Nonisothermal bioreactors in the treatment of vegetation waters from olive oil: laccase versus syringic acid as bioremediation model

Laccase from Trametes versicolor was immobilized by diazotization on a nylon membrane grafted with glycidil methacrylate, using phenylenediamine as spacer and coupling agent. The behavior of these enzyme derivatives was studied under isothermal and nonisothermal conditions by using syringic acid as substrate, in view of the employment of these membranes in processes of detoxification of vegetation waters from olive oil mills. The pH and temperature dependence of catalytic activity under isothermal conditions has shown that these membranes can be usefully employed under extreme pH and temperatures. When employed under nonisothermal conditions, the membranes exhibited an increase of catalytic activity linearly proportional to the applied transmembrane temperature difference. Percentage activity increases ranging from 62% to 18% were found in the range of syringic acid concentration from 0.02 to 0.8 mM, when a difference of 1 degrees C was applied across the catalytic membrane. Because the percentage activity increase is strictly related to the reduction of the production times, the technology of nonisothermal bioreactors has been demonstrated to be an useful tool also in the treatment of vegetation waters from olive oil mills.     

Improved PC1/3 production through recombinant expression in insect cells and larvae

Protein convertase 1/3 is a serine endoproteinase present in the regulated secretory pathway of endocrine and neuroendocrine cells. It is responsible for the processing of numerous prohormones and proneuropeptides into their biologically active moieties, often following cleavage at pairs of basic residues. The determination of its three-dimensional structure, as well as the understanding of its enzymatic properties, would greatly benefit from the production and availability of large amounts of recombinant enzyme. We report herein improvements in the production of PC1/3 by expressing recombinant mutated forms in both insect cells (Spodoptera frugiperda, Sf9 cells) and larvae (Trichoplusia ni commonly referred to as cabbage looper). On one hand, we deleted the last 135 COOH-terminal residues of mPC1/3 and, on the other hand, we replaced the signal peptide of mPC1/3 by the viral glycoprotein gp67 signal peptide. These modifications were shown to improve markedly (up to 125%) the secretion into the Sf9 cells medium and the amount of enzymatic activity recovered when compared to the original vector. Moreover, intracoelemic injection of the vectors into insect larvae led to the production and purification of enzymatically active enzyme at a level of 30 microg/larva in the case of mPC1/3 and to the production of a high amount of another enzymatically active convertase, PC7. The optimal viral titer for infection of larvae was determined to be 10(6)pfu/ml. Taking into account the purification protocol combined with the ease and efficiency of using larvae, it should now be possible to meet the needs for biochemical and structural studies.     

A cohort study of thyroid cancer and other thyroid diseases after the Chornobyl accident: objectives, design and methods

The thyroid gland in children is one of the organs that is most sensitive to external exposure to X and gamma rays. However, data on the risk of thyroid cancer in children after exposure to radioactive iodines are sparse. The Chornobyl accident in Ukraine in 1986 led to the exposure of large populations to radioactive iodines, particularly (131)I. This paper describes an ongoing cohort study being conducted in Belarus and Ukraine that includes 25,161 subjects under the age of 18 years in 1986 who are being screened for thyroid diseases every 2 years. Individual thyroid doses are being estimated for all study subjects based on measurement of the radioactivity of the thyroid gland made in 1986 together with a radioecological model and interview data. Approximately 100 histologically confirmed thyroid cancers were detected as a consequence of the first round of screening. The data will enable fitting appropriate dose-response models, which are important in both radiation epidemiology and public health for prediction of risks from exposure to radioactive iodines from medical sources and any future nuclear accidents. Plans are to continue to follow-up the cohort for at least three screening cycles, which will lead to more precise estimates of risk.     

