Transgenic tobacco transformed with the Trichoplusia ni granulovirus enhancin gene has been demon... more Transgenic tobacco transformed with the Trichoplusia ni granulovirus enhancin gene has been demonstrated to enhance baculovirus infection in larvae. In this paper we describe the effect of the long-term feeding of lyophilized transgenic tobacco material to Pseudaletia separata and Spodoptera exigua larvae. Our results demonstrated that the baculovirus enhancin gene products have potential for use in insect pest management.
Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is of great interest f... more Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is of great interest for developing a bioinsecticide to control mosquitoes. Therefore, it is very important to characterize the functional motif of Cry4Aa that is responsible for its mosquitocidal activity. In this study, to characterize a potential receptor binding site, namely, loops 1, 2, and 3 in domain II, we constructed a series of Cry4Aa mutants in which a residue in these three loops was replaced with alanine. A bioassay using Culex pipiens larvae revealed that replacement of some residues affected the mosquitocidal activity of Cry4Aa, but the effect was limited. This finding was partially inconsistent with previous results which suggested that replacement of the Cry4Aa loop 2 results in a significant loss of mosquitocidal activity. Therefore, we constructed additional mutants in which multiple (five or six) residues in loop 2 were replaced with alanine. Although the replacement of multiple residues a...
Ion channel proteins play important roles in various cell functions, making them attractive drug ... more Ion channel proteins play important roles in various cell functions, making them attractive drug targets. Artificial lipid bilayer recording is a technique used to measure the ion transport activities of channel proteins with high sensitivity and accuracy. However, the measurement efficiency is low. In order to improve the efficiency, we developed a method that allows us to form bilayers on a hydrogel bead and record channel currents promptly. We tested our system by measuring the activities of various types of channels, including gramicidin, alamethicin, α-hemolysin, a voltage-dependent anion channel 1 (VDAC1), a voltage- and calcium-activated large conductance potassium channel (BK channel), and a potassium channel from Streptomyces lividans (KcsA channel). We confirmed the ability for enhanced measurement efficiency and measurement system miniaturizion.
Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with an aerolysin-type arc... more Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with an aerolysin-type architecture, and it is expected to be used as a novel bioinsecticide. Cry46Ab acts as a functional pore-forming toxin, and characteristics of the resulting channel pores, including ion selectivity, have been analyzed. However, the relationship between channel-pore ion selectivity and insecticidal activity remains to be elucidated. To clarify the effects of charged amino acid residues on the ion permeability of channel-pores and the resulting insecticidal activity, in the present study, we constructed Cry46Ab mutants in which a charged amino acid residue within a putative transmembrane β-hairpin region was replaced with an oppositely charged residue. Bioassays using Culex pipiens mosquito larvae revealed that the mosquitocidal activity was altered by the mutation. A K155E Cry46Ab mutant exhibited toxicity apparently higher than that of wild-type Cry46Ab, but the E159K and E163K mutants exhibited decreased toxicity. Ions selectivity measurements demonstrated that the channel pores formed by both wild-type and mutant Cry46Abs were cation selective, and their cation preference was also similar. However, the degree of cation selectivity was apparently higher in channel pores formed by the K155E mutant, and reduced selectivity was observed with the E159K and E163K mutants. Our data suggest that channel-pore cation selectivity is a major determinant of Cry46Ab mosquitocidal activity and that cation selectivity can be controlled via mutagenesis targeting the transmembrane β-hairpin region. KEY POINTS: • Cry46Ab mutants were constructed by targeting the putative transmembrane β-hairpin region. • Charged residues within the β-hairpin control the flux of ions through channel pores. • Channel-pore cation selectivity is correlated with insecticidal activity.
