Polymerization of guaiacol by lignin-degrading manganese peroxidase from Bjerkandera adusta in aqueous organic solvents

 Lignin-degrading manganese (II) peroxidase (MnP) purified from the culture of a wood-rotting basidiomycete, Bjerkandera adusta, was used in the polymerization of guaiacol. MnP was found to catalyze polymerization of guaiacol in 50% aqueous acetone, dimethyl formamide, methanol, ethanol, dioxane, acetonitrile, ethylene glycol and methylcellosolve. Maximum yield of polyguaiacol was achieved in 50% aqueous acetone. The weight average molecular weight (M _w) of the polymer was estimated to be 30 300 by gel permeation chromatography. However, matrix-assisted laser desorption ionization time of flight mass spectroscopy (MALDI-TOF-MS) analysis gave a more reliable M _w of 1690. IR, ¹³C-NMR, MALDI-TOF-MS and pyrolysis GC-MS analyses showed the presence of C–C and C–O linkages and quinone structure in polyguaiacol. It was also indicated that polyguaiacol has a methoxy-phenyl group as the terminal moiety. This suggests that polyguaiacol is a branched polymer in which guaiacol units are cross-linked at the phenolic group. Thermal gravimetric and differential scanning calorimetric analyses were also carried out. MnP also catalyzed the polymerization of o-cresol, 2,6-dimethoxyphenol and other phenolic compounds and aromatic amines. M _w of these polymers ranged from around 1000 to 1500. Received: 2 August 1999 / Received revision: 10 December 1999 / Accepted: 4 January 2000     

Induced Response of Tomato Plants to Injury by Green and Red Strains of Tetranychus Urticae

We studied the induced response of tomato plants to the green strain and the red strain of the spider mite Tetranychus urticae. We focused on the olfactory response of the predatory mite Phytoseiulus persimilis to volatiles from T. urticae-infested tomato leaves in a Y-tube olfactometer. Tomato leaves attracted the predatory mites when slightly infested with the red strain, or moderately or heavily infested with the green strain. In contrast, neither leaves that were slightly infested with green-strain mites, nor leaves that were moderately or heavily infested with the red strain attracted the predators. We discuss the specific defensive responses of tomato plants to each of the two strains.     

Quercetin metabolites inhibit copper ion-induced lipid peroxidation in rat plasma

The oxidative susceptibility of plasma obtained from rats after intragastric administration of quercetin was studied to know whether or not quercetin acts as an in vivo antioxidant after metabolic conversion. Quercetin was raised in the rat blood plasma essentially as glucuronide and/or sulfate conjugates. The plasma obtained from rats after quercetin administration was more resistant against copper sulfate-induced lipid peroxidation than the control plasma on the basis of the accumulation of cholesteryl ester hydroperoxides and the consumption of α-tocopherol. The results strongly suggest that some conjugated metabolites of quercetin act as effective antioxidants when plasma is subject to metal ion-induced lipid peroxidation.     

Nuclear Localization of Brain-type Glycogen Phosphorylase in Some Gastrointestinal Carcinoma

Our previous reports have demonstrated frequent and strong expression of glycogen phosphorylase (EC 2.4.1.1) activity mainly in the cytoplasm of gastric carcinoma. Although previous studies have suggested the phosphorylase glyco-syltransferase system to be in the nucleus from enzyme histochemical analyses, intranuclear localization of the phosphorylase has not been fully established. The aims of the present study are to investigate the nuclear localization of glycogen phosphorylase and to identify the isoform of phosphorylase in the nucleus of gastrointestinal carcinoma. The activity of glycogen phosphorylase in carcinoma cells corresponding to the nucleus was demonstrated using enzyme cytochemical analysis. The phosphorylase activity coincided with localization revealed by immunocytochemistry using affinity-purified specific anti-human brain-type glycogen phosphorylase antibody. The isoform expressed in the nuclei of carcinoma cells was identified as bei ng only the brain type according to a polymerase chain reaction-based assay using RNA obtained from gastric carcinoma cells and primers specific to muscle, liver and brain types of glycogen phosphorylase. The intranuclear localization of the brain-type isoform was confirmed by immunoelectron microscopical analyses. Further investigation to examine the nuclear localization in human carcinoma tissue (145 and 25 specimens with gastric and colonic carcinoma respectively) was carried out by immunohistochemistry using specific anti-brain-type antibody. Nuclear immunostaining was observed in seven cases out of 145 gastric carcinoma. The present study is the first to clarify the nuclear localization of glycogen phosphorylase with enzymatic activity in gastrointestinal carcinoma. The isoform of the enzyme expressed in the carcinoma was identified as the brain type. These results warrant further studies on the mechanisms for transporting the large molecule of brain-type glycogen phosphorylase to nuclei and its function in the nucleus of carcinoma cells.     

