Genetic and Physiological Characterization of the Intestinal Bacterial Microbiota of Bluegill (Lepomis macrochirus) with Three Different Feeding Habits

Bluegill (Lepomis macrochirus) in Lake Biwa, Japan, feed on benthic invertebrates (benthivorous type), aquatic plants (herbivorous type), and zooplankton (planktivorous type). To evaluate the effect of food on intestinal bacterial microbiota, we characterized and compared the intestinal microbiota of these three types of bluegill in terms of community-level physiological profile (CLPP) and genetic structure. The CLPP was analyzed using Biolog MicroPlates (Biolog, Inc., Hayward, CA, USA), and multivariate analysis of variance revealed that the CLPP of intestinal microbiota differed significantly between any pairs of the three types of bluegill. The genetic profiles were analyzed by temperature gradient gel electrophoresis of polymerase chain reaction (PCR)-amplified 16S rDNA fragments, and multidimensional scaling indicated the existence of specific intestinal bacterial structures for both the benthivorous and the planktivorous types. These results suggest that the host's feeding habit can be one factor controlling the intestinal microbiota of fish in the natural environment.     

Quantitative determination of free-DNA uptake in river bacteria at the single-cell level by in situ rolling-circle amplification

Detection of plasmid DNA uptake in river bacteria at the single-cell level was carried out by rolling-circle amplification (RCA). Uptake of a plasmid containing the green fluorescent protein gene (gfp) by indigenous bacteria from two rivers in Osaka, Japan, was monitored for 506 h using this in situ gene amplification technique with optimized cell permeabilization conditions. Plasmid uptake determined by in situ RCA was compared to direct counts of cells expressing gfp under fluorescence microscopy to examine differences in detection sensitivities between the two methods. Detection of DNA uptake as monitored by in situ RCA was 20 times higher at maximum than that by direct counting of gfp-expressing cells. In situ RCA could detect bacteria taking up the plasmid in several samples in which no gfp-expressing cells were apparent, indicating that in situ gene amplification techniques can be used to determine accurate rates of extracellular DNA uptake by indigenous bacteria in aquatic environments.     

Detection of Bacteria Carrying the stx2 Gene by In Situ Loop-Mediated Isothermal Amplification

A new in situ DNA amplification technique for microscopic detection of bacteria carrying a specific gene is described. Loop-mediated isothermal amplification (LAMP) was used to detect stxA₂ in Escherichia coli O157:H7 cells. The mild permeabilization conditions and low isothermal temperature used in the in situ LAMP method caused less cell damage than in situ PCR. It allowed use of fluorescent antibody labeling in the bacterial mixture after the DNA amplification for identification of E. coli O157:H7 cells with an stxA₂ gene. Higher-contrast images were obtained with this method than with in situ PCR.     

d-Xylose in Glaucidium palmatum

Taste interactions of β-cyclodextrin with naringin or limonin, which are bitter tasting constituents of citrus fruits and their products, were evaluated by psychophysical methods. The mechanism of interaction of β-cyclodextrin with naringin or limonin was explained by both the solubilities of the bitter tasting compounds and the changes brought about by proton shifts within the β-cyclodextrin.

In the presence of 0.5% β-cyclodextrin, the bitterness of naringin or limonin was reduced to approximately half of what it was without β-cyclodextrin. This reduction in bitterness by β-cyclodextrin was also observed in the citrus juice itself.

The solubilities of naringin or limonin increased with the addition of β-cyclodextrin. In a series of ¹H NMR spectra of β-cyclodextrin in D₂O, the low-field triplet signals assigned to the H-3 and H-5 protons, which are located within the cavity of β-cyclodextrin, showed a gradual shift to the higher-field values with increases in the molar ratio of naringin to β-cyclodextrin. It was concluded that this bitterness reduction was due to the formation of an inclusion complex between the β-cyclodextrin and the naringin or limonin.     

Inhibition of adenosine 3',5'-monophosphate phosphodiesterase by nicotinamide and its homologues in vitro.

