Secretory expression in Escherichia coli and single-step purification of a heat-stable alkaline protease

Bacteriocin release proteins (BRPs) can be used for the release of heterologous proteins from the Escherichia coli cytoplasm into the culture medium. The gene for a highly thermostable alkaline protease was cloned from Bacillus stearothermophilus F1 by the polymerase chain reaction. The recombinant F1 protease was efficiently excreted into the culture medium using E. coli XL1-Blue harboring two vectors: pTrcHis bearing the protease gene and pJL3 containing the BRPs. Both vectors contain the E. coli lac promoter-operator system. In the presence of 40 microM IPTG, the recombinant F1 protease and the BRP were expressed and mature F1 protease was released into the culture medium. This opens the way for the large-scale production of this protease in E. coli. The recombinant enzyme was purified through a one-step heat treatment at 70 degrees C for 3h and this method purified the protease to near homogeneity. The purified enzyme showed a pH optimum of 9.0, temperature optimum of 80 degrees C, and was stable at 70 degrees C for 24h in the pH range from 8.0 to 10.0. The enzyme exhibited a high degree of thermostability with a half-life of 4 h at 85 degrees C, 25 min at 90 degrees C, and was inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF).     

Isolation and characterisation of tilapia beta-actin promoter and comparison of its activity with carp beta-actin promoter

The regulatory sequence including proximal promoter, untranslated exon 1 and intron 1 of the beta-actin gene from tilapia (Oreochromis niloticus) has been isolated and spliced to a beta-galactosidase reporter gene to test its activity. Comparisons of promoter activity have been carried out with three different constructs: (1) 1.6 kb tilapia beta-actin regulatory sequence, (2) 1.5 kb carp beta-actin regulatory sequence, and (3) 4.7 kb carp beta-actin regulatory sequence. Although the 1.6 kb tilapia beta-actin regulatory sequence gave slightly different expression patterns in tilapia embryos assayed by in situ X-gal staining, no difference was observed in expression level when the tilapia sequence was compared with the 4.7 kb carp beta-actin regulatory sequence by quantitative assay. In comparison with the 1.5 kb carp beta-actin regulatory sequence, the 1.6 kb tilapia beta-actin regulatory sequence gave higher expression levels in tilapia embryos, while a reverse result was observed in zebrafish embryos. In cell transfection experiments, the 1.6 kb tilapia beta-actin regulatory sequence showed three to four times better activity in blue gill cells than either the 4.7 kb carp beta-actin or the 1.5 kb carp beta-actin regulatory sequences. The 1.6 kb tilapia beta-actin regulatory sequence also drove higher reporter gene activity in somatic cells of tilapia than did the 4.7 kb carp beta-actin regulatory sequence following direct injection of constructs into muscle. Therefore, taken together, the data demonstrate that the tilapia beta-actin promoter can be used as an efficient regulatory sequence to produce autotransgenic tilapia.     

Anthraquinones production,hydrogen peroxide level and antioxidant vitamins in Morinda elliptica cell suspension cultures from intermediary and production medium strategies

The effects of medium strategies [maintenance (M), intermediary (G), and production (P) medium] on cell growth, anthraquinone (AQ) production, hydrogen peroxide (H₂O₂) level, lipid peroxidation, and antioxidant vitamins in Morinda elliptica cell suspension cultures were investigated. These were compared with third-stage leaf and 1-month-old callus culture. With P medium strategy, cell growth at 49 g l^–1, intracellular AQ content at 42 mg g^–1 DW, and H₂O₂ level at 9 mol g^–1 FW medium were the highest as compared to the others. However, the extent of lipid peroxidation at 40.4 nmol g^–1 FW and total carotenoids at 13.3 mg g^–1 FW for cultures in P medium were comparable to that in the leaf, which had registered sevenfold lower AQ and 2.2-fold lower H₂O₂ levels. Vitamin C content at 30–120 g g^–1 FW in all culture systems was almost half the leaf content. On the other hand, vitamin E content was around 400–500 g g^–1 FW in 7-day-old cultures from all medium strategies and reduced to 50–150 g g^–1 FW on day 14 and 21; as compared to 60 g g^–1 FW in callus and 200 g g^–1 FW in the leaf. This study suggests that medium strategies and cell growth phase in cell culture could influence the competition between primary and secondary metabolism, oxidative stresses and antioxidative measures. When compared with the leaf metabolism, these activities are dynamic depending on the types and availability of antioxidants.Abbreviations AQ Anthraquinone - DW Dry cell weight - FW Fresh cell weight - G Intermediary medium - M Maintenance medium - MDA Malondialdehyde - P Production medium - ROS Reactive oxygen species - TBA Thiobarbituric acid - t_d Doubling time     