In order to characterize the genome organization of Xestia c-nigrum granulovirus (XcGV), mapping ... more In order to characterize the genome organization of Xestia c-nigrum granulovirus (XcGV), mapping of putative XcGV genes was performed by construction of lambda and M13 phage libraries followed by Southern blot and nucleotide sequencing analyses. Mapping of the lambda (32 clones covering the entire XcGV genome) and M13 (133 clones made by random cloning) phage library clones was carried out by hybridization of the labeled lambda phage clone DNAs to 1) Southern blotted XcGV genomic DNA fragments cleaved with EcoRI, BamHI, or HindIII, and 2) dot blotted M13 clone DNAs. All 133 M13 clone DNAs were sequenced, and coding possibilities were investigated by computer-assisted homology search; in total, about 43 kb of the genome was sequenced. Amino acid sequence homology searches of 67 M13 clones suggested that these GV DNAs coded for previously characterized genes identified in nucleopolyhedroviruses (NPVs) and GVs. These 67 M13 clones were classified into 25 gene homolog groups (including ...
The Plutella xylostella granulovirus (PxGV) genome DNA was sequenced and the predicted open readi... more The Plutella xylostella granulovirus (PxGV) genome DNA was sequenced and the predicted open reading frames (ORFs) were compared to genes of the first-sequenced GV, Xestia c-nigrum GV (XcGV), and those from other baculoviruses and organisms. PxGV DNA has a size of 100,999 bp with a G + C content of 40.7%. The analysis predicted 120 ORFs with a size of 150 nucleotides or larger that showed minimal overlap. Blast searches followed by a comparison of ORF arrangement with those of completely sequenced baculovirus genomes showed the presence of 102 homologs to other genes in the database. Among them, 74 and 100 were homologous to genes of Autographa californica NPV (AcMNPV) and XcGV, respectively. A striking feature of the relationship between the genomes of PxGV and XcGV was the conservation of the order and orientation of homologous genes. Even though the XcGV genome is much larger than that of PxGV (178 vs 101 kb) and had many more predicted ORFs (181 vs 120) with an average amino acid sequence relatedness of 42%, the order and orientation of almost all homologous genes was conserved. The PxGV genome contained four homologous regions (hrs), each with 10 to 23 repeated sequences of 101 to 105 nucleotides containing a 15-bp imperfect palindrome in the center of the repeats.
Cystatin C is a cysteine protease inhibitor produced by a variety of human tissues. The blood con... more Cystatin C is a cysteine protease inhibitor produced by a variety of human tissues. The blood concentration of cystatin C depends on the glomerular filtration rate and is an endogenous marker of renal dysfunction. Recombinant cystatin C protein with high immunogenicity is therefore in demand for the diagnostic market. In this study, to establish an efficient production system, a synthetic cystatin C gene was designed and synthesized in accordance with the codon preference of Escherichia coli genes. Recombinant cystatin C was expressed as a fusion with a peptide-tag, 4AaCter, which facilitates formation of protein inclusion bodies in E. coli cells. Fusion with 4AaCter-tag dramatically increased the production level of cystatin C, and highly purified protein was obtained without the need for complicated purification steps. The purity and yield of the final product was estimated as 87 ± 5% and 7.1 ± 1.1 mg/l culture, respectively. The recombinant cystatin C prepared by our method was as reactive against anti-cystatin C antibodies as native human cystatin C. Our results suggest that protein production systems using 4AaCter-tag could be a powerful means of preparing significant amounts of antigen protein.
