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.     

Antioxidant and cytotoxic activities of naphthalene derivatives from <Emphasis Type="Italic">Diospyros kaki</Emphasis>

4,8-Dihydroxy-5-methoxy-2-naphthaldehyde (Compound I) was isolated from blackened heartwood of Diospyros kaki and was methylated with diazomethane. Antioxidant and cytotoxic activities of Compound I and two methylated derivatives [4-hydroxy-5,8-dimethoxy-2-(2-oxopropyl)-naphthalene (D-1) and 2-glycidyl-4-hydroxy-5,8-dimethoxy naphthalene (D-2)] were evaluated. Compound I showed higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and reducing power than D-1 and D-2. However, D-1 and D-2 exhibited slightly stronger 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)⁺ (ABTS⁺) radical scavenging activity than Compound I. Compound I also exhibited stronger cytotoxic activity than D-1 and D-2 against the growth of HT-29 colon cancer cells. The results supported the hypothesis that methylation of naphthalene derivatives slightly increased ABTS⁺ radical scavenging activity, but significantly decreased DPPH radical scavenging activity, reducing power, and cytotoxic activity.     

Foliar absorption of nutrients: I

Recently, studies on foliar application have made splendid development in the field of agronomy and in the fertilizer industry. This fact has some important meanings. First, the conception of fertilizer has gradually changed from the original meaning. "Non-root" parts of plants do take up nutrients from spray and other applications. This conception could definitely be proved by the use of radioactive isotopes as the tracer. Many investigations by use of radioactive isotopes, ¹⁴C ( 1 and 2 ) ³²P (1, 3, 4, 5, 6, 7 and 8) ⁴²K (9 and 10) ⁴⁵Ca (10) etc. have since been conducted showing the foliar absorption of nutrients. The second important meaning is the improvement of applica tion methods of fertilizers. By the foliar application method, excellent effects which could not be carried out by the soil application method have been displayed. When fertilizers are applied to leaves of plants in critical times as a supplementary fertilizer, they can be absorbed quickly plentifully into plants. Foliar application of micronutrients, for instance, Mg, Fe, Zn, and Mn have been used succesfully to the deficiencies in plants. Among the macronutrients, phosphorus could perhaps offer more promise as a foliar applied fertilizer, because when phosphatic fertilizers are applied to soil, a large percentage of phosphorus usually becomes fixed in unavailable form to growing plants. On the other hand, especially in a high rainfall area, the application of soluble nitrogen fertilizer to the soil has resulted in low efficiency due to leaching loses (19). So, studies on foliar absorption of urea, phosphorus and micro-nutrients have especially been investigated by many workers. In other cases, there are possibilities that fertilizers can be applied to plants with germicides, insecticides, etc.     

Composition of culture medium influences zoosporogenesis and differentiation of <Emphasis Type="Italic">Aphanomyces cochlioides</Emphasis>

A modified medium was used to culture mycelium and produce a large quantity of zoospores of Aphanomyces cochlioides, a principal pathogen of damping-off disease of sugar beet and root rot disease of spinach. The semisolid medium consisted of 17 g corn meal agar (CMA) added with 4 g of yeast extract (YE) per liter of 50 mM phosphate buffer (pH 6.8–7.0). This medium supported the production of ca. 10⁶ zoospores ml⁻¹ in 6-day-old cultures, approximately 11-fold higher than the commonly used CMA (17 g CMA per liter of water, pH 6.0 ± 0.2). Although morphological characters of the zoospores produced from the hyphae grown on CMA and CMA + YE were almost similar, they contrasted their developmental strategy after encystment induced by mechanical agitation. Cystospores originating from the zoospores on CMA regenerated zoospores (>80%), while those from CMA + YE germinated (ca. 80%) and produced hyphae. Furthermore, 4–10% of the germinated cystospores on CMA + YE had double germ tubes. The soluble protein profiles of zoospores produced on CMA and on CMA + YE demonstrated that several proteins were either different or expressed differently. Our results suggest that the culture medium directly influences zoosporogenesis in A. cochlioides hyphae and the developmental strategy of the produced zoospores.     

