Purification and kinetics of two novel thermophilic extracellular proteases from Lactobacillus helveticus, from kefir with possible biotechnological interest

Two thermophilic extracellular proteases, designated Lmm-protease-Lh (29 kDa) and Hmm-protease-Lh (62 kDa), were purified from the Lactobacillus helveticus from kefir, and found active in media containing dithiothreitol; the activity of Lmm-protease-Lh was increased significantly in media containing also EDTAK₂. Both novel proteases maintained full activity at 60 °C after 1-h incubation at 10 °C as well as at 80 °C, showing optimum k_cat/K_m values at pH 7.00 and 60 °C. Only irreversible inhibitors specific for cysteine proteinases strongly inhibited the activity of both novel enzymes, while they remained unaffected by irreversible inhibitors specific for serine proteinases. Both enzymes hydrolyzed the substrate Suc-FR-pNA via Michaelis–Menten kinetics; conversely, the substrate Cbz-FR-pNA was hydrolyzed by Lmm-protease-Lh via Michaelis–Menten kinetics and by Hmm-protease-Lh via substrate inhibition kinetics. Valuable rate constants and activation energies were estimated from the temperature-(k_cat/K_m) profiles of both enzymes, and useful results were obtained from the effect of different metallic ions on their Michaelis–Menten parameters.     

Computational model of particle deposition in the nasal cavity under steady and dynamic flow

A computational model for flow and particle deposition in a three-dimensional representation of the human nasal cavity is developed. Simulations of steady state and dynamic airflow during inhalation are performed at flow rates of 9–60 l/min. Depositions for particles of size 0.5–20 μm are determined and compared with experimental and simulation results from the literature in terms of deposition efficiencies. The nasal model is validated by comparison with experimental and simulation results from the literature for particle deposition under steady-state flow. The distribution of deposited particles in the nasal cavity is presented in terms of an axial deposition distribution as well as a bivariate axial deposition and particle size distribution. Simulations of dynamic airflow and particle deposition during an inhalation cycle are performed for different nasal cavity outlet pressure variations and different particle injections. The total particle deposition efficiency under dynamic flow is found to depend strongly on the dynamics of airflow as well as the type of particle injection.     

In situ fixation of grape berries

Summary Usual immersion protocols in aldehyde solutions fail to fully preserve the fine structure of both exocarp and mesocarp cells of grape berries, especially for theveraison (onset of ripening) and post-veraison stages. In exocarp cells, fixative diffusion is hampered by the thick polysaccharide cell walls. In mesocarp cells, plasma membrane and tonoplast are disrupted before aldehyde crosslinking occurs, owing to the high osmotic pressure and cell wall texture. The fixative was therefore injected under pressure as small droplets in the outer and inner parts of the fruit, with limited changes in the steady-state organization of fruit tissues. Compared to a selective range of immersion protocols, a striking improvement in cell preservation was observed for all berry tissues, allowing new information on various compartments of grape berry cells. The preservation of organ integrity and local concentration of aldehyde molecules are the most critical parameters of improved fixation. This technique may be applicable to a large array of fleshy fruits containing mainly cells comprising a high volumetric proportion of vacuoles accumulating large amounts of organic acids and sugars and bounded by thick-walled exocarp cells.     

Heterologous expression and biochemical characterization of a polyamine oxidase from Arabidopsis involved in polyamine back conversion

Polyamine oxidase (PAO) is a flavin adenine dinucleotide-dependent enzyme involved in polyamine catabolism. Animal PAOs oxidize spermine (Spm), spermidine (Spd), and/or their acetyl derivatives to produce H2O2, an aminoaldehyde, and Spd or putrescine, respectively, thus being involved in a polyamine back-conversion pathway. On the contrary, plant PAOs that have been characterized to date oxidize Spm and Spd to produce 1,3-diaminopropane, H2O2, and an aminoaldehyde and are therefore involved in the terminal catabolism of polyamines. A database search within the Arabidopsis (Arabidopsis thaliana) genome sequence showed the presence of a gene (AtPAO1) encoding for a putative PAO with 45% amino acid sequence identity with maize (Zea mays) PAO. The AtPAO1 cDNA was isolated and cloned in a vector for heterologous expression in Escherichia coli. The recombinant protein was purified by affinity chromatography on guazatine-Sepharose 4B and was shown to be a flavoprotein able to oxidize Spm, norspermine, and N1-acetylspermine with a pH optimum at 8.0. Analysis of the reaction products showed that AtPAO1 produces Spd from Spm and norspermidine from norspermine, demonstrating a substrate oxidation mode similar to that of animal PAOs. To our knowledge, AtPAO1 is the first plant PAO reported to be involved in a polyamine back-conversion pathway.     

Prevalence of obesity in elementary schoolchildren living in Northeast Attica, Greece

Objective: To assess the prevalence of obesity and obesity trend in schoolchildren living in Northeast Attica, Greece. Research Methods and Procedures: Mean (standard deviation) and median weight and BMI were calculated in 4131 (2054 boys and 2077 girls) 6‐ to 11‐year‐old Greek schoolchildren living in Northeast Attica between November 2003 and April 2004. Two hundred thirty‐six (95 boys, 141 girls) immigrant children also participated in the study. The secular trend for obesity was determined comparing our data with those of a similar study performed in 1994. Results: Of boys, 27.8% were overweight, and 12.3% were obese. For girls, the corresponding values were 26.5% for overweight and 9.9% for obesity. There was an increase in the prevalence of overweight and obesity in the last 10 years in both sexes. For boys, overweight increased by 4.2% and obesity by 2.9%, whereas, for girls, overweight increased by 3.8% and obesity by 1.6%. Overweight and obesity were less prevalent in the immigrant children compared with their Greek peers. For immigrant boys, overweight was 15.9% and obesity was 7.9%, and for immigrant girls, overweight was 15.2% and obesity was 8.7%. Discussion: Greek schoolchildren living in Northeast Attica present a high prevalence of overweight and obesity and a positive secular change in the prevalence of obesity.     

