Isolation and measurement of quercetin glucosides in flower buds of Japanese butterbur (Petasites japonicus subsp. gigantea Kitam.)

Three quercetin glucosides were isolated from flower buds of Japanese butterbur (Petasites japonicus subsp. gigantea Kitam.) together with caffeic acid as the ingredients that had DPPH radical scavenging activity, using the DPPH-HPLC method for measuring the radical scavenging activity. These quercetin glucosides were identified as quercetin 3-O-beta-D-glucoside, quercetin 3-O-beta-D-6'-O-acetylglucoside, and rutin, and the amounts of the glucosides in flower buds were also examined by HPLC. The flower buds were harvested from four different sites, the total amount of quercetin glucosides in each site was 100-170 mg/100 g fr. wt., and there were no great differences of the amounts between growing fields.     

A preliminary report of phylogenetic diversity of bacterial strains isolated from marine creatures

Bacterial diversity among marine creatures, especially molluscs, as a source for searching out novel lineages of bacteria, was studied. Marine creatures were collected at the coasts of the Kanto area in Japan. A total of 116 strains of bacteria were isolated from the intestines of 19 species of marine creatures includings molluscs, pisces and protochordata. Partial sequencing of 16S rDNA revealed that most of the isolates belonged to the gamma subclass of the Proteobacteria and Cytophaga-Flavobacterium-Bacteroides group. The BLAST searches revealed that the complete 16S rDNA sequence of 17 strains out of 116 isolates showed less than 94% similarity with 16S rDNA sequences deposited in the database. Four strains out of the 17 isolates belonged to the Rhodobacter group, 8 strains to the Alteromonas group, and the remaining 5 strains to the Cytophaga-Flavobacterium-Bacteroides group. Phylogenetic positions of 6 strains belonging to the Alteromonas group, which were isolated from different marine creatures, were close to each other, and represented a novel 16S rDNA lineage within the gamma subclass of Proteobacteria. Therefore, it may be inferred that these 6 strains belong to a new genus of Proteobacteria. Phylogenetic positions of the other strains are also independent from neighboring taxa, and they were suggested to respectively form a novel lineage. From these results, it is clear that the biodiversity of bacteria in marine creatures is much wider than was previously thought, and unknown microbiological resources are buried in these organisms.     

Lysosomal acid lipase regulates fatty acid channeling in brown adipose tissue to maintain thermogenesis

Lysosomal acid lipase (LAL) is the only known enzyme, which hydrolyzes cholesteryl esters and triacylglycerols in lysosomes of multiple cells and tissues. Here, we explored the role of LAL in brown adipose tissue (BAT). LAL-deficient (Lal?/?) mice exhibit markedly reduced UCP1 expression in BAT, modified BAT morphology with accumulation of lysosomes, and mitochondrial dysfunction, consequently leading to regular hypothermic events in mice kept at room temperature. Cold exposure resulted in reduced lipid uptake into BAT, thereby aggravating dyslipidemia and causing life threatening hypothermia in Lal?/? mice. Linking LAL as a potential regulator of lipoprotein lipase activity, we found Angptl4 mRNA expression upregulated in BAT. Our data demonstrate that LAL is critical for shuttling fatty acids derived from circulating lipoproteins to BAT during cold exposure. We conclude that inhibited lysosomal lipid hydrolysis in BAT leads to impaired thermogenesis in Lal?/? mice.     

Paradorippe granulata – A crab with external fertilization and a novel type of sperm storage organ challenges prevalent ideas on the evolution of reproduction in Eubrachyura (Crustacea: Brachyura: Dorippidae)

