Heterologous expression of gassericin A,a bacteriocin produced by Lactobacillus gasseri LA39

Gassericin A, a bacteriocin produced by Lactobacillus gasseri LA39, has a cyclic structure linking N‐ and C‐terminal amino acids. Gassericin A was expressed in Escherichia coli JM109 as a biotinylated fusion protein on the basis of the DNA sequence of mature bacteriocin. A positive clone accumulated the bacteriocin, with no activity, as a soluble fusion protein in the cytoplasm. After release of an N‐terminal tag with factor Xa protease, gassericin A was converted into an active peptide having N‐ and C‐termini. The total amount of purified bacteriocins (expressed and native) was 480 µg/L and 370 µg/L, respectively. However, the specific activity of expressed gassericin A was 15 AU/mg lower than that of native bacteriocin (2600 AU/mg). Although the actual Mr (molecular weight) of the expressed bacteriocin should be 5666, the peptide showed the same mobility (Mr 3800) in sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) as native cyclic gassericin A, suggesting that the expressed peptide retains compact folding of the molecule similar to that of native gassericin A.     

鹅掌楸组织培养技术初探

鹅掌楸外植体是以种子发芽后的无菌苗为材料。通过实验结果分析，利用基本培养基MS(以下简称MS)添加适当的细胞分裂素可以促进种子的发芽。在初步培养中，以WPM 0．5mg／L(单位下同)BAP 0．1NAA培养基分化最好。在继代培养中，以MS 0．2BAP 0．05NAA培养基中不定芽诱导情况最好。不定芽在接入生根培养基20d后，其高度平均可长高1．86cm。在生根培养中，以1／2MS 0．2IBA 0．1NAA 0．1ABT较好，其生根率达到了60％左右。     

A New and Simple Technique for the Isolation of Plasma Membrane Lipids from Root-Tips

It has been suggested that plasma membrane (PM) lipids play a major role in aluminum (Al) tolerance; however, no direct investigations have been carried out using PM lipids from root-tips. Here we report a new technique for PM isolation as an alternative to the laborious two-polymer phase partitioning method that is commonly applied, as follows: 1) separation of protoplasts from 1-cm root-tip portions by enzymatic digestion, 2) attachment of the purified protoplasts to glass plates coated with polylysine, 3) preparation of PM ghosts by successive burst of the attached protoplasts using three separate buffer solutions (25 mM PIPES, 5 mM EDTA, and 2 mM MgCl₂, at pH 7.0) with slow stirring for 60 s. The PMs were confirmed to be devoid of organelle membranes by fluorescence microscopy, thin layer chromatography (TLC) and western blot analysis. The PM lipids obtained were found to be useful for studies on their differential permeability and lipid composition between lines of triticale or cultivars of maize under Al stress.     

Population and distribution of sclerotia of Rhizoctonia solani kühn in sugar beet field soil

The population and distribution of sclerotia of Rhizoctonia solani Kühn in two sugar beet field soils was determined at harvest by a sieving-flotation method. In rhizosphere soil (RS) and non-rhizosphere soil (NRS) from the most heavily infected roots of sugar beets, 1.43–2.5 and 0.83–1.0 sclerotia g^?1 dry soil were detected, respectively. In the soil around healthy sugar beet, these values were 0.04–0.12 and 0.03–0.04 sclerotia g^?1 dry soil. More sclerotia were always obtained from RS than from NRS. More than 80% of the sclerotia were in the upper 10 cm of soil and within 10 cm of diseased roots. Therefore, there is a non-uniform distribution of sclerotia of R. solani in soil.The sclerotial population in soil increased significantly with disease severity and a good correlation was obtained between the number of sclerotia and the disease severity on infected plants. Most of the sclerotia collected from the field soil ranged in size from 0.5 to 2.0 mm diameter.Viability of sclerotia increased as severity of crown rot increased and as the size of the sclerotia increased. Conversely, there was a progressive decrease in sclerotial germination with increasing depth in soil and increasing distance from the infected root.     

Development of immune assay system for both CpG and non-CpG DNA from lactic acid bacteria using a transfectant of swine Toll-like receptor 9

Bacterial DNA is expected to be a potent immune stimulating agent to Toll‐like receptor (TLR) 9 expressed cells such as macrophages, monocytes, B lymphocytes, NK cells and dendritic cells. In the present study, we constructed a transfectant of swine TLR9 with mammalian cells. We demonstrated that the transfectant, recognizing both CpG and non‐CpG oligonucleotides from lactic acid bacteria, induced NF‐κB activation by gene reporter assay. These findings indicate that the swine TLR9 transfectant will be highly useful for the screening of immunostimulatory DNA from lactic acid bacteria.     

Chemical characterization of milk oligosaccharides of an African lion (Panthera leo) and a clouded leopard (Neofelis nebulosa)

The Carnivora include the superfamilies Canoidea and Feloidea. In species of Canoidea other than the domestic dog, Canis lupus, the milk contains only traces of lactose and much larger concentrations of oligosaccharides. In this study, lactose was found to be the dominant saccharide in the milk or colostrum of two species of Feloidea, namely the African lion (Panthera leo) and the clouded leopard (Neofelis nebulosa). In addition to lactose, the following oligosaccharides were characterized in the milk of a lion; Neu5Gc(α2‐3)Gal(β1‐4)Glc (3′‐NGc‐SL), Fuc(α1‐2)Gal(β1‐4)Glc (2′‐fucosyllactose) and GalNAc(α1‐3)[Fuc(α1‐2)]Gal(β1‐4)Glc (A‐tetrasaccharide). The colostrum of a clouded leopard contained 3′‐NGc‐SL, Gal(α1‐3)Gal(β1‐4)Glc (isoglobotriose) and A‐tetrasaccharide. These oligosaccharides differ in some respects from those previously identified in another species of Feloidea, the spotted hyena (Crocuta crocuta). These milks contained 3′‐NGc‐SL and A‐tetrasaccharide, while spotted hyena colostrum did not; however, it contained Neu5Ac(α2‐3)Gal(β1‐4)Glc (3′‐NAc‐SL) and Gal(α1‐3)[Fuc(α1‐2)]Gal(β1‐4)Glc (B‐tetrasaccharide).     