Histochemical analysis of Bacillus thuringiensis CrylA toxin binding to midgut epithelial cells of Bombyx mori

We analyzed the binding of the Bacillus thuringiensis insecticidal toxins, CrylAa, CrylAb and CrylAc, to midgut tissue of the silkworm, Bombyx mori with ligand blot analysis and histochemical observations. CrylAa, CrylAb and CrylAc bound to unique sets of proteins in various subcellular fractions prepared by centrifugation. CrylAa bound to various proteins in all subcellular fractions, whereas CrylAb bound to a single protein of ∼180 kDa in all fractions as shown by Western blot analysis. Cry1Ac bound to proteins which were primarily ∼100-120 kDa in all fractions. CrylA toxins were labeled with fluorescent dye and Cy3-labeled CrylAa, CrylAb and CrylAc were shown to localize primarily to the apical membrane region. However, they also localized to basement or basolateral membranes. The distribution of a 252-kDa membrane protein (P252) of the B. mori midgut, which was recently identified as a plausible candidate for receptor of CrylA toxins were also examined with histochemical methods. Substantial signals of FITC-labeled antibody against P252, even though not all, were evident in the apical cells, and these were coincident with Cy3-CrylAa and Cy3-CrylAc signals.     

The Complete Nucleotide Sequence of Potato virus X Strain OS: The First Complete Sequence of a Japanese Isolate

The complete nucleotide sequence of the genomic RNA of a Japanese isolate of Potato virus X (PVX), strain OS (PVX-OS), was determined. The PVX-OS genome is 6435 nucleotides in length, excluding the 3′ poly(A) sequence, and encodes for five open reading frames (ORFs), similar to other reported PVX strains. Comparisons of the nucleotide sequence of PVX-OS with those of European and South American PVX strains revealed that the PVX-OS genomic sequence showed 95-96% homology with European strains and 77-78% with South American strains. Phylogenetic analysis of PVX-OS and other PVX strains found that PVX-OS clusters with European strains. This is the first report on the complete nucleotide sequence of a PVX strain from Japan. Received 10 May 2001/ Accepted in revised form 26 October 2001     

Isolation and Sequence of a Gene, celD Xo Involved in Cellobiose Utilization in Xanthomonas oryzae pv. oryzae

A genomic library of Xanthomonas oryzae pv. oryzae (X. o. pv. oryzae) T7174 was screened for 4-methylumbelliferyl β-D-glucoside-hydrolyzing (MUGase) activity. In subcloning of one of the MUGase-positive clones, an approximately 4.2-kb SacI-SphI fragment conferred not only MUGase activity but also 4-methylumbelliferyl β-D-cellobioside-hydrolyzing (MUCase) activity. Sequence analysis showed that the fragment contained an ORF of 2951 bp. The conceptual ORF product was significantly homologous with 1,4-β-D-glucan glucohydrolase D (CELD) from Pseudomonas fluorescens subsp. cellulosa, and was named CELD_Xo. Cell fractionation experiments suggested that CELD_Xo is localized in the cell-envelope fraction. We constructed a CELD_Xo-deficient mutant (74ΔCELD) from X. o. pv. oryzae. Little MUCase activity was detected in the cell-envelope fraction prepared from the mutant. The mutant 74ΔCELD did not grow in synthetic medium containing cellobiose as the sole sugar source. On the other hand, growth in rice leaves and pathogenicity of the mutant and the parental strain did not differ. These results suggested that CELD_Xo is involved in cellobiose utilization of X. o. pv. oryzae but that the gene is not required for bacterial growth in rice leaves. Received 16 February 2001/ Accepted in revised form 11 April 2001     

Genetic characterization and gonad development of artificially produced interspecific hybrids of the abalones, Haliotis discus discus Reeve, Haliotis gigantea Gmelin and Haliotis madaka Habe

Hybridization among abalone species has been suggested as a possible means to increase their growth rates for aquaculture. As a first step to test the usefulness of the hybrids of Japanese abalone species (Haliotis discus discus, Haliotis gigantea and Haliotis madaka) for aquaculture, we characterized the genetic background and gonad development of hybrids that were produced by artificial insemination. The hybrid status of the resulting offspring was confirmed by assaying 14 allozymes and by RFLP analysis of the 16s rRNA and cytochrome oxidase I (COI) regions of mtDNA using 13 restriction enzymes. Histological examination of the gonads of the hybrids was conducted in comparison with those of the parental species. Cross‐breeding among the three species was conducted successfully in all combinations although with lower fertilization rates (means of 1.3–60.8%) than the parental species (34.3–90%). Crosses between H. discus discus and H. madaka had higher fertilization rates (22.4–60.8%) than those involving H. gigantea (1.3–19.9%). The hybrids were ascertained by the presence of both parental genotypes at the LDH‐A, ME‐A, MDH‐A and GPI loci. The maternal origin of the hybrid mtDNA was confirmed by digestion with DdeI, TaqI, HpaII of the COI region. No polymorphism was observed in the 16S rRNA region. The hybrids had gonadal development and maturity stages similar to the parental species up to fully mature oocytes and sperm. They spawned upon stimulation and produced viable offspring with high fertilization rates and successful development to the juvenile stage in back‐ and homologous hybrid crosses.