Two fundamentally different sperm storage organs occur in Brachyura. The probably paraphyletic podotremes show intersegmental spermathecae, which are distant from oviducts and coxal gonopores. Hence, fertilization is external. In contrast to this, the seminal receptacles of Eubrachyura are directly connected with the ovaries. Thus, at least initial fertilization is internal. This pattern has been interpreted as an apomorphy of Eubrachyura. To test this hypothesis, we studied the morphology of the reproductive organs of Paradorippe granulata, a representative of the putatively early diverging eubrachyuran lineage Dorippoidea. Applying histology, 3D-reconstructions and micro-computed-tomography we revealed a novel type of sperm storage organ. Female P.granulata lack the characteristic eubrachyuran seminal receptacle. Instead sperm is stored in four cuticle-lined bursae, two on each side of the paired oviducts. The elaborate bulbous male gonopod with several terminal processes is adapted to transferring sperm into the female twin bursae. Since oviducts and twin bursae are not directly connected, spermatozoa and oocytes mix when gametes pass through the sternal vulva. Thus, fertilization in P.granulata is external. Our finding of a eubrachyuran crab that lacks seminal receptacles and exhibits external fertilization calls prevailing concepts on the evolution of sperm storage in Eubrachyura into question.     

Mode of Infection of Phyllosticta maculata on Banana as Revealed by Scanning Electron Microscopy

The initial infection stages of Phyllosticta maculata on banana were studied using scanning electron microscopy. Conidial germination on the banana leaf surface commenced within 3 h postinoculation to produce a long and slender germ tube. The hyphae developed secondary branches and mostly grew randomly across the leaf surface. Appressoria were formed at the apex of the germ tubes within 18 h postinoculation and were variable in shape. A layer of an extracellular matrix surrounded the appressoria at the pathogen–host interface. On the fruit surface, conidia germinated to produce predominantly swollen germ tubes which functioned as lateral appressoria together with some slender ones. These germ tubes were formed within 3 h postinoculation. There was no stomatal penetration apparent on the leaf; instead, direct penetration through the cuticle with and without the formation of appressoria was observed. Cuticular degradation on the leaf surface was evident with a circular, darkened area around the point of penetration by hyphae or appressoria. The significant role of pycnidia and conidia in the epidemiology of the disease was further demonstrated in naturally infected leaf samples.     

Trypanosoma cruzi: skin-penetration kinetics of vector-derived metacyclic trypomastigotes

In order to investigate the natural route of infection of nude and normal BALB/c mice with Trypanosoma cruzi via the skin, a drop of vector faeces/urine containing metacyclic trypomastigotes was placed onto the puncture site of a bite from Triatoma infestans. The periods of exposure, i.e. until removal of flagellates from the skin, and the time elapsed until surgical removal of the skin around the puncture were varied. After 15 min of exposure, T. cruzi developed in all nude mice without surgery, and in four of 10 mice if the puncture region of the skin was removed directly after exposure. In a shaved puncture region, 5 min of exposure were sufficient to infect all normal BALB/c mice without surgery and one of four mice with direct removal of the puncture region. Longer periods of exposure or time until removal of the skin only sometimes resulted in higher infection rates. Prepatent periods and the development of parasitaemia varied irrespective of the period of exposure or the period until skin removal at the puncture site. The importance of these findings is that they clearly prove that T. cruzi can rapidly invade the host via the puncture site of the bite of the vector and that at least some parasites are immediately transported away from this site.     

Riverine life history of the amphidromous goby Sicyopterus japonicus (Gobiidae: Sicydiinae) in the Ota River, Wakayama, Japan

To understand the riverine life history of the amphidromous goby, Sicyopterus japonicus, studies were conducted in the Ota River, Wakayama, Japan. They were distributed from 3 to 23 km upstream from the river mouth, and abundance was higher in the middle reaches than in the upper and lower reaches. Fish were not observed in rapids during winter, suggesting a seasonal change of habitat. Body length ranged from 24 to 120 mm SL. Both females and males ranged from 1 to 6 years old, and males had larger asymptotic length than females. Condition factor showed two peaks in July and November, which appeared to correspond to the spawning season and preparation for over wintering. Gonad somatic index increased in summer with a peak in August indicating a summertime spawning season that was confirmed by collections of the newly hatched larvae migrating downstream. Eggs of 0.5 mm diameter were attached to stones and newly hatched larvae made continuous vertical movements of sinking and upward swimming and the distance of each movement was longer in freshwater than in seawater. The newly hatched larvae were collected at nighttime (19:00–24:00) mainly in August. Those larvae were very small (mean: 1.4 mm TL) with a yolk sac and no eye pigmentation. Low wintertime temperatures are likely an important determinant of the spawning and recruitment seasons and seasonal changes of activity of this species that lives at much higher latitudes than all other species of the subfamily.     

