States of myosin subfragment-1 studied by catalyzed ascorbate reduction of bound spin label.

The second-order rate constant, k, whereby ascorbate reduces spin label, N-(1-oxyl-2, 2,6,6-tetramethyl-4-piperidyl) iodoacetamide, bound to the fast-reacting (SH₁) thiol groups of heavy meromyosin (HMM) has been compared with the k whereby ascorbate reduces free spin label in the same solvent. It is clear that the k of protein-bound spin label is primarily determined by conditions “on-board” subfragment-1 (S-1), rather than by properties of the solvent. First, in saturating [STP] the k of HMM-bound spin label was much greater than the k of free spin label, and both k's were independent of [KCl], from 0.05 to 1 m. Second, in the absence of ATP, or even in the presence of ADP, the k of HMM-bound spin label was less than the k of free spin label at l m KCl, and much more in a 0.05 m KCl. The organized structure of S-1 is required for observing the change of k with ATP, because the change of k disappeared on denaturing HMM with either guanidine hydrochloride or urea.Measuring k can be a “probe” to specify HMM states. However, the parameter, k, is conceptually dissimilar to measuring peak heights on an EPR spectrum. Experimentally we have observed that when [KCl] is increased, while [MgATP] = 0, spectral peak height is constant, but k varies remarkably. At no [KCl] did excess F-actin affect k. Quantitative examination of metal contamination (e.g., Cu, Fe) in HMM showed that changes in the k of HMM-bound spin label cannot arise from changes in proximity to contaminating metal redox catalysts bound to HMM.An intramolecular participant in the reaction of ascorbate with bound nitroxyl half inhibits the Ca²⁺-ATPase of spin labeled HMM, so signal annihilation and ATPase activity are closely correlated in time. The rate of signal annihilation is unaffected by prior reaction of the “SH₂” thiols with N-ethylmaleimide.     

The pH jump study of enzyme proteins. I. Liquefying alpha-amylase from Bacillus subtilis.

Rapid conformational changes due to pH jump were studied kinetically at 25 degrees mainly by the stopped-flow method using liquefying alpha-amylase from Bacillus subtilis [EC 3.2.1-.1, liquefying]. First, the conformational change due to a pH jump produced by mixing with alkali was monitored as a function of time at 245 nm through the ionization of phenolic hydroxyl groups of tyrosine residues which were originally buried and finally become exposed due to the pH jump. Three distinct phases of conformational change were clearly recognized by this method by varying the final pH values. Each phase involved the exposure of an essentially definite number of tyrosine residues, whose rate constant was crucially dependent on pH. Second, these phases of conformational change were subjected to examination in terms of the optical rotation change at 411 nm and the reversibility upon reverse pH jump with respect to conformational reconstitution, as observed through the protonation ofphenolic hydroxyl groups of ionized tyrosine residues and the enzyme activity. The first phase, which occurs above pH 12.5, involves no change in the optical rotation and is reversible as observed by the above two monitoring methods. In contrast, the other two phases, which are observed above pH 12.7, are accompanied by an optical rotation change and no appreciable reversibility was detected by these methods.     

Genetic polymorphisms of blood proteins in the troops of Japanese macaques,Macaca fuscata: IV. Erythrocyte esterase polymorphism inMacaca fuscata

Genetic variation at the locus controlling A₁ band of erythrocyte esterase was found in the Japanese macaque,Macaca fuscata. Existence of four alleles,Es-A ₁ ¹ ,Es-A ₁ ² ,Es-A ₁ ³ , andEs-A ₁ ⁴ , controlling the mobility of the band and codominance relation between them were postulated. A majority of the troops examined were monomorphic inEs-A ₁ 1-1 phenotype, and the variant phenotypes were observed to occur only in Yugawara-Ihama, Arashiyama, and Koshima areas.     

Development of microbodies in Candida tropicalis during incubation in a n-alkane medium

Development of microbodies in Candida tropicalis pK 233 was studied mainly by electron microscopical observation. The yeast cells, precultured on malt extract, scarcely contained microbodies and showed very low catalase activity. When the precultured cells were transferred to a n-alkane medium and incubated with shaking, the number of microbodies increased and concomitantly the activity of catalase was enhanced. That is, both the area ratio of microbodies in the cell and the ratio of microbodies to cytoplasm in area increased significantly during the utilization of n-alkanes for 8 hrs. Localization of catalase in the microbodies was demonstrated cytochemically by use of 3,3'-diaminobenzidine, but other organella in the cell, except for vacuoles appearing in the early growth phase and mitochondria, were not stained with this reagent. Microbodies seemed to grow by division. Biogenesis of microbodies in the yeast cells is also discussed.     

Detection of Cryptomeria japonica roots with ground penetrating radar

Abstract

Coarse tree roots, which are responsible for most root carbon storage, are usually measured by destructive methods such as excavation and coring. Ground penetrating radar (GPR) is a non-destructive tool that could be used to detect coarse roots in forest soils. In this study, we examined whether the roots of Cryptomeria japonica, a major plantation species in Japan, can be detected with GPR. We also looked for factors that impact the analysis and detection of roots. Roots and wooden dowels of C. japonica were buried 30 cm deep in sandy granite soil. From GPR measurements with a 900 MHz antenna, the distribution and diameter of samples in several transects were recorded. The buried roots were detected clearly and could be distinguished at diameters of 1.1–5.2 cm. There were significant positive relationships between root diameter and parameters extracted from the resultant GPR waveform. The difference in water content between roots and soil is a crucial factor impacting the ability to detect roots with GPR. We conclude that GPR can be used as a non-destructive tool, but further investigation is needed to determine optimal conditions (e.g. water content) and analytical methods for using GPR to examine roots in forest sites.     

Homogeneous Fluorescence Assays for RNA Diagnosis by Pyrene-Conjugated 2′- O -Methyloligoribonucleotides

We developed a bispyrene-conjugated 2 ′-O-methyloligoribonucleotide as an RNA-specific RNA-probe. The probe hybridized with the complementary RNA, greatly enhancing fluorescence and discriminating RNA from DNA. The assay was carried out in homogeneous aqueous media without removing the unbound probe from the detection solution. This homogeneous fluorescence assay also discriminated mismatch sequences in the target RNA. These pyrene probes could possess high potential to detect RNA in biological specimens simply.     

Preparation of 8-Chloropurine Nucleosides Through the Reaction Between their C-8 Lithiated Species and p-Toluenesulfonyl Chloride

Abstract

Chlorination of purine nucleosides protected with tert-butyldimethylsilyl (TBDMS) group was examined by the reaction of the C-8 lithiated species, generated by LDA, with p-toluenesulfonyl chloride as an electrophile. This provides a new method for the preparation of 8-chloropurine nucleosides.