Different behavior of artemisinin and tetraoxane in the oxidative degradation of phospholipid.

The reaction of trioxane and tetraoxane endoperoxides with unsaturated phospholipid 1 in the presence of Fe(II) was investigated in the absence of oxygen by means of tandem ESI-MS analysis. The spectral analyses for the reaction mixtures showed that artemisinin 2 with a trioxane structure gave no peak except that for the remaining intact phospholipid 1 (m/z 758.9), indicating that there was no structural change to 1. On other hand, the reaction mixture of 1 with tetraoxanes 3 and 4 afforded a number of new peaks (m/z 620-850) that were presumably assigned to oxidative degradation products originating from phospholipid 1. Since this reaction was completely inhibited by the addition of a phenolic antioxidant, the process was considered to involve some free radical species. The newly discovered marked differences in reactivity between the trioxane and the tetraoxanes possibly reflects their different anti-malarial mechanisms, and this reactivity may contribute to the classification of a number of anti-malarial endoperoxides whose mode of action is based on phospholipid oxidation.

Synthesis of novel conjugates of tetraoxane endoperoxide with bis(quaternary ammonium salts).

Novel water-soluble conjugates of 1,2,4,5-tetraoxane bis(quaternary ammonium salts) were synthesized in a relatively stable crystalline form via four steps starting from methyltrioxorhenium-catalyzed endo-peroxidation of ethyl 4-oxocyclohexanecarboxylate with hydrogen peroxide in hexafluoro-2-propanol. The assay for the in vitro toxicity of water-soluble tetraoxanes 5a-5d to malaria parasites indicate that they were inactive against the Plasmodium falciparum FCR-3 strain.

Histological skeletal muscle damage and surface EMG relationships following eccentric contractions.

This study examined the effects of a different number of eccentric contractions (ECs) on histological characteristics, surface electromyogram (EMG) parameters (integral EMG, iEMG; muscle fiber conduction velocity, MFCV; and action potential waveform), and isometric peak torque using the rat EC model. Male Wistar rats (n = 40) were anesthetized, and ECs were initiated in the tibialis anterior muscle via electrical stimulation while the muscle was being stretched by electromotor. The rats were grouped according to the number of ECs (EC1, EC5, EC10, EC20, EC30, EC40, and EC100). Three days after the ECs, surface EMG signals and isometric peak torque were measured during evoked twitch contractions via electrical stimulation of the peroneal nerve. The muscle damage was evaluated from hematoxylin-eosin (HE) stained cross sections as a relative number of damaged fibers to intact fibers. Intense histological muscle damage (approximately 50% to 70% of the fiber), loss of isometric peak torque, disturbance of action potential waveform, and depression of iEMG (approximately -60% to -70%) were observed at EC20, EC30, EC40, and EC100. On the other hand, the MFCV did not change in any EC group. Although muscle damage and pathological surface EMG signals were not found at EC10, isometric peak torque was reduced significantly. In conclusion, the extent of histological muscle damage is not proportionally related to the number of ECs. Muscle damage was reflected by iEMG and action potential waveforms, but not by MFCV, which remained unaffected even though approximately 50% to 70% of the fiber demonstrated injury.

Development of novel yeast cell surface display system for homo-oligomeric protein by coexpression of native and anchored subunits.

Streptavidin derived from Streptomyces avidinii was displayed on the cell surface of the yeast Saccharomyces cerevisiae by cell-surface engineering using two types of plasmid for the expression of a native subunit and an anchored subunit fused with the C-terminus of 318 amino acids of Flo1p containing a glycosylphosphatidylinositol anchor attachment signal. The displayed streptavidin had the binding ability for biotinylated compounds. This was confirmed by fluorescence microscopy after the adsorption of yeast cells displaying streptavidin and biotinylated fluorescein isothiocyanate. On the other hand, streptavidin produced by cells harboring only the plasmid for the expression of the anchored subunit showed a very low binding activity for biotinylated compounds. Cells displaying streptavidin may constitute novel whole-cell affinity adsorbents widely used for immunoassay and biosensing. This coexpression method will ensure that proteins, such as homo- and hetero-oligomeric proteins, are displayed on the cell surface in an active form.

