Seasonal Changes in Nitrogen Fractions of Coptis japonica, a Temperate Forest Evergreen Chamaephyte, and their Ecological Implications

Seasonal changes in several forms of nitrogen were investigatedin Coptis japonica, an evergreen rosette hemicryptophyte intemperate deciduous forest. The concentration of total nitrogenin rhizomes and roots decreased during the period of new shootgrowth from winter to spring. In the rhizomes, total solubleprotein stored by early summer decreased gradually until winter,coupled with an increase in free amino acids. Nitrogen was largelystored in free amino acids in the roots, especially during summer.The total soluble protein in current-year leaves decreased fromspring to summer and then increased during winter. The seasonalchanges in nitrogen components were coincident with the changein light-saturated photosynthetic rates recorded in a previousstudy. The ratio of total soluble protein to total nitrogendecreased from spring to summer and then increased from latesummer to winter in the current-year leaves. In contrast, chlorophyllcontent and the ratio of chlorophyll to total nitrogen werehigher in summer than in other seasons. The results indicatethat nitrogen was used in a manner that better utilizes thevery weak light in summer and the higher light intensities inother seasons. The major component of the free amino acid poolwas asparagine, in every organ throughout the season, exceptfor the senescent leaves. Since asparagine has a high N:C ratio,we suppose that the asparagine-dominated amino acid pool isadvantageous in the carbon-limited environment of the forestfloor.Copyright 1994, 1999 Academic Press Free amino acid composition, total nitrogen, total soluble protein, photosynthesis, evergreen hemicryptophyte     

Identification of human ST2 protein in the sera of patients with autoimmune diseases.

Soluble human ST2 protein (IL1RL1-a) in the sera of patients with various autoimmune diseases was identified by a newly developed procedure using specific monoclonal antibodies. After immunoprecipitation and subsequent immunoblotting, a glycosylated protein of about 60 kDa was detected in the sera of SLE patients, but not in the sera of healthy controls. The experiments using gel filtration and SDS-PAGE under a nonreducing condition indicated the existence of the ST2 multimer in serum. The mobility of the natural protein was slower than that of the recombinant human ST2 protein produced by COS7 cells in SDS-PAGE, suggesting a difference of glycosylation between humans and monkeys. The identification of the natural human ST2 protein should be important both to fundamental researches and the further clarification of the clinical implications of the ST2 protein.     

Molecular cloning of a non-isopeptide-selective human endothelin receptor.

We isolated several complementary DNA (cDNA) clones encoding a non-isopeptide-selective human endothelin receptor (ETBR) from a human placenta cDNA library. The clones, different in the length of their 3'-untranslated regions, encoded the same 442-amino acid protein with a transmembrane topology similar to that of other G protein-coupled receptors. The rank order of the binding of ET isopeptides (ET-1, ET-2 and ET-3) to the receptor expressed in COS-7 cells was ET-1 = ET-2 = ET-3. Northern blot analysis identified three mRNA species, 4.3 kb, 2.7 kb and 1.7 kb in size, probably generated by their use of alternative polyadenylation sites. These mRNAs were expressed in a wide variety of human tissues, at the highest level in the brain and at a significant level in cultured endothelial cells.     

Protective effect of sulfhydryl compounds on acute toxicity of morphinone

The ability of sulfhydryl compounds to provide protection against the acute toxicity of morphinone was investigated in mice. Subcutaneous administration of morphinone produced a reduction of hepatic non-protein sulfhydryl concentration. Pretreatments of mice with glutathione or cysteine significantly increased the survival rate of mice given a lethal dose of morphinone, whereas morphinone lethality was markedly potentiated by diethyl maleate. On the other hand, the administration of morphine produced a dose dependent reduction of hepatic non-protein sulfhydryl contents. However, neither glutathione nor cysteine protected mice from the acute toxicity of morphine. A possible explanation for these observations was proposed as follows: morphine is oxidized by morphine 6-dehydrogenase to morphinone, and the morphinone thus produced decreases the sulfhydryl contents in the liver. This mechanism is supported by the fact that morphinone reacts easily with glutathione and cysteine $\text{in vitro}$.     

