Effect of shear stress on cultivation of Bacillus thuringiensis for thuringiensin production

Cultivation of Bacillus thuringiensis for thuringiensin production is a mixed-growth-associated system. Cultivation conditions should be different during the cell growth stage and production stage. In this study, agitation speed and aeration rate were varied during the exponential growth phase and stationary phase in order to investigate the effect of shear stress via agitation on cultivation of B. thuringiensis for thuringiensin production. It was found that shear stress had a significant effect on thuringiensin production during the stationary phase. By decreasing the agitation speed during the stationary phase, product formation was increased up to 43%.     

Somatic cell nuclear transfer in the pig: control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy

To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic embryos.     

Comparative structure analysis of tyrosine and valine residues in unprocessed silk fibroin (silk I) and in the processed silk fiber (silk II) from Bombyx mori using solid-state (13)C,(15)N, and (2)H NMR

The solid-state (13)C CP-MAS NMR spectra of biosynthetically labeled [(13)C(alpha)]Tyr, [(13)C(beta)]Tyr, and [(13)C(alpha)]Val silk fibroin samples of Bombyx mori, in silk I (the solid-state structure before spinning) and silk II (the solid-state structure after spinning) forms, have been examined to gain insight into the conformational preferences of the semicrystalline regions. To establish the relationship between the primary structure of B. mori silk fibroin and the "local" structure, the conformation-dependent (13)C chemical shift contour plots for Tyr C(alpha), Tyr C(beta), and Val C(alpha) carbons were generated from the atomic coordinates of high-resolution crystal structures of 40 proteins and their characteristic (13)C isotropic NMR chemical shifts. From comparison of the observed Tyr C(alpha) and Tyr C(beta) chemical shifts with those predicted by the contour plots, there is strong evidence in favor of an antiparallel beta-sheet structure of the Tyr residues in the silk fibroin fibers. On the other hand, Tyr residues take a random coil conformation in the fibroin film with a silk I form. The Val residues are likely to assume a structure similar to those of Tyr residues in silk fiber and film. Solid-state (2)H NMR measurements of [3,3-(2)H(2)]Tyr-labeled B. mori silk fibroin indicate that the local mobility of the backbone and the C(alpha)-C(beta) bond is essentially "static" in both silk I and silk II forms. The orientation-dependent (i.e., parallel and perpendicular to the magnetic field) solid-state (15)N NMR spectra of biosynthetically labeled [(15)N]Tyr and [(15)N]Val silk fibers reveal the presence of highly oriented semicrystalline regions.     

Structural analysis of an insect lysozyme exhibiting catalytic efficiency at low temperatures

Bombyx mori lysozyme (BmLZ), from the silkworm, is an insect lysozyme. BmLZ has considerable activity at low temperatures and low activation energies compared with those of hen egg white lysozyme (HEWLZ), according to measurements of the temperature dependencies of relative activity (lytic and glycol chitin) and the estimation of activation energies using the Arrhenius equation. Being so active at low temperatures and low activation energies is characteristic of psychrophilic (cold-adapted) enzymes. The three-dimensional structure of BmLZ has been determined by X-ray crystallography at 2.5 A resolution. The core structure of BmLZ is similar to that of c-type lysozymes. However, BmLZ shows some distinct differences in the two exposed loops and the C-terminal region. A detailed comparison of BmLZ and HEWLZ suggests structural rationalizations for the differences in the catalytic efficiency, stability, and mode of activity between these two lysozymes.     