Development of simple sequence repeat markers for the classification of the clubroot pathogen <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

Clubroot disease caused by Plasmodiophora brassicae is one of the most serious diseases in cruciferous crops. To classify isolates, we developed simple sequence repeat (SSR) markers for P. brassicae. Twenty-four Japanese isolates were used in this study: 12 isolates of an unknown pathotype from the Kyoto Prefecture, as well as 12 isolates of known pathotypes, including three single-spore lines. From the 12 isolates from Kyoto Prefecture, 11 were classified into either pathotype 2 (three isolates) or 4 (eight isolates). We designed 23 SSR markers based on the P. brassicae genome, of which 11 markers from intergenic regions showed polymorphisms in the 24 isolates. Many haploid isolates belonging to pathotypes 2 and 4 were monomorphic, and typical alleles were detected in some isolates not belonging to pathotype 4. Two bands were detected for eight SSR loci in five isolates, indicating that different genotypes were mixed in these isolates. We constructed a phylogram based on the 11 polymorphic SSRs. Pathotypes 2 and 4 formed a cluster, from which pathotypes 3 and 1 were successively placed. These results strongly suggest a close genetic relationship between isolates in pathotypes 2 and 4, consistent with our finding that isolates in these two pathotypes were found at one collection site. In combination with pathotype classification and other marker systems, the SSR markers can be used for more detailed analyses to improve the control of clubroot disease.     

Electroretinography using contact lens electrode with built-in light source in dogs

Electroretinography (ERG) is an effective method for the diagnosis of retinal disease. In the dog, dependable ERG recording is difficult without the use of an expensive device like a Ganzfeld full-field stimulator. The International Society for Clinical Electrophysiology of Vision has defined the standard flash stimulus condition (SF) and evaluation of the retina using the b/a ratio in humans. In dogs, evaluation using the b/a ratio has not been reported, whereas the intensity of SF has been defined. In this study, we performed a convenient ERG recording method using a contact lens electrode with a built-in light source (LED-electrode), and confirmed SF as reported previously. ERG recordings were performed on 15 healthy beagle dogs under sedation. We performed bilateral ERG at 12 different intensities after 30 min dark adaptation. After 10 min light adaptation, we recorded single flash cone and flicker cone response using the SF determined in this study. In this study, SF of 3.0 cd/m(2)/sec (6,000 cd/m(2), 0.5 msec) resulted in b/a=2. The intensity for rod response that recorded only the b-wave was 0.0096 cd/m(2)/sec (80 cd/m(2), 0.12 msec). We could achieve ERG for each response easily and smoothly under sedation, and without general anesthesia. Using an LED-electrode, we could perform more quantitative and reproducible ERG examinations than with traditional methods. We propose that the b/a ratio is the most useful parameter in ERG reporting for evaluating retinal function.     

Interaction between manganese and zinc in growth of rice plants

Many instances of antagonistic relationships among various micro-elements and between micro- and macro-elements in their absorption, translocation, and functions in plant growth have been reported (1). In a series of our researches on the effect of micro-elements on the growth of rice plants, it was observed that manganese toxicity symptom of rice plants was markedly reduced by increasing the concentration of zinc in the nutrient sdution. Although it has been reported that manganese toxicity symptoms of crops were reduced by addition of Fe (2,3), A1 (4), Ca (5) and silicate (6) to the medium, there have been few reports of the effect of Zn on Mn toxicity of rice plants. In this paper, the interaction between Mn and Zn in the growth of rice plants was reported.     

Food preservative potential of gassericin A‐containing concentrate prepared from cheese whey culture supernatant of Lactobacillus gasseri LA39

Gassericin A (GA) is a circular bacteriocin produced by Lactobacillus gasseri LA39. In this study, GA‐containing concentrate was prepared using a cross‐flow membrane filtration device (30 kDa cut‐off) from the culture supernatant of Lb. gasseri LA39 cultivated in a cheese whey‐based food‐grade medium. The bacteriocin activity titer in the concentrate was 16 times as high as that of the culture supernatant and was completely maintained through each incubation at 4°C for 3 months, 37°C for 2 months, 60°C for 5 h, and 100°C for 30 min. The GA‐containing concentrate was used with glycine powder to make custard creams, and then four representative strains of custard cream spoilage bacteria (Bacillus cereus, Lactococcus lactis subsp. lactis, Achromobacter denitrificans and Pseudomonas fluorescens) were individually inoculated at c. 10³ colony forming units/g in the custard creams. Throughout 30 days of incubation at 30°C, all of the inoculated bacteria were completely inhibited by the combination of 5% (w/w) of the GA‐containing concentrate and 0.5% (w/w) glycine. This is the first highly practical application of GA to foods as a biopreservative, and the concentration method and the bacteriocin concentrate would contribute to biopreservation of several foods.