Classification and ecological characterization of coniferous forest phytogeocoenoses of Hokkaido,Japan

Coniferous forest phytogeocoenoses of Hokkaido Island, Japan, were studied to classify them based on vegetation characteristics, to analyse their soils, to correlate the vegetation and soil characteristics, and to provide some ecological interpretation for the phytogeocoenosis differentiation and establishment. Five forest types were distinguished based on the vegetation structure, each of which was comparable to plant association of Krajina (1960); 1. the moss type, 2. the Sasa kurilensis type, 3. the Sasa senanensis type, 4. the Carex sachalinensis type, and 5. the Dryopteris crassirhizoma type. Soil base status indicated by pH, electric conductivity, amount of calcium and magnesium, and base saturation showed a fair correlation with the forest types. The forest types were, therefore, arranged along a soil nutritional gradient. The moss type developed in the least fertile habitats whereas the Dryopteris crassirhizoma type in the most fertile habitats, and others were in between the two. It was suggested that in the island, where climate was humid with excess of soil water throughout a year, soil nutritional regime, more specifically availability of bivalent cations which tended to be removed by the excessive soil water, seemed to be a critical factor to differentiate and establish the forest types.This study was financially supported by the Science Research Grant of the (Monbusho) Japanese Goverment (Grant No. 60540422).     

Purification of two chitinases from Rhizopus oligosporus and isolation and sequencing of the encoding genes.

Two chitinases were purified from Rhizopus oligosporus, a filamentous fungus belonging to the class Zygomycetes, and designated chitinase I and chitinase II. Their N-terminal amino acid sequences were determined, and two synthetic oligonucleotide probes corresponding to these amino acid sequences were synthesized. Southern blot analyses of the total genomic DNA from R. oligosporus with these oligonucleotides as probes indicated that one of the two genes encoding these two chitinases was contained in a 2.9-kb EcoRI fragment and in a 3.6-kb HindIII fragment and that the other one was contained in a 2.9-kb EcoRI fragment and in a 11.5-kb HindIII fragment. Two DNA fragments were isolated from the phage bank of R. oligosporus genomic DNA with the synthetic oligonucleotides as probes. The restriction enzyme analyses of these fragments coincided with the Southern blot analyses described above and the amino acid sequences deduced from their nucleotide sequences contained those identical to the determined N-terminal amino acid sequences of the purified chitinases, indicating that each of these fragments contained a gene encoding chitinase (designated chi 1 and chi 2, encoding chitinase I and II, respectively). The deduced amino acid sequences of these two genes had domain structures similar to that of the published sequence of chitinase of Saccharomyces cerevisiae, except that they had an additional C-terminal domain. Furthermore, there were significant differences between the molecular weights experimentally determined with the two purified enzymes and those deduced from the nucleotide sequences for both genes. Analysis of the N- and C-terminal amino acid sequences of both chitinases and comparison of them with the amino acid sequences deduced from the nucleotide sequences revealed posttranslational processing not only at the N-terminal signal sequences but also at the C-terminal domains. It is concluded that these chitinases are synthesized with pre- and prosequences in addition to the mature enzyme sequences and that the prosequences are located at the C terminal.     

A protease inhibitor produced by Streptomyces lividans 66 exhibits inhibitory activities toward both subtilisin BPN' and trypsin.

A proteinaceous protease inhibitor was isolated from the culture broth of Streptomyces lividans 66 by a series of purification steps (salting out by ammonium sulfate, ion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on Phenyl-Sepharose, and gel-filtration on Sephacryl S-200), and was named S. lividans protease inhibitor (SLPI). The purified SLPI existed in a dimeric form consisting of two identical subunits, each of which was composed of 107 amino acids. SLPI exhibited strong inhibitory activity toward subtilisin BPN'. These features were similar to those of protein protease inhibitors produced by other Streptomyces (SSI family inhibitor). In addition, SLPI was capable of inhibiting trypsin with an inhibitor constant (Ki) of about 10(-9) M. The primary structure of SLPI and location of two disulfide bridges were homologous to those of the other serine protease inhibitors of Streptomyces. The reactive site of SLPI was found to be Arg67-Glu68 from the sequence analysis of cleaved SLPI which was produced by acidification of subtilisin-SLPI complex. An Arg residue at the P1 site was consistent with the trypsin-inhibitory property of SLPI. Sequence comparison with other members of the SSI family revealed that amino acid replacements in SLPI were mainly localized on the surface of the SLPI molecule, and many of the amino acid residues in beta-sheets and hydrophobic core were well conserved.     