Photo-Irradiation of Proanthocyanidin as a New Disinfection Technique via Reactive Oxygen Species Formation

In the present study, the bactericidal effect of photo-irradiated proanthocyanidin was evaluated in relation to reactive oxygen species formation. Staphylococcus aureus suspended in proanthocyanidin aqueous solution was irradiated with light from a laser at 405 nm. The bactericidal effect of photo-irradiated proanthocyanidin depended on the concentration of proanthocyanidin, the laser irradiation time, and the laser output power. When proanthocyanidin was used at the concentration of 1 mg/mL, the laser irradiation of the bacterial suspension could kill the bacteria with a >5-log reduction of viable cell counts. By contrast, bactericidal effect was not observed when proanthocyanidin was not irradiated. In electron spin resonance analysis, reactive oxygen species, such as hydroxyl radicals, superoxide anion radicals, and hydrogen peroxide, were detected in the photo-irradiated proanthocyanidin aqueous solution. The yields of the reactive oxygen species also depended on the concentration of proanthocyanidin, the laser irradiation time, and the laser output power as is the case with the bactericidal assay. Thus, it is indicated that the bactericidal effect of photo-irradiated proanthocyanidin is exerted via the reactive oxygen species formation. The bactericidal effect as well as the yield of the oxygen radicals increased with the concentration of proanthocyanidin up to 4 mg/mL, and then decreased with the concentration. These findings suggest that the antioxidative activity of proanthocyanidin might prevail against the radical generation potency of photo-irradiated proanthocyanidin resulting in the decreased bactericidal effect when the concentration is over 4 mg/mL. The present study suggests that photo-irradiated proanthocyanidin whenever used in an optimal concentration range can be a new disinfection technique.     

Trypanosoma cruzi Infection in Neotropical Wild Carnivores (Mammalia: Carnivora): At the Top of the T. cruzi Transmission Chain

Little is known on the role played by Neotropical wild carnivores in the Trypanosoma cruzi transmission cycles. We investigated T. cruzi infection in wild carnivores from three sites in Brazil through parasitological and serological tests. The seven carnivore species examined were infected by T. cruzi, but high parasitemias detectable by hemoculture were found only in two Procyonidae species. Genotyping by Mini-exon gene, PCR-RFLP (1f8/Akw21I) and kDNA genomic targets revealed that the raccoon (Procyon cancrivorus) harbored TcI and the coatis (Nasua nasua) harbored TcI, TcII, TcIII-IV and Trypanosoma rangeli, in single and mixed infections, besides four T. cruzi isolates that displayed odd band patterns in the Mini-exon assay. These findings corroborate the coati can be a bioaccumulator of T. cruzi Discrete Typing Units (DTU) and may act as a transmission hub, a connection point joining sylvatic transmission cycles within terrestrial and arboreal mammals and vectors. Also, the odd band patterns observed in coatis’ isolates reinforce that T. cruzi diversity might be much higher than currently acknowledged. Additionally, we assembled our data with T. cruzi infection on Neotropical carnivores’ literature records to provide a comprehensive analysis of the infection patterns among distinct carnivore species, especially considering their ecological traits and phylogeny. Altogether, fifteen Neotropical carnivore species were found naturally infected by T. cruzi. Species diet was associated with T. cruzi infection rates, supporting the hypothesis that predator-prey links are important mechanisms for T. cruzi maintenance and dispersion in the wild. Distinct T. cruzi infection patterns across carnivore species and study sites were notable. Musteloidea species consistently exhibit high parasitemias in different studies which indicate their high infectivity potential. Mesocarnivores that feed on both invertebrates and mammals, including the coati, a host that can be bioaccumulator of T. cruzi DTU’s, seem to take place at the top of the T. cruzi transmission chain.