Studies to analyse the relationship between IFNα2b gene dosage and its expression,using a Pichia pastoris‐based expression system

Human interferon α2b (hIFNα2b) is the most important member of the interferon family. Escherichia coli, yeasts, mammalian cell cultures and baculovirus‐infected insect cells have been used for expressing recombinant human interferon. Recently a Pichia pastoris‐based expression system has emerged as an attractive system for producing functional human recombinant IFNα2b. In this regard, gene dosage is considered an important factor in obtaining the optimum expression of recombinant protein, which may vary from one protein to another. In the present study we have shown the effect of IFNα2b gene dosage on extracellular expression of IFNα2b recombinant protein from P. pastoris. Constructs containing from one to five repeats of IFNα2b‐expressing cassettes were created via an in vitro multimerization approach. P. pastoris host strain X‐33 was transformed using these expression cassettes. Groups of P. pastoris clones transformed with different copies of the IFNα2b expression cassette were screened for intrachromosomal integration. The IFNα2b expression level of stable transformants was checked. The copy number of integrated IFNα2b was determined by performing qPCR of genomic DNA of recombinant P. patoris clones. It was observed that an increase in copy number generally had a positive effect on the expression level of IFNα2b protein. Regarding the performance of multicopy strains, those obtained from transformation of multicopy vectors showed relatively high expression, compared to those generated using transformation vector having only one copy of IFNα2b. It was also observed that an increase in drug resistance of a clone did not guarantee its high expression, as integration of a marker gene did not always correlate with integration of the gene of interest. Copyright © 2013 John Wiley & Sons, Ltd.     

Establishment of a simple method for measurement of chronic blood flow in uterine artery of pregnant cows

Blood flow to the gravid uterine horn of seven multiparous Holstein cows (mean +/- SD, BW = 625.5 +/- 82.4 kg; age = 4.7 +/- 1.7 yr; parity = 2.9 +/- 1.1 yr) was measured from d 225 of gestation to parturition using transit-time ultrasonic blood flow probes placed around the middle uterine artery. Surgery was conducted on d 215 of gestation. The cows were sedated with xylazine and local anesthesia (procaine or bupivacain hydrochloride) during surgery. The surgical operations were conducted at the flank of standing cows. A transit time ultrasonic flow probe ("S" series, diameter 12 or 14 mm, Transonic Systems Inc., Ithaca, NY) was fitted surgically around the uterine artery of each cow. Surgery was completed within 2 h of anesthesia, and the animals recovered rapidly following surgery. Uterine blood flow (UBF, L/min) was recorded at 10-s intervals for 1395 min; these values were averaged to determine UBF. Cows exhibited normal gestation lengths (279.1 +/- 7.4 d), gave birth to normal healthy calves (birth weight = 40.6 +/- 6.6 kg), and had no retained placentas. The UBF increased significantly (P < 0.01) from d 225 (6.67 +/- 2.47) to d 249 (8.23 +/- 2.89) of gestation, but the latter UBF was similar to that of d 266 (8.38 +/- 2.70). The increased UBF after d 225 indicates increased demand of nutrients of fetus with the progress of gestation. The range of mean UBF varied widely among individual cows from 4.1 L/min to 12.2 L/min. Our method is useful for chronic measurement of UBF in cows for nutritional or physiological studies and does not require sophisticated facilities or special surgical technique.     

Inactivation of Bacillus spores by the combination of moderate heat and low hydrostatic pressure in ketchup and potage

The combination effect of moderate heat and low hydrostatic pressure (MHP) on the reduction of Bacillus subtilis, Bacillus coagulans and Geobacillus stearothermophilus spores in food materials (potage and ketchup) was investigated. These bacterial spores were suspended in potage (pH 7), acidified potage (pH 4), neutralized ketchup (pH 7) and ketchup (pH 4). The suspensions were treated with and without pressure (100 MPa) and temperatures of 65-85 degrees C for 3 to 12 h. The bacterial spores were inactivated by 4-8 log cycles during MHP treatment in potage, acidified potage and ketchup, whereas the spores were highly resistant to long time heat treatment in potage and neutralized ketchup. The degrees of spore destruction were mostly dependent on pH and medium composition during MHP treatment. The inactivation effect in MHP treatment was higher at the pH 7 than at pH 4 both in ketchup and potage. The bacterial spores showed higher inactivation in potage than ketchup during MHP treatment.     