Regulation of yeast actin behavior by interaction of charged residues across the interdomain cleft

His(73) participates in the regulation of the nucleotide binding cleft conformation in yeast actin. Earlier molecular dynamics studies suggested that Asp(184) interacts with His(73) thereby stabilizing a "closed-cleft" G-actin. However, beta-actin in the open-cleft state shows a closer interaction of His(73) with Asp(179) than with Asp(184). We have thus assessed the relative importance of Asp(184) and Asp(179) on yeast actin stability and function. Neutral substitutions at 184 or 179 alone had little adverse effect on the monomer and polymerization behavior of actin. Arg or His at 184 in H73E actin partially rescued the monomeric properties of H73E actin, as demonstrated by near-normal thermostability and wild-type (WT)-like protease digestion patterns. ATP exchange was still considerably faster than with WT-actin although slower than that of H73E alone. However, polymerization of H73E/D184R and H73E/D184H is worse than with H73E alone. Conversely, D179R rescued all monomeric properties of H73E to near WT values and largely restored polymerization rate and filament thermostability. These results and new simulations of G-actin in the "open" state underscore the importance of the His(73)-Asp(179) interaction and suggest that the open and not the closed state of yeast actin may be favored in the absence of the methyl group of His(73).     

The action of Cu2+ on Bacillus thuringiensis growth investigated by microcalorimetry

By using an LKB-2277 Bioactivity Monitor, ampoule mode, the heat output of Bacillus thuringiensis growth metabolism has been determined at 28 degrees C and effect of Cu2+ on B. thuringiensis growth was studied. Copper has been regarded as an essential trace element for life. Its deficiency may be the cause of diseases. Cu2+ of different concentration have different effects on B. thuringiensis growth metabolism, Cu2+ of low concentration (0-30 micrograms/ml) can promote the growth of B. thuringiensis, and Cu2+ of high concentration (40-120 micrograms/ml) is able to inhibit its growth and B. thuringiensis can't grow at all when the concentration of Cu2+ is up to 130 micrograms/ml.     

Epigenetic regulation of Igf2/H19 imprinting at CTCF insulator binding sites

The mouse insulin-like growth factor II (Igf2) and H19 genes are located adjacent to each other on chromosome 7q11-13 and are reciprocally imprinted. It is believed that the allelic expression of these two genes is regulated by the binding of CTCF insulators to four parent-specific DNA methylation sites in an imprinting control center (ICR) located between these two genes. Although monoallelically expressed in peripheral tissues, Igf2 is biallelically transcribed in the CNS. In this study, we examined the allelic DNA methylation and CTCF binding in the Igf2/H19 imprinting center in CNS, hypothesizing that the aberrant CTCF binding as one of the mechanisms leads to biallelic expression of Igf2 in CNS. Using hybrid F1 mice (M. spretus males x C57BL/6 females), we showed that in CNS, CTCF binding sites in the ICR were methylated exclusively on the paternal allele, and CTCF bound only to the unmethylated maternal allele, showing no differences from the imprinted peripheral tissues. Among three other epigenetic modifications examined, histone H3 lysine 9 methylation correlated well with Igf2 allelic expression in CNS. These results suggest that CTCF binding to the ICR alone is not sufficient to insulate the Igf2 maternal promoter and to regulate the allelic expression of the gene in the CNS, thus challenging the aberrant CTCF binding as a common mechanism for lack of Igf2 imprinting in CNS. Further studies should be focused on the identification of factors that are involved in histone methylation and CTCF-associated factors that may be needed to coordinate Igf2 imprinting.     

Bioactive sucrose esters from Bidens parviflora

An investigation on Bidens parviflora led to the isolation of three sucrose esters and a substituted truxillate. Their structures were elucidated as (6-O-(E)-p-coumaroyl)-beta-D-fructofuranosyl-(2-->1)-alpha-D-glucopyranoside, (6-O-(E)-p-coumaroyl)-beta-D-fructofuranosyl-(2-->1)-(6-O-(E)-p-coumaroyl)-alpha-D-glucopyranoside II, 6,6'-sucrose ester of (1alpha,2alpha,3beta,4beta)-3,4-bis(4-hydroxyphenyl)-1,2-cyclobutanedicarboxylic acid, dimethyl ester of (1alpha,2alpha,3alpha,4alpha)-2,4-bis(3,4-dihydroxyphenyl)-1,3-cyclobutanedicarboxylic acid on the basis of spectral and chemical evidence. These compounds were subjected to the following bioassays: the histamine release inhibition of rat mast cells induced by antigen-antibody reaction and the inhibitory activity of PGE(2) production by macrophages.