ABSTRACT Abstract Since the first report of Bacillus sotto by Ishiwata in 1901, thousands of rel... more ABSTRACT Abstract Since the first report of Bacillus sotto by Ishiwata in 1901, thousands of related papers about Bacillus thuringiensis have been documented. In the field of biocontrol of insect pests by this bacterium, after the initial discovery of several B. thuringiensis isolates specific for lepidopteran insects, the isolation of B. thuringiensis israelensis, specific to dipteran larvae by Goldberg and Margalit, and B. thuringiensis tenebrionis, specific to some group of coleopteran insects by Krieg et al. were epoch making advances. In 1992, Ohba et al. isolated B. thuringiensis ja ponensis strain Buibui, which was specific to only scarabaeid larvae. This isolate is the main target of our discussion in this review. These discoveries by which not only B. thuringiensis sciences, but also applied biological control strategies have been enriched, which inspirit us to screen novel isolates.On the other hand, the fields of molecular biology and biochemistry studies on the structural elucidation of toxin proteins and mechanism of action have also tremendously progressed. But the complete mechanism has yet to be solved. For instance, interaction between receptor proteins locating on the plasma membrane of insect midgut epithelial cells and insecticidal proteins has not been fully sketched- In a way, this field is still in chaos. Addressing these exciting and enigmatic subjects will eventually lead to the construction of sustainable agriculture in the 21st century.
Two novel parasporin (PS) genes were cloned from Bacillus thuringiensis B0462 strain. One was 100... more Two novel parasporin (PS) genes were cloned from Bacillus thuringiensis B0462 strain. One was 100 % identical even in nucleotide sequence level with that of parasporin-1Aa (PS1Aa1) from B. thuringiensis A1190 strain. The other (PS1Ac2) showed significant homology (99 % identity) to that of PS1Ac1 from B. thuringiensis 87-29 strain. The 15 kDa (S(113)-R(250)) and 60 kDa (I(251)-S(777)) fragments consisting of an active form of PS1Ac2 were expressed as His-tag fusion. Upon purification under denaturing condition and refolding, the recombinant polypeptides were applied to cancer cells to analyze their cytotoxicities. 3-(4,5-Dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay revealed that either of 15 or 60 kDa polypeptide exhibited no cytotoxicity to HeLa cells, but they became cytotoxic upon mixed together. Our results suggested that PS1Ac2 was responsible for the cytotoxicity of B. thuringiensis B0462 strain, and that the formation of hetero-dimer of 15 and 60 kDa polypeptide was required for their cytotoxicity.
Transgenic tobacco transformed with the Trichoplusia ni granulovirus enhancin gene has been demon... more Transgenic tobacco transformed with the Trichoplusia ni granulovirus enhancin gene has been demonstrated to enhance baculovirus infection in larvae. In this paper we describe the effect of the long-term feeding of lyophilized transgenic tobacco material to Pseudaletia separata and Spodoptera exigua larvae. Our results demonstrated that the baculovirus enhancin gene products have potential for use in insect pest management.
Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is of great interest f... more Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is of great interest for developing a bioinsecticide to control mosquitoes. Therefore, it is very important to characterize the functional motif of Cry4Aa that is responsible for its mosquitocidal activity. In this study, to characterize a potential receptor binding site, namely, loops 1, 2, and 3 in domain II, we constructed a series of Cry4Aa mutants in which a residue in these three loops was replaced with alanine. A bioassay using Culex pipiens larvae revealed that replacement of some residues affected the mosquitocidal activity of Cry4Aa, but the effect was limited. This finding was partially inconsistent with previous results which suggested that replacement of the Cry4Aa loop 2 results in a significant loss of mosquitocidal activity. Therefore, we constructed additional mutants in which multiple (five or six) residues in loop 2 were replaced with alanine. Although the replacement of multiple residues a...
Ion channel proteins play important roles in various cell functions, making them attractive drug ... more Ion channel proteins play important roles in various cell functions, making them attractive drug targets. Artificial lipid bilayer recording is a technique used to measure the ion transport activities of channel proteins with high sensitivity and accuracy. However, the measurement efficiency is low. In order to improve the efficiency, we developed a method that allows us to form bilayers on a hydrogel bead and record channel currents promptly. We tested our system by measuring the activities of various types of channels, including gramicidin, alamethicin, α-hemolysin, a voltage-dependent anion channel 1 (VDAC1), a voltage- and calcium-activated large conductance potassium channel (BK channel), and a potassium channel from Streptomyces lividans (KcsA channel). We confirmed the ability for enhanced measurement efficiency and measurement system miniaturizion.
Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with an aerolysin-type arc... more Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with an aerolysin-type architecture, and it is expected to be used as a novel bioinsecticide. Cry46Ab acts as a functional pore-forming toxin, and characteristics of the resulting channel pores, including ion selectivity, have been analyzed. However, the relationship between channel-pore ion selectivity and insecticidal activity remains to be elucidated. To clarify the effects of charged amino acid residues on the ion permeability of channel-pores and the resulting insecticidal activity, in the present study, we constructed Cry46Ab mutants in which a charged amino acid residue within a putative transmembrane β-hairpin region was replaced with an oppositely charged residue. Bioassays using Culex pipiens mosquito larvae revealed that the mosquitocidal activity was altered by the mutation. A K155E Cry46Ab mutant exhibited toxicity apparently higher than that of wild-type Cry46Ab, but the E159K and E163K mutants exhibited decreased toxicity. Ions selectivity measurements demonstrated that the channel pores formed by both wild-type and mutant Cry46Abs were cation selective, and their cation preference was also similar. However, the degree of cation selectivity was apparently higher in channel pores formed by the K155E mutant, and reduced selectivity was observed with the E159K and E163K mutants. Our data suggest that channel-pore cation selectivity is a major determinant of Cry46Ab mosquitocidal activity and that cation selectivity can be controlled via mutagenesis targeting the transmembrane β-hairpin region. KEY POINTS: • Cry46Ab mutants were constructed by targeting the putative transmembrane β-hairpin region. • Charged residues within the β-hairpin control the flux of ions through channel pores. • Channel-pore cation selectivity is correlated with insecticidal activity.
In order to characterize the genome organization of Xestia c-nigrum granulovirus (XcGV), mapping ... more In order to characterize the genome organization of Xestia c-nigrum granulovirus (XcGV), mapping of putative XcGV genes was performed by construction of lambda and M13 phage libraries followed by Southern blot and nucleotide sequencing analyses. Mapping of the lambda (32 clones covering the entire XcGV genome) and M13 (133 clones made by random cloning) phage library clones was carried out by hybridization of the labeled lambda phage clone DNAs to 1) Southern blotted XcGV genomic DNA fragments cleaved with EcoRI, BamHI, or HindIII, and 2) dot blotted M13 clone DNAs. All 133 M13 clone DNAs were sequenced, and coding possibilities were investigated by computer-assisted homology search; in total, about 43 kb of the genome was sequenced. Amino acid sequence homology searches of 67 M13 clones suggested that these GV DNAs coded for previously characterized genes identified in nucleopolyhedroviruses (NPVs) and GVs. These 67 M13 clones were classified into 25 gene homolog groups (including ...
The Plutella xylostella granulovirus (PxGV) genome DNA was sequenced and the predicted open readi... more The Plutella xylostella granulovirus (PxGV) genome DNA was sequenced and the predicted open reading frames (ORFs) were compared to genes of the first-sequenced GV, Xestia c-nigrum GV (XcGV), and those from other baculoviruses and organisms. PxGV DNA has a size of 100,999 bp with a G + C content of 40.7%. The analysis predicted 120 ORFs with a size of 150 nucleotides or larger that showed minimal overlap. Blast searches followed by a comparison of ORF arrangement with those of completely sequenced baculovirus genomes showed the presence of 102 homologs to other genes in the database. Among them, 74 and 100 were homologous to genes of Autographa californica NPV (AcMNPV) and XcGV, respectively. A striking feature of the relationship between the genomes of PxGV and XcGV was the conservation of the order and orientation of homologous genes. Even though the XcGV genome is much larger than that of PxGV (178 vs 101 kb) and had many more predicted ORFs (181 vs 120) with an average amino acid sequence relatedness of 42%, the order and orientation of almost all homologous genes was conserved. The PxGV genome contained four homologous regions (hrs), each with 10 to 23 repeated sequences of 101 to 105 nucleotides containing a 15-bp imperfect palindrome in the center of the repeats.
