Determination of non-protein-bound iron in human synovial fluid by high-performance liquid chromatography with electrochemical detection

Non-protein-bound iron in human synovial fluid was determined using high-performance liquid chromatography with electrochemical detection. The procedure was based on the separation of the iron—diethylenetriaminepentaacetic acid (DPTA) complex formed directly on a chromatographic column containing an anion-exchange resin followed by electrochemical detection. The method enabled more than 0.1 μM Fe(III) to be determined with an injection volume of 10 μl. A mixture of synovial fluid, 20 μM DTPA and acetate buffer was incubated in the presence and absence of superoxide (O⁻₂) generated by a xanthine—xanthine oxidase system and was ultrafiltered through a 30 000 molecular mass cut-off filter. No iron was detected in the ultrafiltrate at physiological pH. However, the presence of iron was observed in the ultrafiltrate at low pH, and O⁻₂ and decreased pH, iron may be released into the synovial fluid.     

Seed dispersal and vegetation development on a debris avalanche on the Ontake volcano,Central Japan

Seed dispersal and early revegetation processes were studied in an area devastated by a debris avalanche that occurred on volcano Ontake, Central Japan in 1984. The avalanche was initiated by a volcanic earthquake, and a 3.4 x 10⁷ m³ land mass destroyed the vegetation over 700 ha. The revegetation of the first five years was slow in the area of higher elevation (subalpine area). Both the speed of revegetation and the species richness of trapped seeds varied with the elevation. The post-disturbance vegetation on the deposit with surface soil or plant fragments was richer in species than that without any surface soil. Almost all of the seeds caught by sticky traps and most of the species with frequent occurrence in the vegetation after disturbance were wind dispersed. Wind dispersal was especially important on the deposit without surface soil or plant fragments.     

Regulation of L-alanine-initiated germination ofBacillus subtilis spores by alanine racemase

Summary Germination ofBacillus subtilis spores was initiated by L-Ala and competitively inhibited by D-Ala, suggesting the presence of an alanine receptor. The spores showed alanine racemase activity in the spore coat. To investigate the role of alanine racemase (L D) on germination, net racemase activity was determined using diphenylamine as a germination inhibitor and germination was measured using D-penicillamine as a racemase inhibitor. Apparent affinity of L-Ala to the germinant receptor was more than 1000 times higher than that to the racemase. Germination increased in the presence of D-penicillamine, when the concentration of L-Ala was low and that of spores was high. Racemase activity was optimal at 65°C at pH 9.0 and germination at 43°C at pH 7.2. Under unfavorable growth conditions such as high population of spores in limited nutrients, high temperature and high pH, spore alanine racemase converted the germinant actively to the inhibitor and this conversion may regulate germination for survival of the population.     

Response of Japanese beech (Fagus japonica Maxim.) sprouts to canopy gaps

The response of Japanese beech (Fagus japonica Maxim.) sprouts to canopy gaps in natural beech forest in central Japan was studied using two contrasted gaps in which tree-ring chronologies of regenerating stems were analyzed. The gaps were created by uprooting of a single Quercus mongolica var. grosseserrata stem (diameter: 50 cm; gap size: 40 m²; 23 years old) and by concurrent uprootings of four F. japonica stools (gap size: 180 m²; 30 years old). Japanese beech sprouts emerged before and after the gap formation and dominated stem populations in both gaps. In gaps, growth of F. japonica sprouts was equal or lower than growth of stems of seed origin, but most sprouts (F. japonica, Acer mono var. marmoratum) appeared a few years before emergence of seedlings. The small gap created by single stem fall was dominated by some beech sprouts from stools adjacent to the gap. The multiple gap was not closed by beech sprouts from stools surrounding the gap, but some dominant beech stems were resprouts from the uprooted beech stools. The existence of a sprout bank under the canopy may play an important role in the closing process of gaps in natural Japanese beech forest.     

Ethylene is a positive regulator of root hair development in Arabidopsis thaliana

Evidence is provided that ethylene is a positive regulator of hair cell development in the root epidermis of Arabidopsis thaliana. Treatment of seedlings with increasing concentrations of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC) results in progressively more root hair cells developing in positions normally occupied by non-hair cells. Consistent with these findings are observations that treatments that block either ethylene synthesis or its perception reduce the number of root hairs. A model is proposed in which either ethylene or ACC is a signal involved in specifying the pattern of cell differentiation in the Arabidopsis root epidermis.     

α-and β-Glycosidases in maize roots

Four glycosidases were analyzed in 10 mm apical segments prepared from growing roots (15 mm) of Zea mays L. The pH optima were found to be 5.8 for -glucosidase, 4.4 for -galactosidase, 6.4 for -glucosidase and 6.0 for -galactosidase. The -glucosidase showed 4-fold higher activity than the -galactosidase. The distribution of the -glucosidase activity was signifcantly different from that of the -galactosidase, -glucosidase and -galactosidase.Abbreviations -Glu -glucosidase - -Gal -galactosidase - -Glu -glucosidase - -Gal -galactosidase     

