• BMB Reports
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• v.34 no.5
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• pp.402-407
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• 2001

Based on the currently proposed toxicity model for the different Bacillus thuringiensis Cry $\delta$-endotoxins, their pore-forming activity involves the insertion of the ${\alpha}4-{\alpha}5$ helical hairpin into the membrane of the target midgut epithelial cell. In this study, a number of polar or charged residues in helix 4 within domain I of the 65-kDa dipteranactive Cry11A toxin, Lys-123, Tyr-125, Asn-128, Ser-130, Gln-135, Arg-136, Gln-139 and Glu-141, were initially substituted with alanine by using PCR-based directed mutagenesis. All mutant toxins were expressed as cytoplasmic inclusions in Escherichia coli upon induction with IPTG. Similar to the wild-type protoxin inclusion, the solubility of each mutant inclusion in the carbonate buffer, pH 9.0, was relatively low When E. coli cells, expressing each of the mutant proteins, were tested for toxicity against Aedes aegypti mosquito-larvae, toxicity was completely abolished for the alanine substitution of arginine at position 136. However, mutations at the other positions still retained a high level of larvicidal activity Interestingly, further analysis of this critical arginine residue by specific mutagenesis showed that conversions of arginine-136 to aspartate, glutamine, or even to the most conserved residue lysine, also abolished the wild-type activity The results of this study revealed an important determinant in toxin function for the positively charged side chain of arginine-136 in helix 4 of the Cry11A toxin.

• BMB Reports
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• v.40 no.1
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• pp.58-64
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• 2007

Similar to the other known structures of Bacillus thuringiensis Cry $\delta$-endotoxins, the crystal structure of the 65-kDa activated Cry4Ba toxin comprises three domains which are, from the N- to C-terminus, a bundle of $\alpha$-helices, a three-$\beta$-sheet domain, and a $\beta$-sandwich. To investigate the properties of the C-terminal domain III in isolation from the rest of the toxin, the cloned Cry4Ba-domain III was over-expressed as a 21-kDa soluble protein in Escherichia coli, which cross-reacted with anti-Cry4Ba domain III monoclonal antibody. A highly-purified domain III was obtained in a monomeric form by ion-exchange and size-exclusion FPLC. Circular dichroism spectroscopy indicated that the isolated domain III fragment distinctly exists as a $\beta$-sheet structure, corresponding to the domain III structure embodied in the Cry4Ba crystal structure. In vitro binding analysis via immuno-histochemical assay revealed that the Cry4Ba-domain III protein was able to bind to the apical microvilli of the susceptible Stegomyia aegypti larval midguts, albeit at lower-binding activity when compared with the full-length active toxin. These results demonstrate for the first time that the C-terminal domain III of the Cry4Ba mosquito-larvicidal protein, which can be isolated as a native folded monomer, conceivably participates in toxin-receptor recognition.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.6
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• pp.423-427
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• 2004

A simple purification procedure of bioactive human granulocyte macrophage colony stimulating factor (hGM-CSF) secreted in rice cell suspension culture has previously been described. In this study the protein was purified to apparent homogeneity with an overall yield of $80.1\%$ by ammonium sulfate precipitation and a single chromatographic step involving FPLCanion exchange chromatography. The purified hGM-CSF revealed at least five glycosylated forms ranging from $21.5{\~}29$ kDa, and its biological activity was independent of the glycosylation pattern. This is the first purification report of recombinant hGM-CSF to apparent homogeneity from rice cell suspension cultures.

• Journal of Microbiology and Biotechnology
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• v.10 no.3
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• pp.287-292
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• 2000

Recombinant Aspergillus niger was constructed to express and secrete a biologically active murine granulaocyte macrophage-colony stimulating factor (mGM-CSF). A 500 bp fragment encoding the signal peptide and terminator of glyceraldehyde-3-phosphate dehydrogenase (gpd). The hygromycin phosphotrasferase gene (hph) was used as a selection marker for the fungal transformants. An expression vector was introduced into A. niger ATCC 9642, and a Northern blot analysis indicated the presence of a considerable amount of transcripts from the introduced mGM-CSF. The biological activity of recombinant mGM-CSF (rmGM-CSF) isolated from the culture filtrate was confirmend by measuring the proliferationof the GM-CSF dependent FDC-P1 cell line. It appeared that rmGM-CSF was amenable to the proteolytic activity produced by A. niger, since biological actibity was only observed when the transformants were grown in a protease-repressing medium, and the activity of rmGM-CSF dramatically decreased with an increase of age of the culture. The yield of rmGM-CSF, as determined by ELISA. was 640 ng/l of culture filtrate. Accordingly, its specific activity is estimated to be approximately two-and-a-half times higher than that of a commercial preparation from E. coli.

• International Journal of Industrial Entomology and Biomaterials
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• v.11 no.2
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• pp.119-124
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• 2005

To determine the level of heterosis, higher cocoon shell weight multivoltine congenic lines (Con. L) and bivoltine syngenic lines (Syn. L) of silkworm were used for crosses. First filial generations $(F_1s)$ expressed heterobeltiotic genetic interaction at significant magnitude (p < 0.01) for single cocoon shell weight (SCSW). The other linked characters viz., single cocoon weight (SCW) and yield by weight per 10, 000 larvae were also significantly higher (p < 0.01) than the better parental lines. All the hybrids showed significant improvement for these aforesaid characters over standard heterosis (Standard check). The reeling parameters viz., filament length, raw silk, neatness, cohesionstrokes etc, also showed improvement among the hybrids than check in congenial environment. Overall results suggested that the cross between congenic and syngenic lines provide better heterosis with good quality silk than conventional hybrids and may be used for commercial exploitation.