A facile and versatile preparation of bilindiones and biladienones from tetraarylporphyrins.

Bilindiones and biladienones carrying aryl groups at the meso positions were prepared using coupled oxidation reactions of iron tetraarylporphyrins in 20-63% yield.

Conversion of isoeugenol into vanillic acid by Pseudomonas putida I58 cells exhibiting high isoeugenol-degrading activity.

Pseudomonas putida I58 was isolated from soil by a conventional enrichment culture method using isoeugenol as a sole carbon source. The strain utilized isoeugenol, vanillin and vanillic acid as carbon sources. On the other hand, the intermediates of the eugenol-degrading pathway, such as eugenol, coniferyl alcohol, coniferyl aldehyde and ferulic acid, were not utilized by this strain, indicating that isoeugenol is directly degraded to vanillin without the formation of ferulic acid. The resting cells of P. putida I58 rapidly converted isoeugenol into vanillic acid via vanillin with a conversion yield of 98% by 40-min incubation.

Ferulic acid production from clove oil by Pseudomonas fluorescens E118.

For the production of the useful antioxidant ferulic acid from clove oil containing abundant eugenol, the growth of eugenol-degrading microorganisms in the presence of clove oil was examined. Pseudomonas fluorescens E118, a clove-oil-tolerant strain, accumulated 6.1 g/l ferulic acid under optimized culture conditions with the intermittent addition of eugenol. When the bacterium was applied to ferulic acid production from clove oil, 5.8 g/l ferulic acid was produced with the intermittent addition of clove oil. Since clove oil is much cheaper than eugenol, ferulic acid production from clove oil by the bacterium is promising for the industrial production of ferulic acid.

Preparation of yeast strains displaying IgG binding domain ZZ and enhanced green fluorescent protein for novel antigen detection systems.

To develop novel immunofluorescence-labeling and antigen-detection systems, the ZZ domain derived from Staphylococcus aureus, which binds to the Fc part of immunoglobulin G, and enhanced green fluorescent protein (EGFP) were displayed on the cell surface of Saccharomyces cerevisiae by cell-surface engineering using the C-terminus 318 amino acids of Flo1 protein. Two systems were constructed, one for co-display of ZZ and EGFP, and one for display of a fusion protein of the two. In both cases, two proteins on the cell surface successfully retain their activities.

Effects of aging on capillary number and luminal size in rat soleus and plantaris muscles.

To clarify aging-related changes in the capillary network in skeletal muscle, we morphometrically examined the capillary supply to individual muscle fibers and capillary luminal size in young (3-month-old) and old (19-month-old) male Wistar rats. All morphometric parameters for capillary and muscle fiber were determined in the cross sections of the perfusion-fixed soleus (SOL) and plantaris (PL) muscles. The range of fiber size was larger in the old muscles because of hypertrophy and atrophy of fibers. However, the capillary supply to individual muscle fibers, assessed as the mean of capillary contacts around a muscle fiber, did not change with aging in SOL muscle (young rats = 7.8 +/- 0.4 vs old rats = 8.1 +/- 0.8) or PL muscle (young rats = 6.4 +/- 0.3 vs old rats = 7.0 +/- 0.9). The ratio of individual muscle fiber area to the number of capillary contacts around a muscle fiber did not differ between young rats (SOL = 361.7 +/- 76.0; PL = 264.7 +/- 20.9) and old rats (SOL = 350.2 +/- 61.3; PL = 296.8 +/- 44.9). The mean capillary luminal diameter did not differ statistically in young and old rats (SOL, young rats = 5.3 +/- 0.5 vs old rats = 5.1 +/- 0.1; PL, young rats = 5.0 +/- 0.3 vs old rats = 5.4 +/- 0.2). In conclusion, the relationship between capillary supply and muscle fiber size is similar for both young and old rats, and the luminal size of each capillary was maintained with advancing age.