Cystatin C is a cysteine protease inhibitor produced by a variety of human tissues. The blood con... more Cystatin C is a cysteine protease inhibitor produced by a variety of human tissues. The blood concentration of cystatin C depends on the glomerular filtration rate and is an endogenous marker of renal dysfunction. Recombinant cystatin C protein with high immunogenicity is therefore in demand for the diagnostic market. In this study, to establish an efficient production system, a synthetic cystatin C gene was designed and synthesized in accordance with the codon preference of Escherichia coli genes. Recombinant cystatin C was expressed as a fusion with a peptide-tag, 4AaCter, which facilitates formation of protein inclusion bodies in E. coli cells. Fusion with 4AaCter-tag dramatically increased the production level of cystatin C, and highly purified protein was obtained without the need for complicated purification steps. The purity and yield of the final product was estimated as 87 ± 5% and 7.1 ± 1.1 mg/l culture, respectively. The recombinant cystatin C prepared by our method was as reactive against anti-cystatin C antibodies as native human cystatin C. Our results suggest that protein production systems using 4AaCter-tag could be a powerful means of preparing significant amounts of antigen protein.
ABSTRACT Abstract Since the first report of Bacillus sotto by Ishiwata in 1901, thousands of rel... more ABSTRACT Abstract Since the first report of Bacillus sotto by Ishiwata in 1901, thousands of related papers about Bacillus thuringiensis have been documented. In the field of biocontrol of insect pests by this bacterium, after the initial discovery of several B. thuringiensis isolates specific for lepidopteran insects, the isolation of B. thuringiensis israelensis, specific to dipteran larvae by Goldberg and Margalit, and B. thuringiensis tenebrionis, specific to some group of coleopteran insects by Krieg et al. were epoch making advances. In 1992, Ohba et al. isolated B. thuringiensis ja ponensis strain Buibui, which was specific to only scarabaeid larvae. This isolate is the main target of our discussion in this review. These discoveries by which not only B. thuringiensis sciences, but also applied biological control strategies have been enriched, which inspirit us to screen novel isolates.On the other hand, the fields of molecular biology and biochemistry studies on the structural elucidation of toxin proteins and mechanism of action have also tremendously progressed. But the complete mechanism has yet to be solved. For instance, interaction between receptor proteins locating on the plasma membrane of insect midgut epithelial cells and insecticidal proteins has not been fully sketched- In a way, this field is still in chaos. Addressing these exciting and enigmatic subjects will eventually lead to the construction of sustainable agriculture in the 21st century.
Two novel parasporin (PS) genes were cloned from Bacillus thuringiensis B0462 strain. One was 100... more Two novel parasporin (PS) genes were cloned from Bacillus thuringiensis B0462 strain. One was 100 % identical even in nucleotide sequence level with that of parasporin-1Aa (PS1Aa1) from B. thuringiensis A1190 strain. The other (PS1Ac2) showed significant homology (99 % identity) to that of PS1Ac1 from B. thuringiensis 87-29 strain. The 15 kDa (S(113)-R(250)) and 60 kDa (I(251)-S(777)) fragments consisting of an active form of PS1Ac2 were expressed as His-tag fusion. Upon purification under denaturing condition and refolding, the recombinant polypeptides were applied to cancer cells to analyze their cytotoxicities. 3-(4,5-Dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay revealed that either of 15 or 60 kDa polypeptide exhibited no cytotoxicity to HeLa cells, but they became cytotoxic upon mixed together. Our results suggested that PS1Ac2 was responsible for the cytotoxicity of B. thuringiensis B0462 strain, and that the formation of hetero-dimer of 15 and 60 kDa polypeptide was required for their cytotoxicity.
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Papers by Tohru Hayakawa