Purification and characterization of human testis aldose and aldehyde reductase

1. Aldose reductase and aldehyde reductase were purified to homogeneity from human testis. 2. The molecular weight of aldose reductase and aldehyde reductase were estimated to be 36,000 and 38,000 by SDS-PAGE, and the pI values of these enzymes were found to be 5.9 and 5.1 by chromatofocusing, respectively. 3. Aldose reductase had activity for aldo-sugars, whereas aldehyde reductase was virtually inactive for aldo-sugars. The Km values of aldose reductase for D-glucose, D-galactose and D-xylose were 57, 49 and 6.2 mM, respectively. Aldose reductase utilized both NADPH and NADH as coenzymes, whereas aldehyde reductase only NADPH. 4. Sulfate ion caused 3-fold activation of aldose reductase, but little for that of aldehyde reductase. 5. Sodium valproate inhibited significantly aldehyde reductase, but not aldose reductase. Aldose reductase was inhibited strongly by aldose reductase inhibitors being in clinical trials at concentrations of the order of 10(-7)-10(-9) M. Aldehyde reductase was also inhibited by these inhibitors, but its susceptibility was less than aldose reductase. 6. Reaction of aldose reductase with pyridoxal 5'-phosphate (PLP) resulted ca 2.5-fold activation, but aldehyde reductase did not cause the activation. PLP-treated aldose reductase has lost the susceptibility to aldose reductase inhibitor.     

CYP52 (cytochrome P450alk) multigene family in Candida maltosa: molecular cloning and nucleotide sequence of the two tandemly arranged genes

Southern blot analysis under low-stringency conditions using a previously isolated n-alkane-inducible cytochrome P450 (P450alk) gene as a probe revealed the presence of multiple P450alk-related genes in the genome of Candida maltosa. Nine P450alk-related genes (one reported previously and eight in the present report) were isolated from a genomic library constructed from this strain, and these were classified on the basis of sequence similarities into three pairs of putative allelic genes and three nonallelic genes. Two pairs of these alleles were tandemly arranged in the genome. The complete nucleotide sequences of one of these pairs were determined and compared to other members of this P450 family (CYP52) in C. maltosa and C. tropicalis. Northern blot analysis further showed that these genes were regulated by carbon sources. These results provide evidence for a P450alk (CYP52) multigene family in C. maltosa.     

A constitutive form of guinea pig liver cytochrome P450 closely related to phenobarbital inducible P450b(e)

In order to provide evidence that a cytochrome P450 belonging to the IIB subfamily is expressed as a constitutive form in the guinea pig, we tried to purify an isozyme from liver microsomes of untreated guinea pigs by assessing its reactivity with anti-P450b antibody in the present study. One form of cytochrome P450, named P450GP-1, was obtained. The minimum molecular weight of this isozyme was estimated to be 52,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino terminal sequence up to the 33rd amino acid of P450GP-1 was determined. As expected, comparison of the amino acid sequence with those of cytochrome P450 isozymes from other species reported so far indicated that P450GP-1 was highly homologous to P450s categorized in the IIB subfamily; that is, 67% similarity to rat P450b, 82% to rabbit LM2, 76% to dog PBD-2, 70% to mouse pf 3/46, and 73% to human IIB1. On the other hand, P450GP-1 showed only low similarity, less than 41%, to other cytochrome P450s of the II subfamily and those of the I, III, and IV families. Affinity of P450GP-1 to anti-P450b immunoglobulin G was confirmed to be comparable with that of a principal antigen, P450b. Immunoblot analysis revealed that P450GP-1 in the guinea pig liver microsomes was induced by phenobarbital treatment, but the increase was not as large as in the rat. P450GP-1 efficiently catalyzed benzphetamine N-demethylation, strychnine 2-hydroxylation, and testosterone 16 beta-hydroxylation, all of which are also catalyzed by P450b. Based on these results, it was strongly suggested that the IIB-type of cytochrome P450 in guinea pigs, at least one of them, is a constitutive form which is moderately induced by phenobarbital.     

Effects of IAA, Zeatin, Ammonium Nitrate and Sucrose on the Initiation and Development of Floral Buds in Torenia Stem Segments Cultured In Vitro

In Torenia stem segments cultured on a defined medium from whichammonium nitrate and growth regulators were omitted, adventitiousbuds were readily formed from epidermal tissue, with subsequentdifferentiation of floral buds. Using this plant material, thecorrelation between the time of application of various chemicalsand the time-course of floral bud differentiation was investigated.Histological examination showed that adventitious buds werevegetative during the first two weeks of the culture, and floralprimordia appeared after about three to four weeks of culture.We divided the flowering process in Torenia stem segments intothe following 3 phases: the first phase (first 2 weeks) duringwhich adventitious buds are formed, the second phase (3rd and4th weeks) during which floral buds are initiated and the thirdphase (5th to 12th weeks) during which floral buds develop.Then we added IAA, zeatin, ammonium nitrate or a high concentrationof sucrose to the medium during one, two or three of these phases.Ammonium nitrate added during the third phase suppressed floralbud development, but the high concentration of sucrose givenduring this phase stimulated it. These two chemicals influencedonly the development of floral buds previously initiated. Theapplication of IAA during the first phase promoted both theinitiation and development of floral buds. However, its applicationafter 2 weeks of culture failed to promote floral bud formation.Zeatin inhibited floral bud formation in a manner similar toammonium nitrate, but if it was added to the medium only duringthe first phase, it slightly promoted the initiation and developmentof floral buds. (Received July 7, 1981; Accepted October 12, 1981)