Inhibition of cAMP phosphodiesterase from rat liver by nicotinamide and its homologues was studied. Among the several compounds tested, such agents as nicotinamide, N2-ethylnicotinamide, N2-methylnicotinamide, N,N-diethylnicotinamide, 3-acetylpyridine, methylnicotinate, and ethylnicotinate showed potent inhibition of cAMP phosphodiesterase, with an over 90% inhibition by 5 mM ethylnicotinate when cAMP was used as substrate at a 0.48 x 10(-7) M concentration. A comparison of the inhibitory curves of theophylline, papaverine, and ethylnicotinate on enzyme activity showed them to be approximately coincident. Furthermore, the plots with abscissa of equal increments per concentration of ethylnicotinate in the presence of theophylline or papaverine coincided with that in the absence of these agents.     

Aristoloside,an aristolochic acid derivative from stems of Aristolochia manshuriensis

Aristoloside, a new companion aristolochic acid derivative isolated from stems of Aristolochia manshuriensis has been shown to be 6-O-β-d-glucopyranoside of aristolochic acid-D on chemical and physicochemical evidence. Three known acids, aristolochic acids I, IV (both as their corresponding methyl esters), and -D have also been characterized from stems of the plant.     

Replacement of the B protein requirement of the E. coli quinolinate synthetase system by chemically-generated iminoaspartate

Two proteins (A and B) from $\text{Escherichia}\text{,}\text{coli}$ are required for $\text{in}\text{,}\text{vitro}$ synthesis of the NAD⁺ precursor, quinolinate, from L-aspartate and dihydroxyacetone phosphate. The requirement for B protein and L-aspartate in this system can be replaced by millimolar concentrations of oxaloacetate and ammonia if they are added together. This finding supports the concept that the B protein (L-aspartate oxidase) functions to form iminoaspartate which is condensed with dihydroxyacetone phosphate by the A protein to form quinolinate.     

Search for and analysis of single nucleotide polymorphisms (SNPs) in rice (Oryza sativa, Oryza rufipogon) and establishment of SNP markers.

We searched for SNPs in 417 regions distributed throughout the genome of three Oryza sativa ssp. japonica cultivars, two indica cultivars, and a wild rice (O. rufipogon). We found 2800 SNPs in approximately 250,000 aligned bases for an average of one SNP every 89 bp, or one SNP every 232 bp between two randomly selected strains. Graphic representation of the frequency of SNPs along each chromosome showed uneven distribution of polymorphism-rich and -poor regions, but little obvious association with the centromere or telomere. The 94 SNPs that we found between the closely related cultivars 'Nipponbare' and 'Koshihikari' can be converted into molecular markers. Our establishment of 213 co-dominant SNP markers distributed throughout the genome illustrates the immense potential of SNPs as molecular markers not only for genome research, but also for molecular breeding of rice.     

An Endogenous Suppressor of the Defense Response in Pisum sativum

Healthy pea plants contain a substance, tentatively called "endogenoussuppressor", which specifically suppresses the accumulationof pisatin in pea plants that is induced by treatment with CuCl₂or an elicitor from Mycosphaerella pinodes. This suppressorelicits the accumulation of phytoalexins in other legumes, suchas kidney bean, soybean and cowpea. The endogenous suppressorfunctions to delay the accumulation of pisatin, the activationof phenylalanine ammonialyase (PAL) and the accumulation ofmRNAs for PAL and chalcone synthase induced by the elicitorfrom M. pinodes. The substance specifically induces susceptibilityto nonpathogens, such as Mycosphaerella ligulicola and M. melonis,in pea out of four species of legume tested, but the effectis not cultivar-specific. Thus, the endogenous suppressor inhealthy pea plants suppresses a series of self-defense reactionsand induces susceptibility in pea plants in a species-specificmanner, being similar to the exogenous fungal suppressor fromthe pea pathogen, M.pinodes. (Received February 19, 1992; Accepted May 11, 1992)     

Orthovanadate Suppresses Accumulation of Phenylalanine Ammonia-Lyase mRNA and Chalcone Synthase mRNA in Pea Epicotyls Induced by Elicitor from Mycosphaerella pinodes

Orthovanadate delayed accumulation of mRNAs encoding phenylalanineammonia-lyase and chalcone synthase in pea epicotyls inducedby an elicitor from Mycosphaerella pinodes. However, accumulationof mRNA for a putative P-type ATPase was not affected. The relationshipbetweenthe ATPase and defense responses is discussed. ³Present address: Plant Pathology Laboratory, School of Agriculture,Nagoya University, Chikusa, Nagoya, 464-01 Japan.