The bglA gene of Aspergillus kawachii encodes both extracellular and cell wall-bound beta-glucosidases

We cloned the genomic DNA and cDNA of bglA, which encodes beta-glucosidase in Aspergillus kawachii, based on a partial amino acid sequence of purified cell wall-bound beta-glucosidase CB-1. The nucleotide sequence of the cloned bglA gene revealed a 2,933-bp open reading frame with six introns that encodes an 860-amino-acid protein. Based on the deduced amino acid sequence, we concluded that the bglA gene encodes cell wall-bound beta-glucosidase CB-1. The amino acid sequence exhibited high levels of homology with the amino acid sequences of fungal beta-glucosidases classified in subfamily B. We expressed the bglA cDNA in Saccharomyces cerevisiae and detected the recombinant beta-glucosidase in the periplasm fraction of the recombinant yeast. A. kawachii can produce two extracellular beta-glucosidases (EX-1 and EX-2) in addition to the cell wall-bound beta-glucosidase. A. kawachii in which the bglA gene was disrupted produced none of the three beta-glucosidases, as determined by enzyme assays and a Western blot analysis. Thus, we concluded that the bglA gene encodes both extracellular and cell wall-bound beta-glucosidases in A. kawachii.     

Network analysis of potential migration routes for Oriental White Storks (<Emphasis Type="Italic">Ciconia boyciana</Emphasis>)

From 1998 through to 2000, we satellite-tracked the movements of 13 Oriental White Storks (Ciconia boyciana) on their autumnal migration in order to identify their important stopover sites for preserving links from the Russian Far East breeding sites to the wintering sites in south-eastern China. New analytical methods of satellite tracking data were employed to derive robust information on the locations of stay sites, the number of stopovers made during migration, and the distance traveled without making stopovers. Based on the derived information, we modeled a stay site network as an abstraction of the storks potential migration routes from their breeding sites to wintering sites. Using network analysis techniques, we explored how the loss of stopover sites could affect the connectivity of potential migration routes. The results suggested that if the seashore stopover sites facing Bohai Bay in eastern China were lost, the storks wintering sites along the Yangtze River in south-eastern China would be isolated. Among the seashore stopover sites, Jiantuozhi Gley Mire (39.185°N, 118.627°E), located on the northern seashore of Bohai Bay, was considered particularly important for migrating storks, because it was used every year by the storks we tracked. If conservation needs of this critically located site fail to be addressed, the stay site network of storks can create weak links in the chain of migration and, if broken, storks will have great difficulties in completing their autumnal migration.     

Gene cloning and biochemical characterizations of thermostable ribonuclease HIII from Bacillus stearothermophilus

The gene encoding RNase HIII from the thermophilic bacterium Bacillus stearothermophilus was cloned and overexpressed in Escherichia coli, and the recombinant protein (Bst-RNase HIII) was purified and biochemically characterized. Bst-RNase HIII is a monomeric protein with 310 amino acid residues, and shows an amino acid sequence identity of 47.1% with B. subtilis RNase HIII (Bsu-RNase HIII). The enzymatic properties of Bst-RNase HIII, such as pH optimum, metal ion requirement, and cleavage mode of the substrates, were similar to those of Bsu-RNase HIII. However, Bst-RNase HIII was more stable than Bsu-RNase HIII, and the temperature (T(1/2)) at which the enzyme loses half of its activity upon incubation for 10 min was 55 degrees C for Bst-RNase HIII and 35 degrees C for Bsu-RNase HIII. The optimum temperature for Bst-RNase HIII activity was also shifted upward by roughly 20 degrees C as compared to that of Bsu-RNase HIII. The availability of such a thermostable enzyme will facilitate structural studies of RNase HIII.     

Histochemical,ultrastructural, and three-dimensional observation of smooth muscle cells in human gastric mucosa

The present study was undertaken to clarify the histochemical and ultrastructural properties and the three-dimensional distribution of the smooth muscle cells (SMCs) located in the lamina propria (LP) of the human gastric mucosa. Standard paraffin sections obtained from stomachs surgically resected for gastric cancer were immunostained for alpha-smooth muscle actin (alpha-SMA), vimentin, desmin, laminin, and type IV collagen. In addition, 100-m-thick sections were fluorostained for alpha-SMA and CD34, while three-dimensional images were prepared by confocal laser scanning microscope. Ultrastructural studies were carried out using normal gastric biopsy specimens. The results indicated that SMCs in the LP differed between the upper and lower regions, SMCs in the lower LP being fairly typical SMCs, whereas those in the upper LP had apparently lost reactivity for desmin but gained that for vimentin. The basal lamina became sparser, but a fibronexus was occasionally seen in SMCs in the upper LP. Three-dimensional images revealed bundles of SMCs in the upper LP encircling several foveolae to form acinus-like structures and, in the upper LP, SMCs branching into fine fibrils with a brush-like (corpus) or besom-like (i.e., a twiggy witchs broom) appearance (antrum).     

