Occurrence of actinosporean stages of myxosporeans in an inflow brook of a salmon hatchery in the Mena River System,Hokkaido, Japan

Actinospore infection of oligochaetes living in the mud and on the roots of vegetation in an inflow brook of a Hokkaido salmon hatchery was studied within the framework of a Japanese-Hungarian research program. Two triactinomyxon types, 1 echinactinomyxon, and 1 neoactinomyxum type were isolated from the oligochaete Rhyacodrilus komarovi Timm, 1990 collected during the survey. The aurantiactinomyxons were recorded over a period of 3 mo starting from the day after oligochaete collection. The oligochaetes released actinospores for several weeks from the first day of the study. Spore excretion of individual oligochaetes was not synchronous. Of the oligochaetes examined, 0.7, 7, 3 and 3%, were infected with the echinactinomyxon, neoactinomyxum and the 2 types of triactinomyxon spores, respectively. Actinospore infection was intense in the positive oligochaetes in all 4 types. Of the 4 actinospore types presented here, 3 are described for the first time.     

Alpha-glucosidase inhibitor from the seeds of balsam pear (Momordica charantia) and the fruit bodies of Grifola frondosa

Alpha-glucosidase inhibitory activities were found in aqueous methanol extracts of the seeds of Momordica charantia and the fruit bodies of Grifola frondosa. An active principle against the enzyme prepared from rat small intestine acetone powders was isolated and characterized. The structure of the isolated compound was identified as D-(+)-trehalose by FDMS, 1H-, 13C-NMR, and [alpha]D measurements. The inhibitory activity of trehalose was compared with 1-deoxynojirimycin. Trehalose showed 45% inhibitory activity at the concentration of 2 x 10(-3) m, but 1-deoxynojirimycin had 52% inhibitory activity at 1 x 10(-7) M.     

DPPH radical scavengers from dried leaves of oregano (Origanum vulgare)

1,1-Dipehnyl-2-picrylhydrazyl (DPPH) radical scavenging activities were found in the extract of dried leaves of oregano (Origanum vulgare). The water-soluble active ingredients were isolated, and their structures were determined to be 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybenzyl protocatechuate and 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybenzyl 4-O-methylprotocatechuate by (1)H-, (13)C-NMR, DEPT, HMQC, and HMBC spectral analyses, and by NOE experiments. The DPPH radical scavenging activities of these compounds were compared with those of rutin, quercetin and rosmarinic acid at a concentration of 2 x 10(-5) M. The scavenging activity of 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybenzyl protocatechuate was almost the same as that of quercetin and rosmarinic acid, but that of 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybennzyl 4-O-methylprotocatechuate was less than that of quercetin, rosmarinic acid and 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybenzyl protocatechuate. The amount of 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybenzyl protocatechuate was estimated to be 3.8 mg/1 g of dried leaves by an HPLC analysis.     

Inhibition of neuronal nitric oxide synthase by 7-nitroindazole attenuates acute lung injury in an ovine model

Nitric oxide (NO) has been shown to play a major role in acute lung injury (ALI) after smoke inhalation. In the present study, we developed an ovine sepsis model, created by exposing sheep to smoke inhalation followed by instillation of bacteria into the airway, that mimics human sepsis and pneumonia. We hypothesized that the inhibition of neuronal NO synthase (nNOS) might be beneficial in treating ALI associated with this model. Female sheep (n = 26) were surgically prepared for the study and given a tracheostomy. This was followed by insufflation of 48 breaths of cotton smoke (40 degrees C) into the airway of each animal and subsequent instillation of live Pseudomonas aeruginosa [5 x 10(11) colony forming units (CFU)] into each sheep's lung. All sheep were mechanically ventilated using 100% O2. Continuous infusion of 7-nitroindazole (7-NI), an nNOS inhibitor, NG-monomethyl-l-arginine (l-NMMA), a nonspecific NOS inhibitor, or aminoguanidine (AG), an inducible NOS inhibitor, was started 1 h after insult. The administration of 7-NI improved pulmonary gas exchange (PaO2/FiO2; where PaO2 is arterial PO2 and FiO2 is fractional inspired oxygen concentration) and pulmonary shunt fraction and attenuated the increase in lung wet-to-dry weight ratio seen in the nontreated sheep. Histologically, 7-NI prevented airway obstruction. The increase in airway blood flow after injury in the nontreated group was significantly inhibited by 7-NI. The increase in plasma concentration of nitrate and nitrite (NOx) was inhibited by 7-NI as well. Posttreatment with l-NMMA improved the pulmonary gas exchange, but AG did not. The results of the present study show that nNOS may be involved in the pathogenesis of ALI after smoke inhalation injury followed by bacterial instillation in the airway.     