Anti-eye muscle antibodies and hypothyroid Graves' disease: a case report.

We report the case of a 70-year-old man who developed hypothyroidism associated with TSH receptor antibodies and severe ophthalmopathy during lithium therapy. He had received lithium therapy for more than 20 years for manic depression, when ophthalmopathy (class VI of the American Thyroid Association classification) and mild hypothyroidism developed. Orbital magnetic resonance imaging indicated marked enlargement of the superior, medial and inferior rectus muscles in the left eye. He had anti-eye muscle antibodies in his serum, detected by Western blotting and quantified by chromatoscanning, as well as anti-TSH receptor antibodies. He was treated with supplementation of levothyroxine and four cycles of methylprednisolone pulse therapy. After the pulse therapy, both anti-eye muscle antibodies and anti-TSH receptor antibodies decreased and disappeared in parallel with the improvement in eye symptoms and signs. These observations suggest the importance of anti-eye muscle antibodies as clinical markers in the development of thyroid-associated ophthalmopathy.     

Combined mutagenicity of methyl methanesulfonate and ethyl methanesulfonate in Chinese hamster V79 cells.

The combined effects of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) on the induction of 6-thioguanine (6TG)-resistant mutants and chromosome aberrations were examined in Chinese hamster V79 cells. Cells were simultaneously treated with EMS at a concentration of D20 and MMS at various concentrations for 3, 6 or 9 h. In other experiments cells were simultaneously treated with MMS at a concentration of D20 and EMS at various concentrations for 3, 6 or 9 h. The mathematical analysis of the combined effects of both chemicals for cell killing (cytotoxicity) and 6TG-resistant mutations indicates that synergistic interactions were observed for both cell killing and mutations induced by MMS and EMS. The frequency of chromosome aberrations induced by simultaneous treatment with MMS at a concentration of D20 and EMS at various concentrations for 3 h was additive. However, the frequency of chromosome aberrations induced by EMS at a concentration of D20 and MMS at various concentrations for 3 h was not significantly different from those induced by MMS alone.     

Parturition in a free-ranging Japanese monkey (Macaca fuscata)

Parturition behavior of a multiparous female and her interactions with group members throughout the birth process were recorded for a free-ranging Japanese monkey (Macaca fuscata). The female showed evidence of 18 contractions during the 35 min prior to delivery, with a mean duration and a mean intercontraction interval of 30 sec and 96 sec, respectively. These values were similar to those in individually caged Japanese monkeys. Some adult females remained in proximity to the female who was giving birth during the prepartum phase, and her 2-year-old daughter watched the delivery of the infant. Even during the prepartum phase the female moved in order to keep up with the group which traveled from the feeding site to a sleeping site in the forest.     

Existence of lipid vesicles containing platelet-activating factor in endothelial cell lysate

Platelet aggregation activity due to platelet-activating factor (PAF) was detected at high molecular weight (HMW) and low molecular weight fractions after gel-filtration chromatography of cell lysate of endothelial cells. [³H]PAF added to the cell lysate was similarly distributed after chromatography. The radioactivity associated with HMW fraction was not reduced by digesting the lysate with trypsin, suggesting that PAF was not making complexes with proteins but was included in lipid vesicles in cell lysate. Further evidence showed that an unknown specific factor(s) was needed to form these PAF-containing lipid vesicles. Radioactivity was not found in HMW fraction when [³H]PAF was mixed with cell lysate of vascular smooth muscle cells. When monomeric PAF was added to endothelial cell lysate, the specific activity of aggregation decreased to the level exerted by endogenous PAF-containing lipid vesicles due to incorporation into lipid vesicles. PAF in the form of lipid vesicles was more stable in plasma than monomeric form.     

