Maximization of production of secreted recombinant proteins in Pichia pastoris fed-batch fermentation

Pontryagin's Maximum Principle has been applied for optimization of secreted proteins from Pichia pastoris fed-batch fermentation. The objective of this work is to maximize the total accumulated product per unit operation time under different given conditions and system constraints. To obtain optimal solutions, an automated curve-fitting software, Table Curve 2D, was employed to construct the necessary mathematical models and solve the complicated functions. In the solution processes, the end of the glycerol batch phase was defined as the initial state of the system, the end of the methanol fed-batch phase as the final state, the cell mass produced along with product accumulated as state variables, and the specific growth rate (mu) as the control variable. Initially, a relationship between the specific production rate (rho) and mu was established. Then, according to Pontryagin's Maximum Principle, the admissible range of mu and its trajectories for the optimal operations were determined. Four representative cases with different combinations of the operation time along with the initial and final states were evaluated. A close correlation was obtained between the predicted values of the model equation with the experimental results from the Pichia pastoris fed-batch fermentations producing secreted alpha-galactosidase. The approaches proposed here greatly simplify the computational processes and validate the optimization strategy as a generalized approach to maximize the yield from fed-batch fermentations.     

The improvement of sensitivity in lead and cadmium determinations using flame atomic absorption spectrometry

Various designs of quartz tubes of the slotted tube atom trap were examined to improve the sensitivity of flame atomic absorption spectrophotometry. The studied designs include the diameter length and the length of the upper slot of quartz tubes. Tubes having a diameter of 5 and 6 mm and an upper slot length of 2.3 cm produce the best sensitivity—as high as 6- and 10-fold enhancement for lead (Pb) and cadmium (Cd), respectively. The limits of quantitation were found to be 35 and 4 ng ml⁻¹ for Pb and Cd, respectively, by the optimized method. The achieved technique was applied to determine the concentrations of Pb and Cd in cancerous (malign) and noncancerous (adenoma) human thyroid tissues.     

Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovine interferon-tau

It was observed that during fermentative production of recombinant ovine interferon-tau (r-oIFN-tau) in Pichia pastoris, a secreted recombinant protein, the protein was degraded increasingly after 48 h of induction and the rate of degradation increased towards the end of fermentation at 72 h, when the fermentation was stopped. Proteases, whose primary source was the vacuoles, was found in increasing levels in the cytoplasm and in the fermentation broth after 48 h of induction and reached maximal values when the batch was completed at 72 h. Protease levels at various cell fractions as well as in the culture supernatant were lower when glycerol was used as the carbon source instead of methanol. It can be concluded that methanol metabolism along with cell lysis towards the end of fermentation contributes to increased proteolytic activity and eventual degradation of recombinant protein.     

Effect of Diplocystis tipulae Sherlock (Eugregarinida: Apicomplexa), a coelomic gregarine pathogen of tipulids, on the larval size of Tipula paludosa Meigen (Tipulidae: Diptera)

This study demonstrates the debilitative effect of a coelomic gregarine, Diplocystis tipulae, on Tipula paludosa. The larvae were provided contaminated fresh grass leaves from a field where 40.0% of T. paludosa larvae were infected by this pathogen. Resultant infected larvae were separated into four groups according to infection level. Analysing their weights, lengths, and weight/length ratios showed that larval size decreased as infection level increased. Differences, especially at the lower and upper levels of the infection levels, were statistically significant. It was concluded that infection by D. tipulae affected the size of T. paludosa larvae resulting in smaller individuals.     

Effects of selenium on altered mechanical and electrical cardiac activities of diabetic rat

Since selenium compounds can restore some metabolic parameters and structural alterations of diabetic rat heart, we were tempted to investigate whether these beneficial effects extend to the diabetic rat cardiac dysfunctions. Diabetes was induced by streptozotocin (50mg/kg body weight) and rats were then treated with sodium selenite (5 micromol/kg body weight/day) for four weeks. Electrically stimulated isometric contraction and intracellular action potential in isolated papillary muscle strips and transient (I(to)) and steady state (I(ss)) outward K(+) currents in isolated cardiomyocytes were recorded. Sodium selenite treatment could reverse the prolongation in both action potential duration and twitch duration of the diabetic rats, and also cause significant increases in the diminished amplitudes of the two K(+) currents. Treatment of rats with sodium selenite also markedly increased the depressed acid-soluble sulfhydryl levels of the hearts. Our data suggest that the beneficial effects of sodium selenite treatment on the mechanical and electrical activities of the diabetic rat heart appear to be due to the restoration of the diminished K(+) currents, partially, related to the restoration of the cell glutathione redox cycle.     

