Production of lepidimoide by an endophytic fungus from polysaccharide extracted from Abelmoschus sp.: identification of the product and the organism producing it

A highly potent allelopathic factor, lepidimoide, was initially extracted from mucilage of germinated cress seeds. Polysaccharide extracted from okra (Abelmoschus esculentum Moench) is considered to have a similar structure to lepidimoide as its repeating unit. We therefore initiated the screening of enzymes capable of degrading okra polysaccharide into lepidimoide from endophytes. We discovered an endophytic fungal strain AHU9748 isolated from Coleus galeatus, which produced an oligosaccharide having similar properties to lepidimoide on thin layer chromatography. The physico-chemical data from ESI-MS, NMR spectra and other analyses also showed the purified product to be identical to lepidimoide. The strain AHU9748 was identified as a fungus belonging to the coelomycetes, closely related to the genus Colletotrichum, based on morphological characteristics and sequence analysis of the 18S rDNA and ITS region.     

A novel gene delivery system in plants with calcium alginate micro-beads

We have produced micrometer-sized calcium alginate beads referred to as "bio-beads" that encapsulate plasmid DNA molecules carrying a reporter gene. In order to evaluate the efficiency of the bio-beads in mediating genetic transfection, protoplasts isolated from cultured tobacco cells (BY-2) were transfected with bio-beads containing a plasmid that carries the modified green fluorescent protein gene CaMV35S-sGFP. With the bio-beads treatment, approximately ten-fold higher GFP expression was observed after 24 h incubation compared to that with the conventional method using a naked plasmid solution. Transfection was up to 0.22% efficient. These results indicate that bio-beads have a possibility for efficient transformation in plants.     

Steroid hormone profiles of urban and tidal rivers using LC/MS/MS equipped with electrospray ionization and atmospheric pressure photoionization sources

A highly sensitive and uncomplicated method of analyzing steroidal hormones in river and estuarine water samples was developed using a liquid chromatography tandem mass spectrometer equipped with an electrospray ionization (ESI) source and atmospheric pressure photoionization (APPI) source. Steroidal hormones included not only estrogen but also androgen and conjugates of these two. APPI displayed greater sensitivity than ESI for most of the unconjugated steroids examined, with very high sensitivity for testosterone and 4-androstene-3,17-dione in particular. For conjugated hormones, in contrast, ESI was more effective. The method developed was applied to the determination of hormones in the rivers of Osaka City and their estuaries, where the hormones detected were affected by the effluent from municipal wastewater treatment plants (WWTPs), and hormone concentration values were comparable to those reported in previous studies of such effluent. Because of the two-way flow and stagnancy of streams and watercourses, continuous input of steroidal hormones from WWTPs seems to bring about local accumulation. Levels of androgen were 1 order of magnitude lower than those of estrogen. Estrone, estrone 3-sulfate, and 4-androstene-3,17-dione were detected in almost all water samples, with maxima of 51, 5.1, and 6.4 ng L(-1), respectively.     

Gallic acid oxidizes Met residues in peptides released from bovine β-lactoglobulin by in vitro digestion

Phenolic compounds (PCs) are frequently present in foods. However, little is known about the effect of PCs on enzymatic digestion process of food proteins and their products. In this study, the effect of gallic acid (GA) on in vitro digestion of β-lactoglobulin (β-LG) was investigated as a model system for analysis of the interaction between PCs and food proteins. GA showed no effect on the initial rate of β-LG digestion. However, after 1.5 h of digestion, the observed degree of hydrolysis of β-LG was lower in the presence than in the absence of GA. The peptides released from β-LG were characterized by LC/IT-TOF-MS and thirty peptides were identified. In particular, four new peaks were obtained following in vitro digestion of β-LG in the presence of GA. Met(7), Met(24) and Met(145) in the peptides corresponding to these peaks were oxidized to methionine sulfoxide residues.     