Cross reactive antigens of Acholeplasma laidlawii and L-form of Staphylococcus aureus]

We demonstrated that the membrane of Acholeplasma laidlawii PG8 and L-form of Staphylococcus aureus, both of which induce cellular immunity in BALB/c mice, were antigenically related each other. Foodpad responses of the mice immunized with a mixture of either antigen and Freund's complete adjuvant showed clearly a cross reaction when challenged with the other antigen. Cross responses to incorporate 3H-thymidine to the spleen lymphocytes of the mice immunized with either antigen occurred in the presence of the other antigen. Furthermore, the purified T cells, but not B cells, of the spleen were activated in the presence of antigen-presenting cells. These antigens existing in the membrane fractions of both microorganisms were purified by Razin's method. Finally, these membrane components of A. laidlawii and L-form of S. aureus were subjected to gel electrophoresis and transferring to nitrocellulose membrane and used to stimulate the spleen lymphocytes of the mice immunized with A. laidlawii or of non-immunized mice. The fractions representing molecular weights of approximately 45 kD, 25 kD, and 13 kD of both microorganisms consistently stimulated the lymphocytes of the immunized mice but not those of non-immunized mice.     

Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe

Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy- and time-consuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a ‘module’, can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism.     

Microinjection of the monoclonal anti-tubulin antibody YL1/2 inhibits cleavage of sand dollar eggs

Two monoclonal antibodies against alpha-tubulin (YL1/2 and D2D6) were microinjected into the egg of the sand dollar Clypeaster japonicus, and their effects on cleavage of the egg were investigated. They had already been shown by immunoblotting to react specifically with egg tubulin and by immunofluorescence to stain the mitotic apparatus [OKA et al., (1990). Cell Motil. Cytoskel. 16:239-250]. Injection of YL1/2 prevented chromosome movement and cleavage, although the cleavage furrow developed in some cases. In all eggs injected at prometaphase, metaphase, or anaphase, the birefringence of the mitotic apparatus disappeared immediately after injection. Injection of D2D6 had no significant effect on mitosis or cleavage of whole eggs injected after nuclear disappearance, although it prevented the disappearance of the nuclear envelope in 54% of the eggs injected before the disappearance. FITC-conjugated D2D6 did not accumulate in the spindle when injected into the dividing sand dollar egg. These results indicate that YL1/2 disassembled microtubules, whereas D2D6 did not bind to microtubules in the living cell.     

The first isolation of enantiomeric and meso-zeaxanthin in nature

Racemic mixtures of (3RS, 3'RS)-zeaxanthin were separated into the three optical isomers, (3R, 3'R)-zeaxanthin (1), (3R,3'S;meso)-zeaxanthin (2) and (3S,3'S)-zeaxanthin (3), by converting to their corresponding dibenzoates and by using HPLC on an optical resolution column Sumipax OA-2000. According to this procedure, it has been shown that only (1) is isolated from higher plants, shellfish, starfish, sea squirt, sea cucumber and then examined; on the other hand (1), (2) and (3) are isolated from zeaxanthin fraction of shrimp, fish and turtle examined. This is the first isolation of enantiomeric and meso-zeaxanthin in nature.     

Subunit composition and ATP site labeling of the coated vesicle proton-translocating adenosinetriphosphatase

The partially purified proton-translocating adenosinetriphosphatase [(H+)-ATPase] from clathrin-coated vesicles has been reported to contain eight polypeptides of molecular weights 15,000-116,000 [Xie, X.S., & Stone, D.K. (1986) J. Biol. Chem. 261, 2492-2495]. To determine whether these polypeptides form a single macromolecular complex, we have isolated three monoclonal antibodies which recognize the reconstitutively active (H+)-ATPase in the native, detergent-solubilized state. All three monoclonal antibodies precipitate the same set of polypeptides from either the partially purified enzyme or the detergent-solubilized coated vesicle membrane proteins. The immunoprecipitated polypeptides have molecular weights of 100,000, 73,000, 58,000, 40,000, 38,000, 34,000, 33,000, 19,000, and 17,000. These results thus indicate that this set of polypeptides forms a single macromolecular complex and suggest that they correspond to subunits of the coated vesicle (H+)-ATPase. To identify the ATP-hydrolytic subunit of the coated vesicle (H+)-ATPase, the purified enzyme was reacted with N-ethylmaleimide (NEM) and 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl), both of which inhibit activity in an ATP-protectable manner. Labeling was carried out by using [3H]NEM or [14C]NBD-Cl, and the specificity of the reaction was increased by prelabeling of the protein with the nonradioactive reagents in the presence of ATP and by taking advantage of the nucleotide specificity of protection. The principal polypeptide labeled by both [3H]NEM and [14C]NBD-Cl had a molecular weight of 73,000. In addition, this protein was the only polypeptide whose labeling was significantly reduced in the presence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)