Deriving haplotypes through recombination and gene conversion pathways

Retracing the trajectories of past genetic events is crucial to understand the structure of the genome, both in individuals and across populations. A haplotype describes a string of polymorphic sites along a DNA segment. Haplotype diversity is due to mutations creating new variants, and to recombinations and gene conversions that mix and redistribute these variants among individual chromosomes in populations. A number of studies have revealed a relatively simple pattern of haplotype diversity in the human genome, dominated by a few common haplotypes representing founder ancestral ones. New haplotypes are usually rare and have a limited geographic distribution. We propose a method to derive a new haplotype from a set of putative ancestral haplotypes, once mutations in place, through minimal recombination and gene conversion pathways. We describe classes of pathways that represent the whole set of minimal pathways leading to a new haplotype. We show that obtaining this set of pathways can be represented as a problem of finding "secondary structures" of minimum energy. We present a polynomial algorithm solving this folding problem.     

Structural basis of ligand recognition by PABC, a highly specific peptide-binding domain found in poly(A)-binding protein and a HECT ubiquitin ligase

The C-terminal domain of poly(A)-binding protein (PABC) is a peptide-binding domain found in poly(A)-binding proteins (PABPs) and a HECT (homologous to E6-AP C-terminus) family E3 ubiquitin ligase. In protein synthesis, the PABC domain of PABP functions to recruit several translation factors possessing the PABP-interacting motif 2 (PAM2) to the mRNA poly(A) tail. We have determined the solution structure of the human PABC domain in complex with two peptides from PABP-interacting protein-1 (Paip1) and Paip2. The structures show a novel mode of peptide recognition, in which the peptide binds as a pair of beta-turns with extensive hydrophobic, electrostatic and aromatic stacking interactions. Mutagenesis of PABC and peptide residues was used to identify key protein-peptide interactions and quantified by isothermal calorimetry, surface plasmon resonance and GST pull-down assays. The results provide insight into the specificity of PABC in mediating PABP-protein interactions.     

Impact of two fluorescent pseudomonads and an arbuscular mycorrhizal fungus on tomato plant growth,root architecture and P acquisition

The ability of fluorescent pseudomonads and arbuscular mycorrhizal fungi (AMF) to promote plant growth is well documented but knowledge of the impact of pseudomonad-mycorrhiza mixed inocula on root architecture is scanty. In the present work, growth and root architecture of tomato plants (Lycopersicon esculentum Mill. cv. Guadalete), inoculated or not with Pseudomonas fluorescens 92rk and P190r and/or the AMF Glomus mosseae BEG12, were evaluated by measuring shoot and root fresh weight and by analysing morphometric parameters of the root system. The influence of the microorganisms on phosphorus (P) acquisition was assayed as total P accumulated in leaves of plants inoculated or not with the three microorganisms. The two bacterial strains and the AMF, alone or in combination, promoted plant growth. P. fluorescens 92rk and G. mosseae BEG12 when co-inoculated had a synergistic effect on root fresh weight. Moreover, co-inoculation of the three microorganisms synergistically increased plant growth compared with singly inoculated plants. Both the fluorescent pseudomonads and the myco-symbiont, depending on the inoculum combination, strongly affected root architecture. P. fluorescens 92rk increased mycorrhizal colonization, suggesting that this strain is a mycorrhization helper bacterium. Finally, the bacterial strains and the AMF, alone or in combination, improved plant mineral nutrition by increasing leaf P content. These results support the potential use of fluorescent pseudomonads and AMF as mixed inoculants for tomato and suggest that improved tomato growth could be related to the increase in P acquisition.     

Thermal denaturation: is solid-state fermentation really a good technology for the production of enzymes?