Acceleration of cellulose degradation and shift of product via methanogenic co-culture of a cellulolytic bacterium with a hydrogenotrophic methanogen

Although the effects of syntrophic relationships between bacteria and methanogens have been reported in some environments, those on cellulose decomposition using cellulolytic bacteria from methanogenic reactors have not yet been examined. The effects of syntrophic co-culture on the decomposition of a cellulosic material were investigated in a co-culture of Clostridium clariflavum strain CL-1 and the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus strain ΔH and a single-culture of strain CL-1 under thermophilic conditions. In this study, strain CL-1 was newly isolated as a cellulolytic bacterium from a thermophilic methanogenic reactor used for degrading garbage slurry. The degradation efficiency and cell density of strain CL-1 were 2.9- and 2.7-fold higher in the co-culture than in the single-culture after 60?h of incubation, respectively. Acetate, lactate and ethanol were the primary products in both cultures, and the concentration of propionate was low. The content of acetate to total organic acids plus ethanol was 59.3% in the co-culture. However, the ratio decreased to 24.9% in the single-culture, although acetate was the primary product. Therefore, hydrogen scavenging by the hydrogenotrophic methanogen strain ΔH could shift the metabolic pathway to the acetate production pathway in the co-culture. Increases in the cell density and the consequent acceleration of cellulose degradation in the co-culture would be caused by increases in adenosine 5'-triphosphate (ATP) levels, as the acetate production pathway includes ATP generation. Syntrophic cellulose decomposition by the cellulolytic bacteria and hydrogenotrophic methanogens would be the dominant reaction in the thermophilic methanogenic reactor degrading cellulosic materials.     

Use of liposome encapsulated hemoglobin as an oxygen carrier for fetal and adult rat liver cell culture

Engineering liver tissue constructs with sufficient cell mass for transplantation implies culturing large numbers of hepatocytes in a reduced volume; however, providing sufficient oxygen to dense cell cultures is still not feasible using only conventional culture medium. Liposome-encapsulated hemoglobin (LEH), an oxygen-carrying blood substitute originally designed for short-term perfusion, may be a good candidate as an oxygen carrier to cultured liver cells. In this study, we investigated the feasibility of maintaining long term hepatocyte cultures using LEH. Primary fetal and adult rat liver cells were directly exposed to LEH for 6 to 14 days in static culture or in a perfused flat plate bioreactor. The functions and viability of adult rat hepatocytes exposed to LEH were not adversely affected in static monolayer culture and were even improved in the bioreactor. However, some cytotoxicity of LEH was observed with fetal rat liver cells after 4 days of culture. LEH, though a suitable oxygen carrier for long-term culture of mature hepatocytes, is not suitable in its present form for perfusing fetal hepatocyte cultures in direct contact with the liposomes; either the LEH will have to be made less toxic or a more sophisticated bioreactor that prevents the direct contact between hepatocytes and perfusates will have to be designed if fetal cells are to be used for liver tissue engineering.     

Simultaneous induction of calcium transients in embryoid bodies using microfabricated electrode substrates

Precise control of differentiation processes of pluripotent stem cells is a key component for the further development of regenerative medicine. For this purpose, combining a cell-aggregate-size treatment for regulating intercellular signal transmissions and an electrical stimulation technique for inducing cellular responses is a promising approach. In the present study, we developed microfabricated electrode substrates that allow simultaneous stimulation of embryoid bodies (EBs) of P19 cells. Mouse embryonal carcinoma P19 cells can be induced to differentiate into three germ layers and serve as a promising stem cell model. Microcavity–array patterns were fabricated onto indium–tin–oxide (ITO) substrates using a standard photo-lithography technique, and uniform-sized EBs of P19 cells were inserted into each microcavity. Electrical stimulation was applied to the EBs through substrate electrodes and stimulus-induced intracellular calcium transients were monitored. We confirmed that the developed electrode device could simultaneously stimulate smaller (200 μm diameter) and larger (500 μm diameter) EBs inserted in the microcavities and induce specific spatio-temporal patterns of intracellular calcium transients in the EBs with fine reproducibility. We concluded that the developed microcavity array with embedded electrodes could simultaneously and effectively stimulate uniform-sized EBs inserted in it. Therefore, it is a promising experimental tool for precisely controlling cell differentiation processes.     