Effect of rhythmic gymnastics on the rhythm perception of children with deafness

This study was designed to examine the effect of a rhythmic gymnastics program on the rhythm perception of children with deafness. Two groups--control and experiment--of 12 and 17 children, respectively, coming from the same school for the deaf participated in this study. The duration of the program for the individuals in the experiment group was 16 weeks (at a frequency of 3 lessons per week, for 40 minutes each lesson), while children of both groups adhered to their regular school schedules. Five rhythmic patterns in 3 speeds (tempi) were reproduced both by a metronome and each child's performance and were recorded on a digital disk before and after the application of the program. The rate of time deviation (in seconds) between the 2 beats represented the score for each child. The average rate of the 5 rhythmic patterns in each tempo was calculated separately, giving 3 scores (one for every tempo) for each child. Significance was set at p < or = 0.05. The data revealed significant postexercise differences in favor of the experiment group, an improvement of the experiment group in all pre-post values, as well as an improved medium tempo with relation to the control group. The findings show the effectiveness of the specific program in terms of improving rhythm ability, thus indicating its use in educating children with deafness on rhythm instead of preferring the routine of the adapted school program.     

Scope and limitations of the designer proline-rich antibacterial peptide dimer, A3-APO, alone or in synergy with conventional antibiotics

The proline-rich antimicrobial peptide dimer, A3-APO, was designed based on a statistical analysis of native antibacterial peptide and protein sequences. Analysis of a series of structural analogs failed to identify any single or multiple amino acid modification or architectural changes that would significantly improve its potential as a clinical therapeutic. However, a single chain Chex1-Arg20 version, a natural in vivo metabolite, showed a 2 to 8-fold increase in activity against test Enterobacteriaceae strains. In addition to bacterial species close to Escherichia coli in phylogeny, A3-APO analogs were able to effectively kill Pseudomonas aeruginosa and Staphylococcus saprophyticus. Antibacterial efficacy analysis together with biochemical experiments provided further evidence for a multiple mode of action of A3-APO that includes binding and inhibition of the bacterial heat shock protein DnaK. Through inactivating of resistance enzymes, A3-APO was able to recover the lost activity of conventional antibiotics including chloramphenicol, beta-lactams, sulfonamides or trimethoprim against multidrug resistant strains with partial or full synergy. However, the synergy appeared to be individual strain and small molecule drug combination-dependent.     

Comparison between Internalizing Anti-HER2 mAbs and Non-Internalizing Anti-CEA mAbs in Alpha-Radioimmunotherapy of Small Volume Peritoneal Carcinomatosis Using 2 1 2Pb

Background and Purpose

We assessed the contribution of antibody internalization in the efficacy and toxicity of intraperitoneal α-radioimmunotherapy (RIT) of small volume carcinomatosis using ²¹²Pb-labeled monoclonal antibodies (mAbs) that target HER2 (internalizing) or CEA (non-internalizing) receptors.

Materials and Methods

Athymic nude mice bearing 2–3 mm intraperitoneal tumor xenografts were intraperitoneally injected with similar activities (370, 740 and 1480 kBq; 37 MBq/mg) of ²¹²Pb-labeled 35A7 (anti-CEA), trastuzumab (anti-HER2) or PX (non-specific) mAbs, or with equivalent amounts of unlabeled mAbs, or with NaCl. Tumor volume was monitored by bioluminescence and survival was reported. Hematologic toxicity and body weight were assessed. Biodistribution of ²¹²Pb-labeled mAbs and absorbed dose-effect relationships using MIRD formalism were established.

Results

Transient hematological toxicity, as revealed by white blood cells and platelets numbering, was reported in mice treated with the highest activities of ²¹²Pb-labeled mAbs. The median survival (MS) was significantly higher in mice injected with 1.48 MBq of ²¹²Pb-35A7 (non-internalizing mAbs) (MS = 94 days) than in animals treated with the same activity of ²¹²Pb-PX mAbs or with NaCl (MS = 18 days). MS was even not reached after 130 days when follow-up was discontinued in mice treated with 1.48 MBq of ²¹²Pb-trastuzumab. The later efficacy was unexpected since final absorbed dose resulting from injection of 1.48 MBq, was higher for ²¹²Pb-35A7 (35.5 Gy) than for ²¹²Pb-trastuzumab (27.6 Gy). These results also highlight the lack of absorbed dose-effect relationship when mean absorbed dose was calculated using MIRD formalism and the requirement to perform small-scale dosimetry.

Conclusions

These data indicate that it might be an advantage of using internalizing anti-HER2 compared with non-internalizing anti-CEA ²¹²Pb-labeled mAbs in the therapy of small volume xenograft tumors. They support clinical investigations of ²¹²Pb-mAbs RIT as an adjuvant treatment after cytoreductive surgery in patients with peritoneal carcinomatosis.