Spectrophotometrical and immunochemical studies on the conformational changes in poly(dG-dC).poly(dG-dC) after modification by 4-hydroxyaminoquinoline 1-oxide

Poly(dG-dC).poly(dG-dC) was modified by the reaction with 4-hydroxyaminoquinoline 1-oxide (4HAQO) in the presence of seryl-AMP. The conformations of 4HAQO-modified poly(dG-dC).poly(dG-dC) and of poly(dG-dC).poly(dG-dC) were studied by circular dichroism spectra under various salt concentration conditions. 4HAQO residues to guanine bases are inefficient in inducing the transition of poly(dG-dC).poly(dG-dC) from B-form to Z-form conformation. We have elicited monoclonal antibodies against 4HAQO-poly(dG-dC).poly(dG-dC). They were characterized using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and binding to supercoiled DNA. These antibodies reacted with 4HAQO-poly(dG-dC).poly(dG-dC) specifically but not with 4HAQO-modified DNA or poly(dG).poly(dC). However, they cross-reacted with N-acetoxy-2-acetylaminofluorene-modified poly(dG-dC).poly(dG-dC) in Z-form conformation. These monoclonal antibodies may recognize a unique conformation in poly(dG-dC).poly(dG-dC) after 4HAQO modification.     

14-Fluorobacteriorhodopsin and other fluorinated and 14-substituted analogues. An extra, unusually red-shifted pigment formed during dark adaptation

Five vinyl-substituted fluororetinal analogues (8-F, 10-F, 12-F, 14-F, and 13,14-F2) were found to give bacteriorhodopsin analogues with properties similar to those of the parent system. Of these, only 14-fluororetinal was found to give an extra red-shifted BR analogue (lambda max less than or equal to 680 nm) in equilibrium with the normal 587-nm pigment. The 680-nm pigment was enriched upon irradiation. It rearranged to the 587-nm pigment at room temperature (delta E [symbol: see text] = 20.8 kcal/mol). Chromophore extraction experiments revealed the all-trans geometry for the 680-nm pigment. 14-Chlororetinal gave a similarly red-shifted pigment while 14-methylretinal did not. A scheme for dark adaptation of the 14-halogenated bacteriorhodopsins has been proposed in which the new red-shifted pigment was assigned the all-trans, 15-syn geometry.     

AMP synthesis in aqueous solution of adenosine and phosphorus pentoxide

Possible formation of a P₄O₁₀ molecule in magma, the stability of the molecule in hydrous volcanic gas at high temperatures and a possible prebiotic phosphate cycle were discussed in relation to chemical evolution. To demonstrate the utility of phosphorus pentoxide as a phosphorylating agent, aqueous solutions of adenosine (0.02M) and phosphorus pentoxide (0.2M) were incubated at 37°C for 5 months. The pH of the solutions was adjusted every day or every few days to each fixed value (9.0, 10.5, 11.5, 12.5) with 10 N NaOH. The HPLC analysis showed the formation of 2-AMP, 3-AMP, 5-AMP, cyclic (2–3)-AMP and cyclic (3–5)-AMP. The main components of the products were 2- and 3-AMP, though cyclic (2–3)-AMP was the main component in the early period of the incubation at pH 9.0. The yields (conversion rate of adenosine to AMPs) were increased almost linearly with the incubation time for 5 months in the case of pH 9.0. The final yields were about 3% (pH 9.0), 6% (pH 9.0, 1 M NaCl), 5% (pH 9.0, 0.01 M CaCl₂, 0.01 M MgCl₂), 7% (pH 9.0, 0.5 M NaCl, 0.01 M CaCl₂, 0.01 M MgCl₂), 9% (pH 9.0, 1 M NaCl, 0.01 M CaCl₂, 0.01 M MgCl₂), 32% (pH 10.5), 43% (pH 11.5), 35% (pH 12.5).