Expression of mosquitocidal crystal protein genes in non-insecticidal Bacillus thuringiensis subsp. israelensis

Bacillus thuringiensis NTB-1 isolated from soil samples in Korea produces ovoidal parasporal inclusions with proteins of approximately 24–40 kDa in size. Although serological study indicated that the isolate has a flagella (H) antigen identical with subsp. israelensis , it seemed to be non-insecticidal against Lepidoptera and Coleoptera as well as Diptera. To investigate the activity of non-insecticidal B. thuringiensis transformed with insecticidal crystal protein genes, cryIVD and cytA genes of B. thuringiensis subsp. morrisoni PG-14, highly toxic to mosquito larvae, were introduced into the isolate NTB-1. The expression of mosquitocidal crystal protein genes in NTB-1 was characterized by SDS–PAGE analysis and electron microscopy. The results showed that crystalline inclusions of host, CryIVD and CytA were stably expressed in the transformant. However, the mosquitocidal activity of transformant was similar to that of B. thuringiensis subsp. kurstaki Cry⁻ B harbouring cryIVD and cytA genes, demonstrating that a synergistic effect by an interaction of both introduced insecticidal and resident non-insecticidal crystal proteins was not observed.     

Water-holding capacity of a floating peat mat determines the survival and growth ofMenyanthes trifoliata L (bog bean) in an oligotrophic lake

Menyanthes trifoliata is a threatened and endangered species in Korea and USA. To assess the important ecological characteristics for its conservation and restoration, we measured the level of soluble reactive phosphorus (SRP) in the water, total N and C contents in the plants, and the water-holding capacity of the surrounding floating peat mat. These factors, which limit the success of aquatic macrophytes, were examined for their effects on the growth of this species. Leaflet areas and petiole lengths ranged from 6.1 cm² and 12.4 cm, respectively, at a water depth of 5 cm, to 22.7 cm² and 33.0 cm, respectively, at 40 cm. The peat mat was able to retain water at a depth of 5 to 45 cm. SRPs ranged from 10 to 46 ppb, while total C and N contents were between 0.62% (petiole) and 2.76% (leaf), and between 39.7% (petiole) and 43.8% (stem), respectively. The water-holding level was not correlated with SRP, and neither phosphorus nor nitrogen content affected plant growth. However, the capacity of the floating mat to hold water as the depth subsided was positively related to growth. This suggests that such mats are good tools for restoring populations ofM. trifoliata when materials with high water-holding capacities are used.     

Mammalian Hsp70 and Hsp110 proteins bind to RNA motifs involved in mRNA stability.

In this study, in vitro RNA binding by members of the mammalian 70-kDa heat shock protein (Hsp) family was examined. We show that Hsp/Hsc70 and Hsp110 proteins preferentially bound AU-rich RNA in vitro. Inhibition of RNA binding by ATP suggested the involvement of the N-terminal ATP-binding domain. By using deletion mutants of Hsp110 protein, a diverged Hsp70 family member, RNA binding was localized to the N-terminal ATP-binding domain of the molecule. The C-terminal peptide-binding domain did not bind RNA, but its engagement by a peptide substrate abrogated RNA binding by the N terminus of the protein. Interestingly, removal of the C-terminal alpha-helical structure or the alpha-loop domain unique to Hsp110 immediately downstream of the peptide-binding domain, but not both, resulted in considerably increased RNA binding as compared with the wild type protein. Finally, a 70-kDa activity was immunoprecipitated from RNA-protein complexes formed in vitro between cytoplasmic proteins of human lymphocytes and AU-rich RNA. These findings support the idea that certain heat shock proteins may act as RNA-binding entities in vivo to guide the appropriate folding of RNA substrates for subsequent regulatory processes such as mRNA degradation and/or translation.     

Secretion of Recombinant Pediocin PA-1 by Bifidobacterium longum, Using the Signal Sequence for Bifidobacterial α-Amylase

A recombinant DNA, encoding the chimeric protein of the signal sequence for bifidobacterial α-amylase mature pediocin PA-1, was introduced into Bifidobacterium longum MG1. Biologically active pediocin PA-1 was successfully secreted from the strain and showed bactericidal activity against Listeria monocytogenes and the same molecular mass as native pediocin PA-1.     