• The Korean Journal of Mycology
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• v.25 no.1 s.80
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• pp.26-29
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• 1997

Pyrimidine auxotrophic basidiospores of Pleurotus sajor-caju were selected using positive selection method. Wild type basidiospores could not grow on minimal medium containing the pyrimidine analog 5'-fluoro-orotic acid (5'-FOA) whereas pyrimidine auxotrophs grew normally. After treatment of basidiospores with ultraviolet light, a total of 13 pyrimidine auxotrophic basidiospores were isolated among 24 5'-FOA resistant mutants. These mutants require the pyrimidine such as uracil, cytosine, thymine. Mating type group and growth rate of their mutants were determined.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.2
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• pp.177-180
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• 2008

In this study, cattle microsatellite markers recommended for diversity studies of cattle by the EU AIRE 2066 Concerted Action Group were used to study the genetic diversity of 105 Thai swamp buffalo which were randomly selected from eight different research stations of the Department of Livestock Development, Thailand. Of 34 primer pairs, 16 were successfully amplified while the rest showed non-specific amplification. The lowest number of alleles was two while the highest was nine, with an average of 4.7 alleles per locus. The average unbiased heterozygosity for all eight populations was 0.5233, with a low of 0.4772 (Samui) and a high of 0.5616 (Burirum). The genetic distance ranged from 0.0574 to 0.2575. Populations from Lopburi and Burirum showed the closest relationship, whereas Srisagat and Samui were the most divergent. The results generated with the primers recommended by the EU AIRE 2066 Concerted Action Group are at a slight variance from our previous study, possibly as a result of the number of specific amplification products obtained, suggesting that cattle markers may not be optimal for studies of the genetic diversity of the Thai swamp buffalo.

• Molecules and Cells
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• v.26 no.5
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• pp.496-502
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• 2008

Cryparin, encoded as a single copy gene (Crp) of the chestnut blight fungus Cryphonectria parasitica, is the most abundant protein produced by this fungus. However, its accumulation is decreased remarkably in C. parastica strains containing the double-stranded (ds) RNA virus Cryphonectria hypovirus 1. To characterize the transcriptional regulatory element(s) for strong expression and viral regulation, promoter analysis was conducted. Serial deletion of the Crp promoter region resulted in a step-wise decrease in promoter activity, indicating a localized distribution of genetic elements in the cryparin promoter. Promoter analysis indicated two positive and a repressive cis-acting elements. Among them, the promoter region between nt -1,282 and -907 appeared to be necessary for hypoviral-mediated down-regulation. An electrophoretic mobility shift assay (EMSA) on the corresponding promoter region (-1,282/-907) indicated two regions at (-1,257/-1,158) and (-1,107/-1,008) with the characteristic AGGAGGA-N42-GAGAGGA and its inverted repeat TCCTCTC-N54-TCCTCCT, respectively, appeared to be specific binding sites for cellular factors.

• BMB Reports
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• v.33 no.4
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• pp.294-299
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• 2000

Proteolytic processing of the dengue virus serotype 2 polyprotein precursor is catalyzed by a host signal peptidase and a virus encoded two-component protease consisting of the nonstructural proteins, NS2B and NS3. We expressed in Escherichia coli the NS2B, NS3(pro) and NS2B-NS3 proteins from the dengue virus type 2 strain 16681 as N-terminal fusions with a hexahistidine affinity tag under the control of the inducible trc promoter. All fusion proteins were purified to >90% purity by detergent extraction of inclusion bodies and a single step metal chelate chromatography. Proteins were refolded on-column and recovered with yields of 0.5, 6.0 and 1.0 mg/l of E. coli culture that was grown to $OD_{600}=1.0$ for NS2B, NS3(pro) and NS2B-NS3, respectively. Purified proteins gave strong signals in Western blots using $Ni^{2+}-nitrilotriacetic$ acid as a probe for the presence of the polyHis tag. During the purification process, $(His)_{6}NS2B-NS3$ was apparently not autoproteolytically cleaved at the NS2B/NS3 site.

• BMB Reports
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• v.47 no.9
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• pp.506-511
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• 2014

We investigated the effects of high calorie and low calorie diets on skeletal integrity, and whether ${\beta}$-adrenergic blockade (BB) attenuates bone loss induced by dietary calorie alteration. Male 6-week-old C57BL/6 mice were assigned to either an ad-lib fed control diet (CON), a high calorie diet (HIGH), or a low calorie diet (LOW) group. In each diet group, mice were treated with either vehicle (VEH) or propranolol, a ${\beta}$-adrenergic antagonist. Over 12-weeks, ${\beta}$-blockade mitigated body weight and fat mass increases induced by the high calorie diet. Femoral trabecular bone mineral density and the expression levels of osteogenic marker genes in bone marrow cells were reduced in HIGHVEH and LOWVEH mice, and BB significantly attenuated this decline only in HIGH mice. In summary, the magnitude of bone loss induced by low calorie diet was greater than that caused by high calorie diet in growing mice, and ${\beta}$-blockade mitigated high calorie diet-induced bone loss.