Expression and purification of the immunogenically active fragment B of the Park Williams 8 Corynebacterium diphtheriae strain toxin

The construction of a hexahistidine-tagged version of the B fragment of diphtheria toxin (DTB) represents an important step in the study of the biological properties of DTB because it will permit the production of pure recombinant DTB (rDTB) in less time and with higher yields than currently available. In the present study, the genomic DNA of the Corynebacterium diphtheriae Park Williams 8 (PW8) vaccine strain was used as a template for PCR amplification of the dtb gene. After amplification, the dtb gene was cloned and expressed in competent Escherichia coli M15™ cells using the expression vector pQE-30™. The lysate obtained from transformed E. coli cells containing the rDTB PW8 was clarified by centrifugation and purified by affinity chromatography. The homogeneity of the purified rDTB PW8 was confirmed by immunoblotting using mouse polyclonal anti-diphtheria toxoid antibodies and the immune response induced in animals with rDTB PW8 was evaluated by ELISA and dermonecrotic neutralization assays. The main result of the present study was an alternative and accessible method for the expression and purification of immunogenically reactive rDTB PW8 using commercially available systems. Data also provided preliminary evidence that rabbits immunized with rDTB PW8 are able to mount a neutralizing response against the challenge with toxigenic C. diphtheriae.

Hemodynamic mechanisms of the attenuated blood pressure response to mental stress after a single bout of maximal dynamic exercise in healthy subjects

To determine the hemodynamic mechanisms responsible for the attenuated blood pressure response to mental stress after exercise, 26 healthy sedentary individuals (age 29 ± 8 years) underwent the Stroop color-word test before and 60 min after a bout of maximal dynamic exercise on a treadmill. A subgroup (N = 11) underwent a time-control experiment without exercise. Blood pressure was continuously and noninvasively recorded by infrared finger photoplethysmography. Stroke volume was derived from pressure signals, and cardiac output and peripheral vascular resistance were calculated. Perceived mental stress scores were comparable between mental stress tests both in the exercise (P = 0.96) and control (P = 0.24) experiments. After exercise, the blood pressure response to mental stress was attenuated (pre: 10 ± 13 vs post: 6 ± 7 mmHg; P < 0.01) along with lower values of systolic blood pressure (pre: 129 ± 3 vs post: 125 ± 3 mmHg; P < 0.05), stroke volume (pre: 89.4 ± 3.5 vs post: 76.8 ± 3.8 mL; P < 0.05), and cardiac output (pre: 7.00 ± 0.30 vs post: 6.51 ± 0.36 L/min; P < 0.05). Except for heart rate, the hemodynamic responses and the mean values during the two mental stress tests in the control experiment were similar (P > 0.05). In conclusion, a single bout of maximal dynamic exercise attenuates the blood pressure response to mental stress in healthy subjects, along with lower stroke volume and cardiac output, denoting an acute modulatory action of exercise on the central hemodynamic response to mental stress.

A glycoprotein E gene-deleted bovine herpesvirus 1 as a candidate vaccine strain

A bovine herpesvirus 1 (BoHV-1) defective in glycoprotein E (gE) was constructed from a Brazilian genital BoHV-1 isolate, by replacing the full gE coding region with the green fluorescent protein (GFP) gene for selection. Upon co-transfection of MDBK cells with genomic viral DNA plus the GFP-bearing gE-deletion plasmid, three fluorescent recombinant clones were obtained out of approximately 5000 viral plaques. Deletion of the gE gene and the presence of the GFP marker in the genome of recombinant viruses were confirmed by PCR. Despite forming smaller plaques, the BoHV-1△gE recombinants replicated in MDBK cells with similar kinetics and to similar titers to that of the parental virus (SV56/90), demonstrating that the gE deletion had no deleterious effects on replication efficacy in vitro. Thirteen calves inoculated intramuscularly with BoHV-1△gE developed virus neutralizing antibodies at day 42 post-infection (titers from 2 to 16), demonstrating the ability of the recombinant to replicate and to induce a serological response in vivo. Furthermore, the serological response induced by recombinant BoHV-1△gE could be differentiated from that induced by wild-type BoHV-1 by the use of an anti-gE antibody ELISA kit. Taken together, these results indicated the potential application of recombinant BoHV-1 △gE in vaccine formulations to prevent the losses caused by BoHV-1 infections while allowing for differentiation of vaccinated from naturally infected animals.

Enhanced production and organic solvent stability of a protease fromBrevibacillus laterosporus strain PAP04

A bacterial strain (PAP04) isolated from cattle farm soil was shown to produce an extracellular, solvent-stable protease. Sequence analysis using 16S rRNA showed that this strain was highly homologous (99%) to Brevibacillus laterosporus. Growth conditions that optimize protease production in this strain were determined as maltose (carbon source), skim milk (nitrogen source), pH 7.0, 40°C temperature, and 48 h incubation. Overall, conditions were optimized to yield a 5.91-fold higher production of protease compared to standard conditions. Furthermore, the stability of the enzyme in organic solvents was assessed by incubation for 2 weeks in solutions containing 50% concentration of various organic solvents. The enzyme retained activity in all tested solvents except ethanol; however, the protease activity was stimulated in benzene (74%) followed by acetone (63%) and chloroform (54.8%). In addition, the plate assay and zymography results also confirmed the stability of the PAP04 protease in various organic solvents. The organic solvent stability of this protease at high (50%) concentrations of solvents makes it an alternative catalyst for peptide synthesis in non-aqueous media.