Asian Tapirs Are No Elephants When It Comes To Seed Dispersal

The elimination of the largest herbivores (elephants and rhinoceroses) from many forests in tropical East Asia may have severe consequences for plant species that depend on them for seed dispersal. We assessed the capacity of Malayan tapirs Tapirus indicus—the next largest nonruminant herbivore in the region—as a substitute for the lost megafauna in this role by studying their ability to disperse the seeds of nine fleshy‐fruited plants with seeds 5–97 mm in length. We combined information from feeding trials, germination tests, and field telemetry to assess the effect of tapir consumption on seed viability and to estimate how far the seeds would be dispersed. The tapirs (N=8) ingested few seeds. Seed survival through gut passage was moderately high for small‐seeded plants (e.g., 36.9% for Dillenia indica) but very low for medium‐ (e.g., 7.6% for Tamarindus indica) and large‐seeded (e.g., 2.8% for Artocarpus integer) plants. Mean seed gut passage times were long (63–236 h) and only the smallest seeds germinated afterwards. Using movement data from four wild tapirs in Peninsular Malaysia we estimated mean dispersal distances of 917–1287 m (range=22–3289 m) for small‐seeded plants. Malayan tapirs effectively dispersed small‐seeded plants but acted as seed predators for the large‐seeded plants included in our study, suggesting that they cannot replace larger herbivores in seed dispersal. With the absence of elephants and rhinos many megafaunal‐syndrome plants in tropical East Asia are expected to face severe dispersal limitation problems.     

Effect of phenotypic switching on the biological properties and susceptibility to chlorhexidine in Candida krusei ATCC 14243

Phenotypic switching is characterized as a virulence factor of Candida spp. This study was carried out to evaluate the phenotypic switching ability of C.?krusei ATCC 14243 and to determine its effect on the biological properties, adherence capacity and susceptibility towards chlorhexidine digluconate (CHX). To induce switched generations C.?krusei was cultured under nitrogen-depleted growth conditions by adding phloxine B. These phenotypically switched colonies were designated as the 1st generation. Subsequent sub-culturing was performed to produce the 2nd, 3rd and 4th switched generations. The recovery of the 3rd generation was the highest at 85.7% while that of the 4th generation was lower at 70.8%, and the recovery of the 1st and 2nd generations gradually reduced to 46.6% and 36.4%, respectively. All generations of C.?krusei were susceptible towards CHX. The unswitched C.?krusei was the most susceptible but the least adherent to coated hard surfaces. The 2nd generation was the least susceptible, but with the highest adherent ability. The minimum inhibition concentration and minimal fungicidal concentration of C.?krusei of all generations were determined at 0.4?mg?mL(-1) . These observations suggest that the switching activity of C.?krusei induces changes to its biological properties and susceptibility towards CHX.     

Semisynthesis and cytotoxic activities of andrographolide analogues

Andrographolide 1, a diterpenoid lactone of the plant Andrographis paniculata, known to possess antitumour activity in in vitro and in vivo breast cancer models was subjected to semisynthesis leading to the preparation of a number of novel compounds. These compounds exhibited in vitro antitumour activity with moderate to excellent growth inhibition against MCF-7 (breast) and HCT-116 (colon) cancer cells. Compounds 3,19-(2-chlorobenzylidene)andrographolide(5), 3,19-(3-chlorobenzylidene)andrographolide(6), 3,19-(3-fluorobenzylidene) andrographolide(7), 3,19-(4-fluorobenzylidene)andrographolide(8), 3,19-(2-fluorobenzylidene)andrographolide(10), 3,19-(2-chloro-5-nitrobenzylidene)andrographolide (21), 3,19-(4-chlorobenzylidene)andrographolide(30) and 3,19-(2-chloro-4-fluorobenzylidene) andrographolide(31) were also screened against 60 NCI (National Cancer Institute, USA) human tumour cell lines derived from nine cancer cell types.