Nonequivalent binding sites in cystathionase. Nanosecond and steady fluorescence studies.

The analogs P-pyridoxyl-L-alanine and P-pyridoxyl-L-homoserine bind to the apoprotein of the enzyme cystathionase and inhibit the reactivation of enzymatic activity after addition of pyridoxyl-5-P. The binding of the inhibitors was monitored by measuring the fluorescence emitted by the P-pyridoxyl moiety at 395 nm (excitation 325 nm). The fluorometric titration results indicate the presence of nonequivalent binding sites in the apoprotein. A model based on two classes of independent binding sites fits the fluorometric data reasonably well. The presence of nonequivalent fluorescent sites in reduced cystathionase was also detected by nanosecond spectroscopy. In contrast to the model compound P-pyridoxyl-epsilon-lysine (tau equals 2.6 ns), the P-pyridoxyl residues of cystathionase display multiexponential fluorescence decay. Two fluorescence lifetimes (tau2 equals 4.1 ns and tau2 equals 15 ns) fit the deconvoluted decay results obtained by pulse fluorimetry. It is proposed that the P-pyridoxyl chromophores of reduced cystathionase have different environments.     

Interleukin-1-β and Tumor Necrosis Factor-α Increase Peripheral-Type Benzodiazepine Binding Sites in Cultured Polygonal Astrocytes

Peripheral-type benzodiazepine binding sites (PTBBS) are markedly increased in the injured CNS. Astrocytes appear to be the primary cell type which express increased PTBBS. Because certain cytokines within the injured CNS are potent mitogens for astrocytes, we examined the effects of two such cytokines, interleukin (IL)-1 beta and tumor necrosis factor (TNF), on PTBBS in cultured astrocytes using [3H]Ro 5-4864 as the specific ligand. Purified cultures of either polygonal or process-bearing astrocytes were prepared from neonatal rat cerebral hemispheres. At a concentration of 1.8 nM, specific binding of the radioactive ligand to polygonal astrocytes reached equilibrium within 60 min and was half-maximal by 5-10 min. By contrast, specific binding to process-bearing astrocytes barely exceeded background levels. IL-1 and TNF increased PTBBS within polygonal astrocytes in both dose- and time-dependent manners. At 10-50 ng/ml, IL-1 beta and TNF-alpha elevated [3H]Ro 5-4864 binding in polygonal astrocyte cultures 65 and 87%, respectively, above the level in control cultures. However, no changes in PTBBS were seen within polygonal astrocytes after IL-2 treatment. Scatchard analysis of saturation binding experiments suggested that the increase in PTBBS promoted by TNF was due to an increased number of binding sites present in polygonal astrocytes and not due to an increase in receptor affinity. Binding data suggested that PTBBS within cultures of process-bearing astrocytes were virtually absent irrespective of the treatment. These in vitro data suggest that certain cytokines found in the injured brain may be involved in up-regulating PTBBS within a particular subtype of astrocyte.     

Arsenite oxidation by a facultative chemolithotrophic bacterium SDB1 isolated from mine tailing

An arsenite (As[III])-oxidizing bacterium, SDB1, was isolated from mine tailing collected from the Sangdong mine area in Korea and showed chemolithotrophic growth on As[III] and CO₂ as the respective electron and carbon sources. SDB1 is Gram-negative, rod-shaped, and belongs to the Sinorhizobium-Ensifer branch of α-Proteobacteria. Growth and As[III] oxidation was enhanced significantly by the presence of yeast extract (0.005%) in minimal salt medium containing 5 mM As[III]; decreasing the doubling time from 9.8 to 2.1 h and increasing the As [III] oxidation rate from 0.014 to 0.349 pmol As [III] oxidized cell⁻¹ h⁻¹. As[III] oxidation nearly stopped at pH around 4 and should be performed at pH 7∼8 to be most effective. SDB1 was immobilized in calcium-alginate beads and the oxidation capacity was investigated. Specific As[III] oxidation rates obtained with SDB1 (10.1−33.7 mM As[III] oxidized g⁻¹ dry cell h⁻¹) were 10∼16-times higher than those reported previously with a heterotrophic bacterial strain (Simeonova et al., 2005). The stability and reusability of immobilized SDB1 strongly suggested that the immobilized SDB1 cell System can make the As[III] oxidation process technically and economically feasible in practical applications.