Novel reporter gene expression systems for monitoring activation of the Aspergillus nidulans HOG pathway

The Aspergillus nidulans high-osmolarity glycerol response (AnHOG) pathway is involved in osmoadaptation. We found that fludioxonil, a fungicide, causes improper activation of HogA mitogen-activated protein kinase (MAPK) in A. nidulans. Here we present novel reporter systems for monitoring activation of the AnHOG pathway. The promoter region of gfdB (glycerol-3-phosphate dehydrogenase), whose expression depends on the presence of HogA, was fused to a beta-glucuronidase uidA gene (GUS) to construct the reporter, which was introduced into A. nidulans wild type and hogADelta. Increased GUS activity was detected in the wild type only when it was treated with high osmolarity or fludioxonil, while reporter activity was scarcely stimulated in the hogADelta mutant. These results indicate that the reporter activity is controlled via HogA activation. Furthermore, we present possible applications of the reporter systems in screening new antifungal compounds.     

Antioxidant properties of aqueous extracts from red tide plankton cultures

The antioxidant properties of aqueous extracts from the dinophycean flagellates Gymnodinium impudicum and Alexandrium affine and the raphidophycean flagellate Chattonella ovata were examined. An electron spin resonance (ESR)-spin trapping method coupled with steady state kinetic analysis showed that all of the extracts directly scavenge superoxide, and that the superoxide scavenging potential of any of the extracts was comparable to that of L-ascorbic acid. As for hydroxyl radical scavenging, the Fenton reaction and the method of ultraviolet radiation to hydrogen peroxide were used as hydroxyl radical generation systems. All of extracts reduced the level of hydroxyl radicals in both of the systems, indicating that the extracts also directly scavenge hydroxyl radicals. Since the levels of phenolic compounds did not correlate with the antioxidant activities of the extracts, substances other than phenolic compounds also appeared to be attributable to the activities. It is of our interest that the scavenging activities of extract from G. impudicum against superoxide and hydroxyl radicals were increased by heat exposure at 100 degrees C and 200 degrees C respectively. Although the reason for the increased activities of the aqueous extract from G. impudicum is not clear, the heat-resistance of the extract from G. impudicum might make it a desirable antioxidant.     

Evaluation of nutritional availability and anti-tumor activity of selenium contained in selenium-enriched Kaiware radish sprouts

We estimated the nutritional availability of selenium (Se) in Se-enriched Kaiware radish sprouts (SeRS) by the tissue Se deposition and glutathione peroxidase (GPX) activity of rats administered the sprouts, and examined the effect of SeRS on the formation of aberrant crypt foci (ACF) in the colon of mice administered 1,2-dimethylhydrazine (DMH) to evaluate anti-tumor activity. Male weanling Wistar rats were divided into seven groups and fed a Se-deficient basal diet or the basal diet supplemented with 0.05, 0.10, or 0.15 microg/g of Se as sodium selenite or SeRS for 28 d. Supplementation with Se dose-dependently increased serum and liver Se concentrations and GPX activities, and the selenite-supplemented groups showed a higher increase than the SeRS-supplemented groups. The nutritional availability of Se in SeRS was estimated to be 33 or 64% by slope ratio analysis. Male 4-week-old A/J mice were divided into seven groups and fed a low Se basal diet or the basal diet supplemented with selenite, SeRS, or selenite + non-Se-enriched radish sprouts (NonSeRS) at a level of 0.1 or 2.0 microg Se/g for 9 weeks. After 1 week of feeding, all mice were given six subcutaneous injections of DMH (20 mg/kg) at 1-week intervals. The average number of ACF formed in the colon of mice fed the basal diet was 4.3. At a supplementation level of 0.1 mug Se/g, only SeRS significantly inhibited ACF formation. At a supplementation level of 2.0 microg Se/g, both selenite and SeRS significantly inhibited ACF formation. The addition of NonSeRS to the selenite-supplemented diets tended to inhibit ACF formation, but this was not statistically significant. These results indicate that SeRS shows lower nutritional availability but higher anti-tumor activity than selenite.     

The influence of the immunostimulation by bacterial cell components derived from altered large intestinal microbiota on probiotic anti-inflammatory benefits

Using murine macrophage-like J774.1 cells and fecal precipitates prepared from the feces of elderly volunteers whose acute inflammation had been inhibited by LKM512 yogurt consumption, we investigated the likelihood that immunostimulation by altered intestinal bacterial cell components contribute to the anti-inflammatory benefits of this yogurt. Tumor necrosis factor-alpha production due to stimulation by fecal precipitates obtained during LKM512 yogurt consumption tended to be higher than due to stimulation by precipitates obtained from preconsumption (P=0.0827), although acute phase response was suppressed by LKM512 yogurt consumption. We suggest that the anti-inflammatory benefits of LKM512 yogurt on elderly volunteers are independent of direct immunostimulation by the bacterial cell components derived from altered intestinal microbiota.     