Chemical composition effects onto antimicrobial and antioxidant activities of propolis collected from different regions of Turkey

Chrysin, apigenin, flavonoids, flavanones, naringenin, ethyl oleate, 3-4-dimethoxy-cinnamic acid and 9-octadecenoic acid were the predominant components of propolis samples collected from different regions of Turkey. The extracts of P3 from Denizli-Ba?karci, P5 from Denizli and P7 from Tekirda? had effective antibacterial activities on Gram-negatives. Chrysin, which has antibacterial activity, was found to be high concentration. The extracts of P3, P2B from Aydin and P6 from Konya had much more effective antibacterial activities on Gram-positives. The total antioxidant activity increased with the increasing amount of extracts added to linoleic acid emulsion. All doses of propolis ethanol extract displayed antioxidant activity.     

Antioxidant and antimicrobial properties of ethanolic extract fromLepista nuda (Bull.) Cooke

Antioxidant capacity and antimicrobial activities ofLepista nuda (Bull.) Cooke extracts obtained with ethanol were investigated. Four complementary test systems, namely DPPH free radical scavenging, β-carotene/linoleic acid systems, total phenolic compounds and total flavonoid concentration, have been used. Linoleic, acid inhibition values ofL. nuda ethanolic extract, BHA and α-to copherol standards were found to be 84.3% 98.9% and 99.2% respectively in the concentration of 160μg/ml. Total flavonoid amount was 8.21 ± 0.56 μg mg^?1 quercetin equivalent while the phenolic compound amount was 48.01 ± 0.29 μg mg^?1 pyrocatechol equivalent in the extract. The antimicrobial activity ofL. nuda extract was testedin vitro by using the agar-well diffusion method. TheL. nuda extract showed antibacterial activity againstMicrococcus flavus, Micrococcus luteus, Bacillus cereus, Yersinia enterocolitica, Staphylococcus aureus, Salmonella enteritidis andEscherichia coli. TheL. nuda extract did not exhibit antican didal activity againstCandida albicans. The extracts could be suitable as antimicrobial and antioxidativeagents in the food industry.     

Effect of weight loss on P wave dispersion in obese subjects

Objective: The aim of this study was to investigate effect of loss weight on P wave dispersion in obese subjects. Research Methods and Procedures: After a 12‐week weight loss program (diet and medical therapy), a total of 30 (24 women and six men) obese subjects who had lost at least 10% of their original weight were included in the present study. All subjects underwent a routine standard 12‐lead surface electrocardiogram. Electrocardiograms were transferred to a personal computer by a scanner and then magnified 400 times by Adobe Photoshop software (Adobe Systems, Mountain View, CA). P wave dispersion, which is also defined as the difference between the maximum P wave duration and the minimum P wave duration, was also calculated. Results: After a 12‐week weight loss program, BMI (p < 0.001), maximum P wave duration (p < 0.001), and P wave dispersion (p < 0.001) significantly decreased. The mean percentage of weight loss was 13% (10% to 20.3%). The decrease in the level of P wave dispersion (21 ± 10 and 7 ± 12 ms, p < 0.002) was more prominent in Group II (≥12% loss of their original weight) than Group I (<12% loss of their original weight) after the weight loss program. A statistically significant correlation between decrease in the level of P wave dispersion and percentage of weight loss was found (r = 0.624, p < 0.001). Discussion: Substantial weight loss in obese subjects is associated with a decrease of P wave duration and dispersion. Therefore, these observations suggest that substantial weight loss is associated with improvement in atrial repolarization abnormalities in obese subjects.     

Alpha Lipoic Acid Alleviates Oxidative Stress and Preserves Blood Brain Permeability in Rats with Subarachnoid Hemorrhage

The neuroprotective effect of alpha lipoic acid (ALA; 100 mg/kg, po), a dithiol antioxidant, on experimentally induced subarachnoid hemorrhage (SAH) was assessed in Wistar albino rats. Neurological examination scores recorded at the 48th h of SAH induction were increased in SAH groups, which were accompanied with significant increases in the formation of reactive oxygen species, DNA fragmentation ratios, malondialdehyde levels and myeloperoxidase activity, while significant decreases in the brain glutathione content and Na⁺, K⁺-ATPase activity were observed. On the other hand, ALA treatment reversed all these biochemical indices as well as SAH-induced histopathological alterations. Increased brain edema, impaired blood-brain-barrier permeability and neurological scores were also improved by ALA treatment. The results demonstrate that ALA exerts neuroprotective effects via the enhancement of endogenous antioxidant enzyme activity, the inhibition of neutrophil accumulation and free radical generation, suggesting a therapeutic potential in reducing secondary injury after SAH in patients.