Microbiological spoilage of vacuum and modified atmosphere packaged Vietnamese Pangasius hypophthalmus fillets

This study investigated the identity, growth and metabolite production of micro-organisms causing spoilage of Pangasius hypophthalmus fillets packaged in air, vacuum and modified atmospheres (MAP) (MAP 1: 50%CO(2)-50%N(2) and MAP 2: 50%CO(2)-50%O(2)) during storage at 4 °C. Based on the time it took for psychrotrophic total colony counts to exceed 7 log cfu g(-1), the shelf life of the fillets packaged in air, vacuum, MAP 1 and MAP 2 was estimated to be 7, 10, 12 and 14 days respectively. The longest lag phases were observed in the samples packaged in MAP 2 (50%CO(2)-50%O(2)). In the fillets packaged in air and under vacuum, the dominant flora identified by partial 16S rDNA sequencing at the end of the shelf life generally consisted of Gram-negative bacteria mostly belonging to the genera Serratia and Pseudomonas. In contrast, lactic acid bacteria (Carnobacterium maltaromaticum and Carnobacterium divergens) and Brochothrix thermosphacta were identified as the dominant spoilage flora in the samples packaged under the two MAPs investigated. By means of solid-phase microextraction gas chromatography mass spectrometry (SPME GC-MS) analysis, volatile organic compounds in the headspace of the samples at the end of the shelf life were identified for each packaging condition. Based on these results, a selective ion flow tube mass spectrometry (SIFT-MS) method was developed to quantify the production of volatile metabolites during storage of the fillets. The results of these analyses indicated that several compounds contributed to the bacterial spoilage of Pangasius fillets e.g., ethanol, 2,3-butanediol, diacetyl, acetoin, ethyl acetate, acetic acid and sulfur compounds. It also emerged that the production of these compounds was dependent on the packaging condition applied.     

Immunoconcentration of Shiga toxin-producing Escherichia coli O157 from animal faeces and raw meats by using Dynabeads anti-E. coli O157 and the VIDAS system

To identify the reservoirs and routes of transmission of Shiga toxin-producing Escherichia coli (STEC) O157, sensitive detection and isolation methods are necessary. The sensitivity of traditional culture methods can be improved significantly by the inclusion of an immunoconcentration step, resulting in less false-negatives. In this report, we evaluated the results of two commercially available test systems: Dynabeads anti-E. coli O157 and the Vitek Immunodiagnostic Assay System (VIDAS) Immuno-Concentration E. coli O157 (ICE) kit. Additionally, we compared two selective isolation media for STEC O157. Statistical analysis of the results obtained for animal faecal samples (n=637) examined by both immunoconcentration methods showed that by the manual Dynabeads anti-E. coli O157 procedure systematically more samples were identified as positive than by the VIDAS ICE. In case of meat samples (n=360), no difference between the results of the two methods was found. In addition to being accurate, the Dynabeads anti-E. coli O157 method is a less expensive method than the VIDAS ICE. But, the Dynabeads method is laborious and there is a risk of cross-contamination. The VIDAS ICE procedure on the other hand is fully automated with a standardised performance; fast and safe for the user. Irrespective of the type of sample (faeces or meat) and the immunoconcentration technique applied (Dynabeads anti-E. coli O157 or VIDAS ICE) more samples were found positive after plating onto CHROMagar O157 with cefixime (0.025 mg l(-1)) and tellurite (1.25 mg l(-1)) than after plating onto sorbitol-MacConkey agar with cefixime (0.05 mg l(-1)) and tellurite (2.5 mg l(-1)). However, only in case of meat samples examined by the VIDAS ICE the difference between the isolation media was not statistically significant.