In-depth proteomic profiling of the normal human kidney glomerulus using two-dimensional protein prefractionation in combination with liquid chromatography-tandem mass spectrometry

The kidney glomerulus plays a pivotal role in ultrafiltration of plasma into urine and also is the locus of kidney disease progressing to chronic renal failure. We have focused proteomic analysis on the glomerulus that is most proximal to the disease locus. In the present study, we aimed to provide a confident, in-depth profiling of the glomerulus proteome. The glomeruli were highly purified from the kidney cortex from a male, 68-year-old patient who underwent nephroureterectomy due to ureter carcinoma. The patient was normal in clinical examinations including serum creatinine and urea levels and liver function, and did not receive any chemotherapy and radiotherapy. The cortical tissue was histologically normal, and no significant deposition of immunoglobulins and complement C3 was observed. We employed a novel strategy of protein separation using 1D (SDS-PAGE) and 2D (solution-phase IEF in combination with SDS-PAGE) prefractionation prior to the shotgun analysis with LC-MS/MS. The protein prefractionation produced 90 fractions, and eventually provided a confident set of identified proteins consisting of 6686 unique proteins (3679 proteins with two or more peptide matches and 3007 proteins with one peptide match), representing 2966 distinct genes. All the identified proteins were annotated and classified in terms of molecular function and biological process, compiled into 1D and 2D protein arrays, consisting of 15 and 75 sections, corresponding to the protein fractions which were defined by MW and pI range, and deposited on a Web-based database (http://www.hkupp.org). The most remarkable feature of the glomerulus proteome was a high incidence of identification of cytoskeleton-related proteins, presumably reflecting the well-developed, cytoskeletal organization of glomerular cells related to their physiological functions.     

Application of a single‐colony coculture technique to the isolation of hitherto unculturable gut bacteria

Molecular studies have led to postulation of a relationship between gut microbiota and certain diseases. However, because studies of hitherto uncultured species in vivo are essential for characterizing the biology and pathogenic properties of gut bacteria, techniques for culturing and isolating such bacteria must be developed. Here, a technique is described that partially overcomes the obstacles that prevent detection of interbacterial communication in vitro and are thus responsible for the failure to culture certain bacterial species. For this purpose, a ring with a membrane filter at the bottom was designed and a relatively simple nutrient medium was used instead of conventional media. Gut bacteria were cocultivated in soft agar separated by the membrane filter to simulate interbacterial communication in vitro. Use of this soft agar coculture technique led to the successful isolation of hitherto uncultured bacteria and the demonstration of multistage interbacterial communication among gut bacteria in vitro. Cultivation and isolation of single colonies of bacteria that require other bacteria for growth will enhance efforts to better understand the physiological and pathogenic roles of gut microbiota.     

KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis

Humans are unable to synthesize l-ascorbic acid (AsA), yet it is required as a cofactor in many critical biochemical reactions. The majority of human dietary AsA is obtained from plants. In Arabidopsis thaliana, a GDP-mannose pyrophosphorylase (GMPP), VITAMIN C DEFECTIVE1 (VTC1), catalyzes a rate-limiting step in AsA synthesis: the formation of GDP-Man. In this study, we identified two nucleotide sugar pyrophosphorylase-like proteins, KONJAC1 (KJC1) and KJC2, which stimulate the activity of VTC1. The kjc1kjc2 double mutant exhibited severe dwarfism, indicating that KJC proteins are important for growth and development. The kjc1 mutation reduced GMPP activity to 10% of wild-type levels, leading to a 60% reduction in AsA levels. On the contrary, overexpression of KJC1 significantly increased GMPP activity. The kjc1 and kjc1kjc2 mutants also exhibited significantly reduced levels of glucomannan, which is also synthesized from GDP-Man. Recombinant KJC1 and KJC2 enhanced the GMPP activity of recombinant VTC1 in vitro, while KJCs did not show GMPP activity. Yeast two-hybrid assays suggested that the stimulation of GMPP activity occurs via interaction of KJCs with VTC1. These results suggest that KJCs are key factors for the generation of GDP-Man and affect AsA level and glucomannan accumulation through the stimulation of VTC1 GMPP activity.     

In vitro protein import of a putative amino acid transporter from Arabidopsis thaliana into chloroplasts and its suborganellar localization

We identified a gene product of At5g19500 (At5g19500p) from Arabidopsis thaliana that is homologous to EcTyrP, a tyrosine-specific transporter from Escherichia coli. Computational analyses of the amino acid sequence of At5g19500p predicted 11 transmembrane domains (TMDs) and a potential plastid targeting signal at its amino terminus. As a first step toward understanding the possible role of At5g19500p in plant cells, we attempted to determine the localization of At5g19500p by an in vitro chloroplastic import assay using At5g19500p translated in a cell-free wheat germ system (Madin et al., Proc. Natl. Acad. Sci. USA, 97, 559-564 (2000)), followed by subfractionation of the chloroplasts. At5g19500p was successfully imported into chloroplasts, and the newly transported mature form of At5g19500p was recovered from the inner envelope membrane.     

A proposal of the method of deer density estimate without fecal decomposition rate: a case study of fecal accumulation rate technique in Japan

The accuracy of estimating deer density using the fecal pellet count method is greatly limited by variability of the fecal decomposition rate. The fecal accumulation rate technique can avoid the issue of decomposition rate. However, the precision of this technique is not clear when the decomposition rate is relatively high, such as in Japanese forests. We estimated deer population densities on Yakushima Island by the fecal accumulation rate technique and compared them between seasons. The estimated densities were similar to reported estimates, and did not differ seasonally, in accord with reports that deer on Yakushima do not migrate seasonally. Thus, we conclude that the fecal accumulation rate technique is applicable in Japanese forests.