Fluorescent in situ hybridization analysis of open lactic acid fermentation of kitchen refuse using rRNA-targeted oligonucleotide probes

Reproducible amounts of lactic acid accumulate in minced kitchen refuse under open conditions with intermittent pH neutralization [Sakai et al., Food Sci. Technol. Res., 6, 140 (2000)]. Here, we showed that such pH-controlled open fermentation of kitchen refuse reproducibly resulted a selective proliferation of a major lactic acid bacterial (LAB) species. In one experiment, the predominant microorganisms isolated during the early phase (6 h) were Gammaproteobacteria. In contrast, those that predominated during the late phase (48 h) were always Lactobacillus plantarum in three independent experiments. To further quantify the microbial community within open lactic acid fermentation, we performed fluorescent in situ hybridization (FISH) analysis targeting 16S (23S) rRNA. We designed two new group-specific DNA probes: LAC722(L) was active for most LAB including the genera Lactobacillus, Pediococcus, Leuconostoc and Weisella, whereas Lplan477 was specific for L. plantarum and its related species. We then optimized sample preparation using lysozyme and hybridization conditions including temperature, as well as the formamide concentration and the salt concentration in the washing buffer. We succeeded in quantification of microorganisms in semi-solid, complex biological materials such as minced kitchen refuse by taking color microphotographs in modified RGB balance on pre-coated slides. FISH analysis of the fermentation of kitchen refuse indicated that control of the pH swing leads to domination by the LAB population in minced kitchen refuse under open conditions. We also confirmed that L. plantarum, which generates lactic acid in high quantities but with low optical activity, became the dominant microorganism in kitchen refuse during the late phase of open fermentation.     

Denatured-jacalin derivatives with selective recognition for O-linked glycosides (ST,T, Tn,and STn Type) on IgA1 hinge region

Immunoglobulin A1 (IgA1) concentration in the plasma of patients with IgA nephropathy (IgAN) as the cause of renal failure is higher than that in the plasma of normal controls. IgA1 with abnormal sugars is considered to deposit in the glomerular mesangium, aggravating nephritis in IgAN. Jacalin is a lectin that recognizes sugars on IgA1. However, its selective-recognition for normal-type (ST type, NeuAc-α(2,3)-Gal-β(1,3)-GalNAc) and abnormal-type (T type, Gal-β(1,3)-GalNAc; Tn type, GalNAc; STn type, NeuAc-α(2,6)-GalNAc) sugars α-O-linked to serine/threonine in IgA1 is weak. Therefore, jacalin cannot be used for recognizing specific sugar types on IgA1. We attempted to develop a new recognition method for specific sugar types on IgA1 by utilizing the multirecognition capability of jacalin. Its binding abilities were regulated by heat denaturation with suitable template sugar (galactose or N-acetylgalactosamine). Further, we successfully prepared denatured-jacalin derivatives, which recognized ST-/T-type sugars on IgA1, by sugar-immobilized affinity chromatography. Enzyme-linked immunosorbent assay of denatured-jacalin derivatives, showed the ratios of abnormal sugars on IgA1 in the plasma of IgAN patients and normal controls to be approximately 60% and 20%, respectively. The results proved that profiling of sugar types in IgAN can successfully be performed by solely using jacalin derivatives.     

Correlation of the structures of agricultural fungicides to gene expression in Saccharomyces cerevisiae upon exposure to toxic doses

Correlations between the chemical structures of agricultural fungicides and mRNA expression levels following exposure of Saccharomyces cerevisiae to toxic doses of thiuram, zineb, maneb, TPN, and PCP were examined. Structurally, thiuram, zineb, and maneb are dithiocarbamate fungicides, whereas TPN and PCP are not. To characterize chemical toxicity, genes expression was classified according to the functional groups used by the MIPS database. However, no correlations between the classification scheme and chemical structures were found. Hierarchical clustering of gene expression profiles was performed to characterize the effects of the five chemicals. According to this analysis the similarity of gene expression profiles depended on the similarity of chemical structures. These results suggest that DNA microarray technology has potential for predicting the major chemicals which will cause environmental toxicity and will provide information on new biomonitoring methods.     

Determination of benzimidazole anthelmintics in livestock foods by HPLC

A simple and rapid liquid chromatographic method was developed for the simultaneous determination of twelve benzimidazole anthelmintics in livestock foods using reversed-phase high-performance liquid chromatography with photodiode array detection (PDA). A sample was homogenized with acetonitrile and n-hexane, and centrifuged. The acetonitrile phase was isolated and evaporated. The residue was dissolved in 0.1 mol/L carbonate buffer solution (pH = 9.1), sonicated, and then subjected to clean-up on a Bond Elut LRC-C18 cartridge. The benzimidazole compounds were separated isocratically on a Capcell Pak C18 UG 120 (5 microns, 150 x 4.6 mm i.d.) column and detected by PDA at 295 and 313 nm. Mixtures of acetonitrile and 0.05 mol/L ammonium acetate in mixing ratios of (20:80) and (40:60) were used as the mobile phase, and the flow-rate was 1.0 mL/min at 40 degrees C. The mean recoveries (n = 3) from 0.1-0.5 microgram/g added samples were 72.6-97.2% with coefficients of variation of 0.3-8.5%. The detection limits were 0.01-0.05 microgram/g.     

Affinity capture and elution/electrospray ionization mass spectrometry assay of phosphomannomutase and phosphomannose isomerase for the multiplex analysis of congenital disorders of glycosylation types Ia and Ib

We report a new application of affinity capture-elution electrospray mass spectrometry (ACESI-MS) to assay the enzymes phosphomannomutase (PMM) and phosphomannose isomerase (PMI), which when deficient cause congenital disorders of glycosylation CDG-type Ia and type Ib, respectively. The novel feature of this mass-spectrometry-based assay is that it allows one to distinguish and quantify enzymatic products that are isomeric with their substrates that are present simultaneously in complex mixtures, such as cultured human cell homogenates. This is achieved by coupled assays in which the PMM and PMI primary products are in vitro subjected to another enzymatic reaction with yeast transketolase that changes the mass of the products to be detected by mass spectrometry. The affinity purification procedure is fully automated, and the mass spectrometric analysis is multiplexed in a fashion that is suitable for high-throughput applications.     

Acid-dyeable acrylic fibres copolymerised with amino alkyl methacrylates for differential cross-dyeing

The equilibrium adsorption of acid dyes on acrylic fibres copolymerised with dimethyl amino ethyl methacrylate has been investigated. A typical characteristic of this type of fibre is its excellent stable dyeability over a wide pH range. This is due to the strong basicity of alkylamino groups in the copolymerised component, which makes it possible to dye the fibre thoroughly even in a weakly acidic dyebath using a normal dyeing processes of acrylic fibre with cationic dye. Consequently it is useful for differential cross-dyeing in a single dyebath.     

Differences in knowledge production between disciplines based on analysis of paper styles and citation patterns

To identify the differences in the knowledge production between disciplines, we analyzed the relation between the average paper length and impact factor of 100 journals from 5 disciplines. We found negative correlation between the average length and the impact factor in the natural sciences, but not in the social sciences. We also analyzed the structures of paper and the citation patterns. These analyses are expanded to the comparison between Mode 1 and Mode 2. All results showed the natural sciences articles could emphasize the differences from previous studies and be diffused effectively by the short standardized style of paper. This research is partly funded by International Program in Special Coordinating Funds for Promoting S&T from Science and Technology Agency of Japan.