Direct ethanol production from barley beta-glucan by sake yeast displaying Aspergillus oryzae beta-glucosidase and endoglucanase

Three beta-glucosidase- and two endoglucanase-encoding genes were cloned from Aspergillus oryzae, and their gene products were displayed on the cell surface of the sake yeast, Saccharomyces cerevisiae GRI-117-UK. GRI-117-UK/pUDB7 displaying beta-glucosidase AO090009000356 showed the highest activity against various substrates and efficiently produced ethanol from cellobiose. On the other hand, GRI-117-UK/pUDCB displaying endoglucanase AO090010000314 efficiently degraded barley beta-glucan to glucose and smaller cellooligosaccharides. GRI-117-UK/pUDB7CB codisplaying both beta-glucosidase AO090009000356 and endoglucanase AO090010000314 was constructed. When direct ethanol fermentation from 20 g/l barley beta-glucan as a model substrate was performed with the codisplaying strain, the ethanol concentration reached 7.94 g/l after 24 h of fermentation. The conversion ratio of ethanol from beta-glucan was 69.6% of the theoretical ethanol concentration produced from 20 g/l barley beta-glucan. These results showed that sake yeast displaying A. oryzae cellulolytic enzymes can be used to produce ethanol from cellulosic materials. Our constructs have higher ethanol production potential than the laboratory constructs previously reported.     

Effects of indigenous starter cultures on the microbial and physicochemical characteristics of Urutan, a Balinese fermented sausage

Urutan is a Balinese traditional dry fermented sausage prepared from lean pork and various kinds of spice. Urutan is different from the European sausages, because it is fermented under warm condition with fluctuating temperatures of approximately 25 degrees C at night to 50 degrees C during sun drying. In this study, two of the 71 strains of lactic acid bacteria (LAB) isolated from natural urutan fermentation were used as starter cultures: Lactobacillus plantarum U201, the dominant LAB, and Pediococcus acidilactici U318, a bacteriocin producer. A soft urutan with yellowish brown color was produced using these strains as multiple starters. The starter cultures grew in characteristic succession which reconstructed the natural fermentation process. Lactobacilli were dominant until 48 h fermentation and pediococci dominated at the later stage of fermentation. Proliferation of starter cultures produced lactic acid which resulted in the decrease in pH and coagulation of soluble protein in urutan. Both strains could eliminate the Enterobacteriaceae in urutan after 24 h fermentation, and could suppress and eliminate the occurrence of micrococci at 120 h fermentation. By using a single starter culture, no succession was observed to occur in urutan and the time of elimination of Enterobacteriaceae was delayed. Thus, the strains of L. plantarum U201 and P. acidilactici U318 have great potential for use as multiple starter cultures in urutan fermentation.     

The effects of di-D-fructofuranose-1,2':2,3'-dianhydride (DFA III) administration on human intestinal microbiota

Di-D-fructofuranose-1,2':2,3'-dianhydride (DFA III) was shown to enhance Ca absorption in rat and human intestine. The effects of DFA III administration (9 g per day for 4 weeks that corresponded to 3-fold the optimal dosage of DFA III) on human intestinal microbiota were studied using denaturing gradient gel electrophoresis (DGGE). The major groups of human intestinal microbiota reported previously: the Bacteroides, the Clostridium coccoides group (Clostridium cluster XIVa), the Clostridium leptum group (Clostridium cluster IV), and the Bifidobacterium group were detected. The similarity of 30 DGGE profiles based on the V3 region (before and after administration to the 15 subjects) of the 16S rDNA were calculated using Pearson's correlation based on numbers, positions and intensity of bands, and then a dendrogram of DGGE profiles was constructed by the unweighted pair group method using arithmetic average (UPGMA) clustering method. By these analyses, no difference in DGGE profiles after DFA III administration was observed in healthy subjects, while two subjects with chronic constipation showed different profiles, namely on numbers, positions and the intensity of some bands. Their stools were softer and stool frequencies increased and they obtained relief from constipation.