Brown Adipose Tissue in Cetacean Blubber

Brown adipose tissue (BAT) plays an important role in thermoregulation in species living in cold environments, given heat can be generated from its chemical energy reserves. Here we investigate the existence of BAT in blubber in four species of delphinoid cetacean, the Pacific white-sided and bottlenose dolphins, Lagenorhynchus obliquidens and Tursiops truncates, and Dall’s and harbour porpoises, Phocoenoides dalli and Phocoena phocoena. Histology revealed adipocytes with small unilocular fat droplets and a large eosinophilic cytoplasm intermingled with connective tissue in the innermost layers of blubber. Chemistry revealed a brown adipocyte-specific mitochondrial protein, uncoupling protein 1 (UCP1), within these same adipocytes, but not those distributed elsewhere throughout the blubber. Western blot analysis of extracts from the inner blubber layer confirmed that the immunohistochemical positive reaction was specific to UCP1 and that this adipose tissue was BAT. To better understand the distribution of BAT throughout the entire cetacean body, cadavers were subjected to computed tomography (CT) scanning. Resulting imagery, coupled with histological corroboration of fine tissue structure, revealed adipocytes intermingled with connective tissue in the lowest layer of blubber were distributed within a thin, highly dense layer that extended the length of the body, with the exception of the rostrum, fin and fluke regions. As such, we describe BAT effectively enveloping the cetacean body. Our results suggest that delphinoid blubber could serve a role additional to those frequently attributed to it: simple insulation blanket, energy storage, hydrodynamic streamlining or contributor to positive buoyancy. We believe delphinoid BAT might also function like an electric blanket, enabling animals to frequent waters cooler than blubber as an insulator alone might otherwise allow an animal to withstand, or allow animals to maintain body temperature in cool waters during sustained periods of physical inactivity.     

Nano-LC-MS/MS for Quantification of Lyso-Gb3 and Its Analogues Reveals a Useful Biomarker for Fabry Disease

Biomarkers useful for diagnosis and evaluation of treatment for patients with Fabry disease are urgently needed. Recently, plasma globotriaosylsphingosine (lyso-Gb3) and lyso-Gb3-related analogues have attracted attention as promising biomarkers of Fabry disease. However, the plasma concentrations of lyso-Gb3 and its analogues are extremely low or below the detection limits in some Fabry patients as well as in healthy subjects. In this paper, we introduce the novel application of a nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) system to the measurement of lyso-Gb3 and its analogues in plasma. Nano-LC-MS/MS requires smaller amounts of samples and is more sensitive than conventional techniques. Using this method, we measured the plasma concentrations of lyso-Gb3 and its analogues in 40 healthy subjects, 5 functional variants (males with E66Q), and various Fabry patients (9 classic Fabry males/9 mutations; 7 later-onset Fabry males/5 mutations; and 10 Fabry females/9 mutations). The results revealed that the mean lyso-Gb3 and lyso-Gb3(-2) concentrations in all the Fabry patient subgroups were statistically higher, especially in the classic Fabry males, than those in the functional variants and healthy subjects. The plasma concentrations of lyso-Gb3 and its analogues in healthy subjects, functional variants, and some Fabry patients with specific mutations (R112H and M296I) that cannot be established by conventional techniques were successfully determined by means of nano-LC-MS/MS. The lyso-Gb3 and lyso-Gb3(-2) concentrations in male patients with these mutations were lower than those in most Fabry patients having other mutations, but higher than those in the functional variants and healthy subjects. This new method is expected to be useful for sensitive determination of the plasma concentrations of lyso-Gb3 and its analogues. This study also revealed that not only lyso-Gb3 but also lyso-Gb3(-2) in plasma is a useful biomarker for the diagnosis of Fabry disease.