan and the results were compared with those from other primary evergreen broad-leaved forests in southwestern Japan, whereD. racemosum was the dominant species. Several common types of gap regeneration behavior were recognized among the major tree species and forests with or withoutD. racemosum consisted of three typical regeneration guilds which could be detected in the principal component analysis.Castanopsis cuspidata frequently regenerated in gaps from saplings recruited before gap formation in the forest withoutD. racemosum, although elsewhere, in forests withD. racemosum, it lacked advanced regeneration and regenerated in gaps from saplings recruited after gap formation. Some evergreenQuercus had their regenerations in gaps of the forest withoutD. racemosum, although elsewhere, in forests withD. racemosum, evergreenQuercus might not regenerate. The results indicate that tree species may change their regeneration behavior depending on the presence or absence of another key dominant species. This suggests that the presence and the dominance of a potential competitor induces shifts in the regeneration niche of other coexisting tree species.     

(2S)-2-Amino-5-chloro-4-hydroxy-5-hexenoic acid,a new chloroamino acid,and related compounds fromAmanita gymnopus

Combining different chromatography systems, unusual nonprotein amino acids were isolated and unequivocally identified from a small amount (less than 100 g fresh weight) ofAmanita gymnopus fruit body. Without obtaining crystals of these amino acids, on the basis of¹H-NMR determination, high resolution mass spectrometry, chlorine analysis and oxidation with L-amino acid oxidase, one of them proved to be a new chloroamino acid, (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid (G2). The other three were (2S)-2-amino-5-hexenoic acid (G1), (2S)-2-amino-4,5-hexadienoic acid (G3) and (2S)-2-amino-5-hexynoic acid (G4). Amino acid (G1) was also encountered for the first time in natural products. Amino acid (G3) has been reported from several kinds of fungi belonging toAmanita, subgenusLepidella. The occurrence of amino acid (G4) was already reported fromCortinarius claricolor.Part 23 in the series Biochemical studies of nitrogen compounds in fungi. Part 22, Hatanaka, S. I. et al. 1985. Trans. Mycol. Soc. Japan26: 61–68.     

Inhibition of endoplasmic reticulum (ER)-to-Golgi transport induces relocalization of binding protein (BiP) within the ER to form the BiP bodies.

Immunofluorescence staining of yeast cells with anti-binding protein (BiP) antibodies shows uniform staining of the endoplasmic reticulum (ER). We have found that overproduction of Sec12p, an ER membrane protein, causes a change of BiP distribution within the cell. Upon induction of Sec12p by the GAL1 promoter, the staining pattern of BiP turns into bright dots scattering in the cell, whereas the staining of Sec12p remains to be the typical ER figure. Overproduction of other ER membrane proteins, HMG-CoA reductase or Sed4 protein, does not induce such relocalization of BiP. Pulse-chase experiments and electron microscopy have revealed that the overproduction of Sec12p inhibits protein transport from the ER to the Golgi apparatus. When the transport is arrested by one of the sec mutations that block the ER-to-Golgi step at the restrictive temperature, the BiP staining also changes into the punctate pattern. In contrast, the sec mutants that block later or earlier steps of the secretory pathway do not induce such change of BiP localization. These observations indicate that relocalization of BiP is caused by the inhibition of ER-to-Golgi transport. Using immunoelectron microscopy, we have found that the punctate staining is because of the accumulation of BiP in the restricted region of the ER, which we propose to call the "BiP body." This implicates existence of ER subdomains in yeast. A vacuolar protein, proteinase A, appears to colocalize in the BiP body when the ER-to-Golgi transport is blocked, suggesting that the BiP body may have a role as the site of accumulation of cargo molecules before exit from the ER.     

Embryonic Development and Postnatal Changes in Free d-Aspartate and d-Serine in the Human Prefrontal Cortex

Abstract: We have analyzed free chiral amino acids (aspartate and serine) in the human frontal cortex at different ontogenic stages (from 14 weeks of gestation to 101 years of age) by HPLC with fluorometric detection after derivatization with N-tert -butyl-oxycarbonyl- l -cysteine and o -phthaldialdehyde. Exceptionally high levels of free d -aspartate and d -serine were demonstrated in the fetal cortex at gestational week 14. The ratios of d -aspartate and of d -serine to the total corresponding amino acids were also high, at 0.63 and 0.27, respectively. The concentration of d -aspartate dramatically decreased to a trace level by gestational week 41 and then remained very low during all postnatal stages. In contrast, the frontal tip contained persistently high levels of d -serine throughout embryonic and postnatal life, whereas the d -amino acid content in adolescents and aged individuals was about half of that in the fetuses. Because d -aspartate and d -serine are known to have selective actions at the NMDA-type excitatory amino acid receptor, the present data suggest that these d -amino acids might play a pivotal role in cerebral development and functions that are related to the NMDA receptor.     

The Role of c-kit Proto-oncogene during Melanocyte Development in Mouse. In vivo Approach by the In utero Microinjection of Anti-c-kit Antibody

In order to investigate the role of the c- kit oncogene in the melanoblast development, a rat monoclonal antibody (ACK2) against the mouse c-kit protein was used to localize cells expressing c-kit during fetal development. ACK2 was also injected directly into the amniotic cavity of mouse fetuses at successive developmental stages. After birth, the offspring were examined to determine the resulting coat color patterns. c-kit positive melanoblasts first appeared in dermis of fetuses at 11.5 days postcoitum (dpc). Subsequently, these cells increased in number and migrated dorsolaterally to the ventral region, and by 12.5 dpc some of them began to invade the epidermis. Treatment of fetuses by ACK2 microinjection appeared to affect the pigmentation in the coat, inducing a variety of spotting patterns in offspring, and the location of the spots was closely correlated with gestational stage. ACK2 injection of early fetuses produced major changes in coat color even though few c-kit positive cells were detectable in the dermal mesenchyme at the time of injection. Large spots were also induced when mid-stage fetuses with a only few c-kit positive cells in the dorsal region were injected. By contrast, except for spot formation in the center of ventral region, ACK2 injection did not appear to affect melanogenesis in late stage fetuses that had many c-kit positive cells.