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.     

Spectrophotometric quantification of lactic bacteria in alginate and control of cell release with chitosan coating

Lactococcus lactis ssp. cremoris was entrapped within a Ca-alginate matrix, and an in situ spectrophotometric method for monitoring cell population in calcium alginate beads described. The intracapsular cell population can be estimated by measuring the optical density of beads containing cells, using cell-free beads as reference, or by measuring absorbance of a liquified bead suspension. Alginate beads, and beads coated with chitosan type I, II, and I and II mixtures, were examined for cell release. Lower viscosity chitosan (type I) coatings reduced cell release by a factor of 100 from10⁵ cfu ml⁻¹ to 10³ cfu ml⁻¹ after 6 h of fermentation. Reuse of chitosan I coated alginate beads also showed a reduction in cell release by a factor of 100. Cell loading and initial cell growth within the beads greatly affected cell release. Reducing the initial cell release would lower the overall levels of cell release throughout the fermentation. Compared to non-immobilized cultures, a 20–40% reduction in the lactic acid production rate was observed for alginate beads and chitosan I coated alginate beads, respectively. This reduction can be compensated for by increasing the intracapsular cell loading during immobilization, or before the onset of fermentation.     

Generation of Lys-gamma 3-melanotropin from pro-opiomelanocortin 1-77 by a bovine intermediate lobe secretory vesicle membrane-associated aspartic protease and purified pro-opiomelanocortin converting enzyme

The ability of bovine intermediate lobe secretory vesicle membrane-associated enzyme(s) and purified, soluble paired basic residue-specific, pro-opiomelanocortin converting enzyme (Loh, Y.P., Parish, D. C., and Tuteja, R. (1985) J. Biol. Chem. 260, 7194-7205) to cleave bovine NH2-terminal pro-opiomelanocortin1-77 (N-POMC 1-77) was investigated. Purified pro-opiomelanocortin converting enzyme and an enzyme activity associated with the secretory vesicle membrane were shown to cleave bovine N-POMC1-77 to two major products: N-POMC1-49 and Lys-gamma 3-melanotropin (MSH), and one minor product, gamma 3-MSH. These products were identified by their retention times on high performance liquid chromatography, immunological characteristics, and for Lys-gamma 3-MSH, amino acid composition. The products generated indicate cleavage preferentially between Arg 49-Lys 50 of bN-POMC1-77 (where b indicates bovine), which is identical to the processing pattern found in the bovine intermediate lobe in situ. The membrane converting activity was shown to be stimulated by 5 mM Ca2+ and has a pH optimum of 4-5 and an inhibitor profile characteristic of an aspartic protease. This suggests that the membrane-associated enzyme involved is very similar or identical to the purified, soluble pro-opiomelanocortin converting enzyme, which has previously been reported to be an acidic, aspartic protease responsible for the initial steps of POMC processing. The results of this study lead to the proposal that the lack of processing of the Arg49-Lys50 site in POMC in the anterior lobe versus the intermediate lobe of the pituitary in vivo may be due to other regulatory mechanisms rather than invoking the existence in the intermediate lobe of another enzyme specific for this site, different from pro-opiomelanocortin converting enzyme.     

When the Background Matters: Using Scenarios from Integrated Assessment Models in Prospective Life Cycle Assessment

Prospective life cycle assessment (LCA) needs to deal with the large epistemological uncertainty about the future to support more robust future environmental impact assessments of technologies. This study proposes a novel approach that systematically changes the background processes in a prospective LCA based on scenarios of an integrated assessment model (IAM), the IMAGE model. Consistent worldwide scenarios from IMAGE are evaluated in the life cycle inventory using ecoinvent v3.3. To test the approach, only the electricity sector was changed in a prospective LCA of an internal combustion engine vehicle (ICEV) and an electric vehicle (EV) using six baseline and mitigation climate scenarios until 2050. This case study shows that changes in the electricity background can be very important for the environmental impacts of EV. Also, the approach demonstrates that the relative environmental performance of EV and ICEV over time is more complex and multifaceted than previously assumed. Uncertainty due to future developments manifests in different impacts depending on the product (EV or ICEV), the impact category, and the scenario and year considered. More robust prospective LCAs can be achieved, particularly for emerging technologies, by expanding this approach to other economic sectors beyond electricity background changes and mobility applications as well as by including uncertainty and changes in foreground parameters. A more systematic and structured composition of future inventory databases driven by IAM scenarios helps to acknowledge epistemological uncertainty and to increase the temporal consistency of foreground and background systems in LCAs of emerging technologies.     

Preclinical Assessment of Adjunctive tPA and DNase for Peritoneal Dialysis Associated Peritonitis

A major complication of peritoneal dialysis is the development of peritonitis, which is associated with reduced technique and patient survival. The inflammatory response elicited by infection results in a fibrin and debris-rich environment within the peritoneal cavity, which may reduce the effectiveness of antimicrobial agents and predispose to recurrence or relapse of infection. Strategies to enhance responses to antimicrobial agents therefore have the potential to improve patient outcomes. This study presents pre-clinical data describing the compatibility of tPA and DNase in combination with antimicrobial agents used for the treatment of PD peritonitis. tPA and DNase were stable in standard dialysate solution and in the presence of antimicrobial agents, and were safe when given intraperitoneally in a mouse model with no evidence of local or systemic toxicity. Adjunctive tPA and DNase may have a role in the management of patients presenting with PD peritonitis.