The potential for thermal denaturation to cause enzyme losses during solid-state fermentation processes for the production of enzymes was examined, using the protease of Penicillium fellutanum as a model system. The frequency factor and activation energies for the first-order denaturation of this enzyme were determined as 3.447 x 10(59) h(-1) and 364,070 Jmol(-1), respectively. These values were incorporated into a mathematical model of enzyme deactivation, which was used to investigate the consequences of subjecting this protease to temporal temperature profiles reported in the literature for mid-height in a 34 cm high packed-bed bioreactor of 150 mm diameter. In this literature source, temperature profiles were measured for 5, 15 and 25 liters per minute of air and enzyme activities were measured as a function of time. The enzyme activity profiles predicted by the model were distributed similarly, one relative to the other, as had been found in the experimental study, with substantial amounts of denaturation being predicted when the substrate temperature exceeded 40 degrees C, which occurred at the lower two airflow rates. A mathematical model of a well-mixed bioreactor was used to explore the difficulties that would be faced at large scale. It suggests that even with airflows as high as one volume per volume per minute, up to 85% of the enzyme produced by the microorganism can be denatured by the end of the fermentation. This work highlights the extra care that must be taken in scaling up solid-state fermentation processes for the production of thermolabile products.     

Production of Botrytis cinerea for potential introduction into a vineyard

Botrytis cinerea was produced in solid-phase fermentation, liquid fermentation and on potato dextrose agar. Stored products were evaluated for grape colonization in grape bioassays and in field trials, and for B. cinerea density using colony forming unit analyses and a nucleic-acid-based method. B. cinerea colony forming unit density was significantly correlated to the probability of successful grape colonization in grape bioassays (p-value=0.0002). Solid fermentation products could be stored longer than liquid fermentation and potato dextrose agar products. There was little difference in the rate of grape colonization in laboratory bioassays among solid-phase fermentation, liquid fermentation and plate culture products. Although the initial B. cinerea colonization rate of field grapes was slightly greater on vines treated with solid-phase fermentation and plate culture products compared to vines treated with product from liquid fermentation, there was no significant difference in final colonization between vines treated with solid-phase fermentation, liquid fermentation and plate culture products and untreated vines.     

Special management of insulin lispro in continuous subcutaneous insulin infusion in young diabetic children: a randomized cross-over study

OBJECTIVE: To compare the safety, efficacy and management of insulin lispro (LP) with regular human insulin (RH) in young diabetic children treated with continuous subcutaneous insulin infusion (CSII). STUDY DESIGN: 27 very young diabetic children (age 4.6 +/- 2.2 years) treated with CSII participated in an open-label, randomized cross-over multicenter study comparing 2 periods of 16 weeks of CSII with LP or RH. RESULTS: Mean daily basal rate was significantly higher during the LP period (p = 0.04). No differences were seen in changes in HbA1c levels, number of hypoglycemic events, cutaneous infections and catheter occlusions. There was no significant difference between the two treatments for preprandial and postprandial glucose values, although prandial glucose excursions tended to be lower with LP (significant at dinner, p = 0.01). Mean blood glucose levels were significantly higher at 0.00 and 3.00 a.m. during LP therapy (p < 0.05). No episode of ketoacidosis occurred during LP treatment. More parents indicated that LP made their own and the child's daily life easier (p = 0.02) and preferred LP (p = 0.01). CONCLUSIONS: LP in CSII therapy in children is safe, as effective as RH, improved postprandial excursions, met the needs of young children in their daily life well, and gained their parents' satisfaction and preference. However, a shorter duration of LP resulted in hyperglycemia during the first part of the night, which must be compensated for by increasing nocturnal basal rates during this time.     

Mia40, a novel factor for protein import into the intermembrane space of mitochondria is able to bind metal ions

Many proteins located in the intermembrane space (IMS) of mitochondria are characterized by a low molecular mass, contain highly conserved cysteine residues and coordinate metal ions. Studies on one of these proteins, Tim13, revealed that net translocation across the outer membrane is driven by metal-dependent folding in the IMS . We have identified an essential component, Mia40/Tim40/Ykl195w, with a highly conserved domain in the IMS that is able to bind zinc and copper ions. In cells lacking Mia40, the endogenous levels of Tim13 and other metal-binding IMS proteins are strongly reduced due to the impaired import of these proteins. Furthermore, Mia40 directly interacts with newly imported Tim13 protein. We conclude that Mia40 is the first essential component of a specific translocation pathway of metal-binding IMS proteins.