Rapid oxidation of dichlorodihydrofluorescin with heme and hemoproteins: formation of the fluorescein is independent of the generation of reactive oxygen species

Oxidative stress and the generation of reactive oxygen species (ROS) have been implicated in the pathogenesis of cellular damage. These events have usually been reported in terms of oxidation of a reporter molecule such as 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA). Treatment of HeLa cells with hemin or metalloporphyrins resulted in a rapid oxidation of DCFH in a time- and dose-dependent manner. This oxidation was inhibited by treatment of the cells with a large amount of superoxide dismutase and catalase, which is different from observations that these enzymes had no effect on the induction of heme oxygenase-1, a stress-induced protein, in hemin-treated cells. To examine the possibility that the oxidation of DCFH is independent of the generation of ROS, the oxidation was measured using hemoglobin-synthesizing erythroleukemia K562 cells. When K562 cells were treated with delta-aminolevulinic acid, a precursor of heme, oxidation of DCFH increased depending on the heme content in cells. Then DCFH-DA was oxidized directly with heme, hemoglobin, myoglobin and cytochrome c. These results suggest that oxidation of DCFH is not always related to the generation of ROS but may be related to heme content in cells.     

Structural changes during the formation of early intermediates in the bacteriorhodopsin photocycle

Early intermediates of bacteriorhodopsin's photocycle were modeled by means of ab initio quantum mechanical/molecular mechanical and molecular dynamics simulations. The photoisomerization of the retinal chromophore and the formation of photoproducts corresponding to the early intermediates were simulated by molecular dynamics simulations. By means of the quantum mechanical/molecular mechanical method, the resulting structures were refined and the respective excitation energies were calculated. Two sequential intermediates were found with absorption maxima that exhibit red shifts from the resting state. The intermediates were therefore assigned to the K and KL states. In K, the conformation of the retinal chromophore is strongly deformed, and the N--H bond of the Schiff base points almost perpendicular to the membrane normal toward Asp-212. The strongly deformed conformation of the chromophore and weakened interaction of the Schiff base with the surrounding polar groups are the means by which the absorbed energy is stored. During the K-to-KL transition, the chromophore undergoes further conformational changes that result in the formation of a hydrogen bond between the N--H group of the Schiff base and Thr-89 as well as other rearrangements of the hydrogen-bond network in the vicinity of the Schiff base, which are suggested to play a key role in the proton transfer process in the later phase of the photocycle.     

Changes in body temperature pattern in vertebrates do not influence the codon usages of alpha-globin genes

Codon usages are known to vary among vertebrates chiefly due to variations in isochore structure. Under the assumption that marked differences exist in isochore structure between warm-blooded and cold-blooded animals, the variations among vertebrates were previously attributed to an adaptation to homeothermy. However, based on data from a turtle species and a crocodile (Archosauromorpha), it was recently proposed that the common ancestors of mammals, birds and extent reptiles already had the "warm-blooded" isochore structure. We determined the nucleotide sequences of alpha-globin genes from two species of heterotherms, cuckoo (Cuculus canorus) and bat (Pipistrellus abramus), and three species of snakes (Lepidosauromorpha), Naja kaouthia from a tropical terrestrial habitat, Elaphe climacophora from a temperate terrestrial habitat, and Hydrophis melanocephalus from a tropical marine habitat. Our purposes were to assess the influence of differential body temperature patterns on codon usage and GC content at the third position of a codon (GC3), and to test the hypothesis concerning the phylogenetic position at which GC contents had increased in vertebrates. The results of principal component analysis (PCA) using the present data and data for other taxa from GenBank indicate that the primary difference in codon usage in globin genes among amniotes and other vertebrates lies in GC3. The codon usages (and GC3) in alpha-globin genes from two heterotherms and three snakes are similar to those in alpha-globin genes from warm-blooded vertebrates. These results refute the influence of body temperature pattern upon codon usages (and GC3) in alpha-globin genes, and support the hypothesis that the increase in GC content in the genome occurred in the common ancestor of amniotes.