Fungicide activity through activation of a fungal signalling pathway

Fungicides generally inhibit enzymatic reactions involved in fungal cellular biosynthesis. Here we report, for the first time, an example of fungicidal effects through hyperactivation of a fungal signal transduction pathway. The OSC1 gene, encoding a MAP kinase (MAPK) related to yeast Hog1, was isolated from the fungal pathogen Colletotrichum lagenarium that causes cucumber anthracnose. The osc1 knockout mutants were sensitive to high osmotic stress and showed increased resistance to the fungicide fludioxonil, indicating that Osc1 is involved in responses to hyperosmotic stress and sensitivity to fludioxonil. The Osc1 MAPK is phosphorylated under high osmotic conditions, indicating activation of Osc1 by high osmotic stress. Importantly, fludioxonil treatment also activates phosphorylation of Osc1, suggesting that improper activation of Osc1 by fludioxonil has negative effects on fungal growth. In the presence of fludioxonil, the wild-type fungus was not able to infect the host plant because of a failure of appressorium-mediated penetration, whereas osc1 mutants successfully infected plants. Analysis using a OSC1-GFP fusion gene indicated that Osc1 is rapidly translocated to the nucleus in appressorial cells after the addition of fludioxonil, suggesting that fludioxonil impairs the function of infection structures by activation of Osc1. Furthermore, fludioxonil activates Hog1-type MAPKs in the plant pathogenic fungi Cochliobolus heterostrophus and Botrytis cinerea. These results strongly suggest that fludioxonil acts as a fungicide, in part, through activation of the MAPK cascade in fungal pathogens.     

Serum concentrations of androstenediol and androstenediol sulfate, and their relation to cytokine production during and after normal pregnancy

Since it is known that androstenediol (ADIOL) has potent immunoregulatory effects, changes in ADIOL levels during and after pregnancy might affect the maternal immune system. We examined serum concentrations of ADIOL and androstenediol 3-sulfate (ADIOLS) together with IFN-gamma and IL-4 production levels during pregnancy and after delivery up to 10-11 months postpartum. The subjects were 73 normal pregnant, 76 normal postpartum, and 28 normal non-pregnant women. ADIOL and ADIOLS were measured using EIA and GC/MS, respectively. The cytokine levels in the supernatant of whole-blood cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin were measured using ELISA. ADIOL levels significantly decreased compared to non-pregnant levels in the first trimester (P < 0.05) and were reversed in the third trimester (P < 0.05). After pregnancy, ADIOL levels gradually declined, and a significant decrease was observed at 10-11 months postpartum (P < 0.05). ADIOLS levels were significantly lower in the third trimester (P < 0.05) and significantly higher at the first month postpartum (P < 0.001) compared to non-pregnant women. IFN-gamma and IL-4 levels decreased during pregnancy and subsequently increased postpartum. On the other hand, we found significant negative correlations between ADIOL concentrations and production levels of IFN-gamma (P < 0.05) or IL-4 (P < 0.05). These findings suggest that ADIOL may be involved in modifying the maternal immune response during and after pregnancy.     

Extract of Perilla frutescens enriched for rosmarinic acid, a polyphenolic phytochemical, inhibits seasonal allergic rhinoconjunctivitis in humans

Extract of Perilla frutescens enriched for rosmarinic acid, a polyphenolic phytochemical, suppresses allergic immunoglobulin responses and inflammation caused by polymorphonuclear leukocytes (PMNL) in mice. However, few placebo-controlled clinical trials have examined the efficacy and safety of polyphenolic phytochemicals for treatment of allergic inflammatory diseases in humans. The present study determined whether oral supplementation with rosmarinic acid is an effective intervention for patients with seasonal allergic rhinoconjunctivitis (SAR). In this 21-day, randomized, double-blind, age-matched, placebo-controlled parallel group study, patients with mild SAR were treated daily with extract of Perilla frutescens enriched for rosmarinic acid (200 mg [n=10] or 50 mg [n=9]) or placebo (n=10). Patients recorded symptoms daily in a diary. Profiles of infiltrating cells and concentrations of eotaxin, IL-1beta, IL-8, and histamine were measured in nasal lavage fluid. Serum IgE concentrations and routine blood tests were also examined. As compared with placebo supplementation, supplementation with extract of Perilla frutescens enriched for rosmarinic acid resulted in a significant increase in responder rates for itchy nose, watery eyes, itchy eyes, and total symptoms (P<0.05). Active treatment significantly decreased the numbers of neutrophils and eosinophils in nasal lavage fluid (P<0.05 vs. placebo). Patients reported no adverse events, and no significant abnormalities were detected in routine blood tests. In conclusion, extract of Perilla frutescens enriched for rosmarinic acid can be an effective intervention for mild SAR at least partly through inhibition of PMNL infiltration into the nostrils. Use of this alternative treatment for SAR might reduce treatment costs for allergic diseases.     

Development of flight performance in the brown booby

How do birds acquire flight skills after fledging? This issue is important, as it is closely related to variation in the duration of offspring care, the causes of which remain unknown. In this study, we raised hatchling brown boobies, Sula leucogaster, and attached an acceleration data logger to each bird at fledging to record its movements. This allowed us to quantify precisely the time spent flapping, gliding and resting. The duration of foraging trips and proportion of time spent gliding during flight increased with the number of days since fledging, whereas the proportion of time spent in flight decreased. This indicates that brown boobies gradually acquire efficient flight skills during the post-fledging period, which might be the proximate cause of the long postfledging care period in this species. To the authors' knowledge, this is the first study to record precisely the ontogeny of flight behaviour in birds.